HGVINE AND RABBiT TO PREGNANT MARE “RUM GONADOTROPHIN Thesis {or ”w Dawn of D’I. D. WCHIGAN STATE UNIVERSITY Jack 11. Ohms 1961 TH EST 5 This is to certify that the thesis entitled THE IMMUNOLOGICAL RESPONSE OF THE BOVINE AND RABBIT TO PREGNANT MARE SE RUM G ONADOTROPHI N presented by Jack I. Ohms has been accepted towards fulfillment of the requirements for PhoDo degree in Dairy Major lessor Date ¥W 2- ”. / ?é/. L I B R A R Y Michigan State University ABSTRACT THE IMMUNOLOGICAL RESPONSE OF THE BOVINE AND RABBIT TO PREGNANT MARE SERUM GONADOTROPHIN by Jack I. Ohms Immunization with heterologous hormones results in hormone-neutralizing antibodies or antihormones. The mechanism of this neutralization is unknown and is con- founded by the presence of heterogeneous antibodies to the impure hormone. The efficacy of emulsions of Freund's adjuvant and pregnant mare serum gonadotrophin (PMS) in producing precipitating and hormone-neutralizing antibodies was demonstrated in cows and rabbits. The precipitating- antibody spectrum was demonstrated by agar-diffusion methods. The neutralization of gonadotrophin activity by the antisera was detected by assay in immature female rats. Weekly injection of emulsions of 5,000 IU of PMS and adjuvant in cows were as effective as daily injections of 1,500 IU of PMS alone. In both regimes, a total of 35,000 IU of the hormone resulted in demonstrable Jack I. Ohms hormone-neutralizing activity in the antisera. This activity resided in the water-soluble euglobulin and both water- soluble and water-insoluble pseudoglobulin fractions obtained by ammonium sulfate salting-out procedures. Fractionation of the water soluble euglobulin, by anion exchange cellulose chromatography with phosphate buffer at pH 8.0 and an ionic strength gradient, separated the hormone-neutralizing fraction at approximately u = 0.1. The hormone-neutralizing activity was not involved with the horse serum concomitants of the PMS. Absorption of the antisera with horse serum and absorption of PMS with rabbit anti-horse serum showed that hormone-neutralizing reaction was unrelated to normal horse serum antigens. A method based on obtaining the dose of antiserum which would inhibit 50%.of the gonadotrophic activity of a standard dose of hormone was found to be a repeatable means for quantitation of the hormone-neutralizing potency of antisera. Agar diffusion studies of bovine and rabbit antisera revealed a similarity in the antibody spectrum to PMS. In general, two to five antibodies were produced by horse serum concomitants in the PMS. Absorption of the antisera with horse serum left one antibody which was peculiar to PMS alone. This single antibody was identical with a single Jack I. Ohms precipitin reaction between the antisera and PMS which had been absorbed with rabbit anti-horse serum. The diffusion coefficient of 4.0 Ficks was obtained for this single antigen in PMS by means of an agar diffusion method. This is comparable with a published value of 4.2 Ficks obtained for a hormonally active fraction of PMS which was homo- geneous by starch electrophoresis. The antiserum was fractionated by anion exchange cellulose chromatography. Fractions which possessed the ability to neutralize the gonadotrophic activity of PMS also gave precipitin reactions in agar-diffusion experiments. It is concluded from the above evidence, although mainly indirect, that immunization with PMS results in an antibody which possesses both an in vitro precipitating and an in vivo neutralizing activity toward the gonadotrophin. These antibodies can be readily produced by immunization with the low sustained levels of PMS as obtained by Freund's adjuvant tedhnique. THE IMMUNOLOGICAL RESPONSE OF THE BOVINE AND RABBIT TO PREGNANT MARE SERUM GONADOTROPHIN BY Jack I? Ohms A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1961 .,. ’A ii ACKNOWLEDGEMENTS The author gratefully acknowledges the guidance and sincere interest of Dr. M. J. Gordon. His many helpful suggestions and constructive criticisms concerning the research have made this endeavor stimulating and rewarding. The generous assistance with the manuscript is also greatly appreciated. The interest and assistance of Dr. H. D. Hafs is also appreciated. The assistance with many laboratory tasks by Alan Hunter and Claude Desjardins is acknowledged. Dr. J. E. Nellor was most helpful and generous in his interest and assistance with the bioassays. The assis- tance of Dr. J. A. Williams and the Department of Veter- inary Pathology with the post-mortem studies of the cows is acknowledged. Michigan Artificial Breeders Cooperative was most helpful by its care and maintenance of the cows. The gift of the pregnant mare serum gonadotrophin (Gonadogen) by The Upjohn Company is greatly appreciated. The author gratefully acknowledges the considerable assistance and encouragement of Lois Ohms. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . ii LIST OF TABLES. . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . Chapter I. INTRODUCTION . . . . . . . . . . . . . . . 1 II. REVIEW OF LITERATURE . . . . . . . . . . . 3 Early Observations and Theories on Hormone-Inhibitory substances 3 The Immunological Nature of Antihormones 11 Response to Antigenic Stimuli of Hormones . 15 General Characteristics of the Antihormone Response 16 The Effect of the Nature of the Injected Animal 21 The Effect of Method of Injection 27 The Antigenicity of Hormones 33 Detection of Antihormones 44 Nature of Antihormones 65 Chemical Properties and Preparation of Antihormones 66 iv Effects of Antihormones in Animals 70 Specificity 78 Mechanism of Inactivation of Hormone by Specific Antibody 91 Pregnant Mare Serum or Equine Gonadotrophin 96 III. MATERIALS AND METHODS . . . . . . . . . . . . 104 Immunization 104 In Vitro Reaction of Antisera and Pregnant Mare Serum 109 In Vivo Reaction of Antisera and Pregnant Mare Serum 115 Fractionation of Antisera 120 Homogenization of Mycobacterium . butyricum 123 IV. RESULTS . . . . . . . . . . . . . . . . . . . 124 Effect of Immunization Regimen 124 Antibody Spectrum to Pregnant Mare Serum 143 Antiserum Fractionation 156 Allison—Humphrey—Diffusion—in-Gel Method 163 Immunoelectrophoresis in an Agar Matrix 163 Immunoelectrophoresis on Cellulose Acetate 166 Complement Fixation Tests 1 167 Bentonite Particle Fixation Test 168 Latex Fixation Test 169 Rat Assays for Hormone—Neutralizing Antibodies A 171 V. DISCUSSION OF RESULTS . . . . . . . . . . . 179 Immunization of Cows 179 Antibody Spectrum to Pregnant Mare Serum 182 Quantitation of the Antigonadotrophic Activity of Antisera 185 Immunodiffusion 189 * Relationship of Antigenic Site to Hormonal Site 191 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . 193 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . 197 Table LIST OF TABLES Specificity of antigonadotrophic sera. . Physical-chemical characteristics and elementary analysis of pregnant mare serum gonadotrophin. . . . . . . . . . . . . . . . Assay of serum from cow 565 for hormone- neutralizing antibodies. . . . . . . . . . . The immunization regimen of cow 3039 and the vivo and in vitro reactions of the resultant antisera . . . . . . . . . . . . . . . . . . The immunization regimen of cow 3044 and the vivo and in vitro reactions of the resultant antisera . . . . . . . . . . . . . .y. . . . The immunization regimen of cow 3053 and the vivo and in vitro reactions of the resultant antisera . . . . . . . . . . . . . . . . . . Assay for hormone neutralizing antibodies in sera of cow 3039 . . . . . . . . . . . . . . in in in vi Page 80 101 125 126 130 134 137 10. 11. 12. 13. 14. Assay of sera of cow 3044 for hormone neutralizing antibodies. . . . . . . .v. . . . . Assay of sera of cow 3053 for hormone neutralizing antibodies. . . . . . . . . . . Assay of hormone-neutralizing antibodies in the water soluble globulin fraction of serum sample 3044-3-9-61 following absorption with horse serum. . . . . . . . . . . . . . . . . . . . . Assay for hormone—neutralizing antibodies in the water soluble globulin of serum 3044-3-9—61 fractionated by anion—exchange-cellulose chromatography. Effect of pH, ionic strength (H) and levels of Tween 80 in unbuffered saline and bovine serum on autoagglutination of unsensitized latex particles. . . . . . Assay of bovine antisera for the effect of serum storage and presence of possible interfering substances . . . . . . . . . . . . . . . . . . . Antihormone assay of dilutions of serum sample 3044-5-26-59 . . . . . . . . . . . . . . . vii 138 139 157 162 170 172 173 15. l6. l7. viii Assay for excess gonadotrophin after in vitro reaction of varying levels of PMS with the water— soluble globulin fraction of 3044—3-9-61 (bovine anti-PMS serum) which had been absorbed with an excess of horse serum . . . . . . . . . . . . . 176 Effect of correction of ovarian weight to an equalized bodyweight of 100 grams. . . . . . . . 178 Data from Hamburger and Ostergaard (1949) on average ovarian weight of immature rats treated with mixtures of pregnant mare serum gonado- trophin and rabbit antiserum in various combina- tions used for calculation of serum antigonado- trOphic potency by the 50% inhibition method . . . . . . . . . . . . . . . . . . . . . 187 Figure ix LIST OF FIGURES Page Template and Pattern for Cutting Wells in Agar-Gel Diffusion Plates. . . . . . . . . . lll Plate A7, Comparison of Rabbit Anti—PMS Serum with Bovine Anti-PMS Sera. Plate T5, Precipitating Antibody Spectrum of Bovine Anti—PMS Serum . . . . . . . . . . 144 Plate V9, Comparison of Rabbit Anti—PMS Sera with Bovine Anti—PMS Sera. Plate C Precipitin Reaction of Rabbit ll' Anti—PMS with Fractions of Horse Serum. Plate D Precipitin Reaction of Rabbit Anti-PMSlwith Fractions of Horse Serum. . . 148 Plate 03,Antibody Spectrum of Bovine Anti- PMS Serum. Plates W and Y, Effect of Several Methods of Disruption of M, butyricum on the Reaction with Antiserum. . . . . . . . . . . 151 Plate F Reaction of Serum of Cows 3044 1a' and 3053 with M, butvricum. Plate U Demonstration of a Specific 14’ Antigen in PMS . . . . . . . . . . . . . . . 154 Plates X10, Y10' and Zlo Reaction of PMS With the Water-Soluble Globulin of 3044-3-9—61 Fractionated by Anion Exchange Cellulose Chromatography . . . . . . . . . . . . c . . 160 Plate X14, Determination of the Diffusion Coefficient of the PMS-Specific Antigen. Plate IE-2, Immunoelectrophoretic Analysis of the Reaction of Horse Serum and Bovine Anti-PMS . . . . . . . . . . . . . . . . . . 164 Estimation of the 50% Inhibiting Dose of Serum Antigonadotrophin. . . . . . . . . . . 175 Estimation of the 50% Inhibiting Dose of Serum Antigonadotrophin. . . . . . . . . . . 188 .0. u~ -. CHAPTER I INTRODUCTION Acquired resistance to injected hormones in animals is often noticed after prolonged administration. This acquired resistance is transferrable through the serum to other animals. The preponderance of evidence pertaining to this phenomenon indicates that the inhibitory substance in the serum is an antibody against the hormone. The means of inhibition of the biological activity of the hormone, as a consequence of combination with its antibody, is not well understood; i.e., the relationship of the antigenic site on the hormone molecule to the site responsi— ble for hormonal activity is unknown. Why is the relationship between the antigenic site and the hormonal site important? Both immunochemical bonding and hormonal activity are thought to be due to complemen- tarysteriochemical structure. This structural feature imparts considerable specificity to both of these reactions. The complementary structure and specificity of the antibody make it a very desirable reagent for the study of many of the physiological reactions of hormones. Immunochemical techniques are sensitive, permitting small amounts of this antibody reagent to be detected. Thus, if the stoichiometry of the reaction at the antigenic site can be correlated with the stoichiometry at the hormonal site, an immunochemical tool is available for quantitative study of the hormonal site. Also immuno- chemical means for study of steriochemistry of the anti- genic site would yield some insight into the sterio- chemistry necessary for hormonal activity if these two sites are coincidental. Considerable preliminary experimentation is necessary in order to initiate the above studies on gonadotrophins. Gonadotrophins, which are available commercially, are impure, and their use for immunization results in a heterogeneous or multiple antibody system. Therefore, in order to correlate the immunochemical reaction with bio- logical activity of gonadotrophin, it is necessary to identify and isolate the immunochemical reaction which is specific for the hormone molecule. Cows and rabbits were immunized with pregnant mare serum gonadotrophin (PMS) and the resultant antiserum studied for its antibody spectrum to this hormone. This antibody spectrum was in turn related to the in vivo hormone—inhibiting activity of the antiserum. CHAPTER II REVIEW OF LITERATURE Early Observations and Theories on Hormone—inhibitory Substances Endocrinologists have often noted that prolonged administration of the protein hormones results in decreased response of the target organ to the administered hormone. Such observations are included in reports of some of the earliest investigations of the protein hormones. These early accounts add little to the understanding of the mech- anism of acquired resistance to hormones. However, the theories advanced to explain this phenomenon and the termi— nology employed in its description provide an insight into the evolution of knowledge of the subject. Mobius (1903) was probably the first to express the notion of hormone—inhibitory substances in the blood when he observed that the serum of thyroidectomized sheep neutralized the action of thyroid hormone. Subsequently, this material was made available by the Merck Labora- tories as, "antithyreioden-Mobius." A similar inhibitory substance in the blood of normal sheep was termed, "Katechin" by Blum (1933). Schafer (1916) postulated the existence of hormone-like substances in the organism possessing inhibitory effects which he termed, "chalones" (Greek, to relax), while reserving the term, "hormone" for the endocrine excitatory principles. When Abderhalden (1918) treated animals with extracts of endocrine glands, he detected an inhibitory substance which he named and thought of as, "Abwehrfermente," a specific hormone- destroying enzyme. De Jongh (1924) coined the term, "anti- insulin" for an inhibitory substance found in certain preliminary stages of insulin preparation. An early use of the term, "antihormone" is found in the account of Wiese in 1928. He observed that testicular extract coagulated the follicular fluid of nymphomaniac cows, but had no effect on the follicular fluid of normal animals. This phenomenon was attributed to the formation of, “antihormones” in the nymphomaniac cow. The administration of testis extracts was found to be effective in increasing ovarian activity in cattle, which the author believed was due to production of "antihormones." Demonstration of an inhibitory substance as the conse- quence of treatment with an endocrine gland and the begin- nings of an immunological concept for its formation origi- rmted with Masay in 1906. He described the induction of a a- hypothyroid state in animals treated with the serum of guinea pigs and rabbits, which had previously been treated with pituitary suspensions. This condition was compared to that following thyroidectomy. Legiardi-Laura (1919, 1923, 1929) attempted to utilize antiserum from horses injected with posterior pituitary for clinical control of diabetes and hypertension. The precipitin reactions of thyroglobulin were studied by Hektoen §§_§1. (1923, 1925, 1927a, 1927b) following immunization of rabbits with rab- bit thyroglobulin, Hicks (1926) applied the precipitin technique to a study of the thyroglobulin content of various body fluids. The first reports of alteration of gonadal response came from the studies of Evans and Long (1921, 1922) on the effect of chronic treatment with extracts of beef anterior hypophysis on growth of rats. Aside from the observation of increased body weight in the injected rats, certain anomalies were noticed in the reproductive system. Estrual changes in the vaginal smear were absent or irreg- ular. The uteri were one-half as heavy as those of the controls. The ovaries, although heavier than those of the controls, were heavily luteinized about unruptured and atretic follicles with a complete absence of ripe, normal follicles. When human chorionic gonadotrophin was administered for long periods to female mice (Zondek, 1931) or rats (Collip, 1932), the weight of the ovaries increased at first but later declined to or below the normal level. A similar effect was observed in rats when they were chronically stimulated by daily implantation of rat pituitary tissue (McPhail, 1933). Treatment of male rats for 6 to 11 weeks with an extract of ox pituitaries resulted in a reduction in the weight of penis, testes, seminal vesicles, and prostate (Korenchevsky, 1930). Collip §t_§1. (1938), in a histological study of the oVaries of rats which were no longer responsive to gonadotrophin, observed that the ova~ ries exhibited the same characteristics as the ovaries of hypophysectomized rats — that is, they were atrophic, and "wheel cells" were present in the theca. In the early 1930's, Collip and his co—workers sought the source and nature of this lack of response or refrac- toriness in animals resistant to thyrotrophic hormone. Collip and Anderson (1934) demonstrated that when thyro- trophin was administered to a test animal the stimulatory effects on metabolism were inhibited by simultaneous administration of blood, as well as liver and spleen from thyrotrophin-refractory animals. Shortly thereafter, Selye et a1. (1934) reported that the sera of animals refractory to human chorionic gonadotrophin prevented the increase in ovarian weight by a test dose of the hormone when the sera and hormone were injected concurrently. In an attempt to explain these observations, Collip advanced his, "antihormone theory" and the, "principle of inverse response" (Collip, 1934a, 1935). His concept, in brief, was that for every hormone in an organism there exists an inhibitory hormone such that both work in a sys- tem of checks and balances for physiological homeostasis. These inhibitory substances or antihormones help to regu- late the action of hormones and to counteract excessive stimulation due to sudden discharge of large doses of a certain hormone into the blood stream. The detection of inhibitory substances was made possible by the lag in the termination of their production and incomplete disappear- ance once homeostasis is reestablished. Thus, he has cho- sen to invoke Schafer's, "chalone" postulate in the termi- nology of, "antihormone," "antigonadotrophin," and “antithyrotrophin,” and to reject the hypothesis that the inhibitory subatances are antibodies. Collip indicated that there was evidence that the inhibitory substances were not comparable to antibodies in the usual immunological sense. This evidence was that the inhibitory substance was detected after administration of homologous hormones. Rats were made resistant to gonado— trophin of rat pituitaries by continued implantation of rat hypophysis (Selye §t_§1,, 1934), and antiserum against sheep pituitary gonadotrophic hormone was obtained in sheep treated with sheep hypophysis extract (Collip, 1937). Also, the serum of certain humans contained a principle which inhibited the action of gonadotrophic preparations, although these patients had not been pretreated with such hormone extracts (Collip, 1935). Other evidence which Collip marshalled to support his theory was the apparent lack of strict parallelism between in vitro immunological data and in vivo inhibition tests or assays in animals. No correlation was found between the precipitin or complement binding properties and the levels of antihormone to human chorionic gonadotrophin. In most cases, these immunological reactions could be attributed to non-specific urinary antigens (Bachman, 1935; Eichbaum and Kindermann, 1935; Brandt and Goldhammer, 1936; Sulman, 1937). Gustus et a1. (1935) presented similar evidence on antiserum from monkeys injected with gonadotrophin from pregnant mare serum. In this same experiment the precipitin reaction could be eliminated by absorption of the antiserum with serum of non-pregnant mares which does not contain gonado- trophin. However, the inhibitory or antihorminal charac- teristic remained. Gegerson gt_§1, (1936) absorbed a bovine pituitary antiserum with bovine serum without remov— ing its antihormonal properties. A last line of evidence which Collip felt supported his theory concerned the lack of hormone specificity observed by some investigators. Parkes and Rowlands (1936) reported that antigonadotrophic serum obtained from rabbits treated with ox pituitary gonadotrophin prevented ovulation when injected into other rabbits. Apparently, the rabbit's endogenous gonadotrophin was inhibited by the rabbit anti— ox pituitary serum. Kupperman §t_§1. (1939) showed that antiserum from rabbits chronically treated with sheep pituitary inhibits the excessive luteinization of the ovary normally resulting from the endogenous gonadotrophic secre— tion of the castrate partner's pituitary gland in para- biotic rats. Collip §£_a1. (1940) felt that the lack of species specificity of the above antigonadotrophic sera was 10 not in agreement with the concept of antihormones as true antibodies. In summary, Collip believed that antihormones were not antibodies on the basis of three types of evidence: (1) the presence of antihormone when homologous hormones were admin— istered; (2) the lack of strict parallelism between the immunological data and the antihormone tests; and (3) the lack of strict species and hormone specificity of the anti- hormone. subsequent investigations on antihormones have rendered possible new interpretations of some of the results used by Collip in support.of his antihormone theory. Since the formulation of this theory, accumulation of evidence along lines other than those chosen by Collip casts predominantly an immunological aspect upon antihormones. Concurrently, an expansion of knowledge in immunochemistry allows alter- nate interpretations for some of the early results. There- fore, contradictory evidence to Collip's theory will be presented where appropriate in the remainder of this review. The current widespread evidence for the immunological basis for antihormones relegates Collip's theory principally to the realm of historical interest. The importance of Collip's work and conclusions is aptly summarized by Zondek and Sulman (1942): 11 "Although Collip's antihormone theory has not been accepted by the majority of authors, its importance for anti- hormone research has been outstanding. It was.this theory which first stirred up discussion of this subject and provided the impetus to more thorough study of the 'antihormone' reaction." The Immunological Nature 9; Antihormones Because the bulk of the literature supports the immunological concept of antihormones, the remainder of this review will be developed on an immunological format. Therefore, it is necessary that a clarification of some of the commonly used terms be made in an immunological sense. Chronic treatment of an animal with protein hor— mones (antigens), especially heterologous hormones, constitutes immunization. The inhibitory substance or antihormone which arises will henceforth be treated as an antibody and its demonstration in another animal in an antihormone assay as a form of passive immunization. 12 The Production of Hormone Antibodies (Antihormones) A consideration of the production of antihormonal anti- bodies requires scrutiny of the literature with respect to three main areas: (1) site of antihormone formation; (2) response to antigenic stimuli; and (3) consideration of hormones as antigens. The earliest investigations on the site of antihormone production dealt with the effect of the removal of organs and tissues on subsequent antihormone production or refrac— toriness to continued hormone injection. Gordon §£_§1. (1937) observed that loss of gonadal response to implanta— tion of rat pituitary into rats was delayed by splenectomy, but Emery (1937) was unable to repeat these results. A later attempt by Gordon and Charipper (1938) to repeat Gordon's earlier results on homozoic pituitary implants in rats was also unsuccessful. However, they were successful in showing that the loss of ovarian response was delayed in splenectomized rats if heterozoic pituitaries (sheep) were administered. The serum from splenectomized rabbits is only slightly lower in antihormone titer to human chorionic gonadotrophin and pregnant mare serum gonadotrophin than normal controls (Ostergaard, 1942), indicating that the spleen in rabbits plays only a minor role in antihormone formation. 13 In contrast, Guercio and Cazzola (1939) were unable to form antibodies in splenectomized rabbits to human chorionic gonadotrophin, but noted that splenectomy of refractory animals caused disappearance of the anti—~ bodies from the blood. Removal of the spleen was without effect on the neutralization of gonadotrophin in an anti— hormone assay. Although Collip and Anderson (1934) showed that saline extracts of the spleen and liver of refractory rats pos- sess antithyrotrophin, subsequent workers have been unable to demonstrate antigonadotrophins in the spleen and liver (Zondek and Sulman, 1942; Ely §t_§l,, 1949), urine (Zondek and Sulman, 1937a; Brandt and Goldhammer, 1936; DeFremery and Scheygrond, 1941; Bussard, 1952), the testis, ovary, or muscle (Zondek and Sulman, 1942). Although antigonadotrophin was not detectable in stillborn goat fetuses, it was found in high levels in the milk of a goat immunized with human chorionic gonadotrophin (DeFremery and Scheygrond, 1941). When the gonads of either sex were removed, no effect on the formation of antigonado- trophin was noted (Bachman §t_g1,, 1934; Brandt and Goldhammer, 1936; Zondek and Sulman, 1937a; Guercio and Cazzola, 1939; Zeldenrust, 1939). Hypophysectomized 14 animals form antigonadotrophin only slightly less readily than normal animals (Bachman gt_al., 1934). The participation of the reticuloendothelial system in the formation of antihormones is to be expected if anti- hormones are antibodies. Gordon gt_al, (1939a) observed that the appearance of gonadotrophin refractoriness was markedly delayed if trypan blue injection was used to blockade the reticuloendothelial system. This technique in conjunction with splenectomy indicates that the pro— duction of the antagonistic principle is not peculiar to the spleen, but is possessed by the reticuloendothelial system as a whole. An investigation that lymphoid tissue as a whole and the lymphocyte in particular is the source of antihormone was made by Ely, gt_§1, (1949). Homogenates of rabbit lymph node, spleen, liver, bone marrow, cecum, and thymus were found to be devoid of antigonadotrophin. Lymph plasma collected from the thoracic duct demonstrated an antigonadotrophic content equivalent to that in blood, whereas the lymphocytes, separated from the thoracic duct lymph in the procurement of the active lymph plasma, were without antigonadotrophic activity. Injection of a crude sheep pituitary extract into the sole of the foot of rats 15 and rabbits resulted, respectively, in increased pop- liteal lymph gland weight and a lack of demonstrable inhibitory substance in the gland. Lymphocytopenia induced by injection of adrenal cortex extracts or lymph node antiserum failed to alter the antihormone level in serum, although increased antibody titers are ordinar- ily observed from this treatment. From the foregoing, it is surmised that the site of antihormone production, like other antibodies in general, is not well understood. However, formation takes place oblivious of the target organ and appears to be primar- ily humoral in existence. Response to Antigenic Stimuli of Hormones The antibody response to injection of hormone antigen is extremely variable. This variability can be categor— ized as that due to the nature of the animal injected, method of injection, and the antigenicity of the hormone. Despite considerable interaction, these variables can be dealt with separately. 16 General Characteristics of the AntihormonefiResponse. Some brief generalizations on the characteristics of the antihormone response appear necessary in order to define the framework within which the several variables to be discussed exert their effects. The antibody response to injection of gonadotrophic hormone antigen is generally characterized by a rapid appearance of antibodies. The titer of antibodies usually rises rapidly to a station- ary level which is low in relation to other commonly studied antigens. In an experiment to determine how early antigonado— trophin could be demonstrated, Hamburger (1938) gave 44 rabbits daily injections of 150 IU of pregnant mare serum. These rabbits were bled at different time inter— vals after the first injection. The gonadotrophic activ- ity of the serum from the various groups of rabbits was tested by injection into infantile female mice. The average ovarian weight obtained in these mice was used as an expression for the hormone content of the serum. Thus it was found that, under the continued daily injec- tions of 150 IU of pregnant mare serum, the gonadotro- phin content of the serum increased and reached its maximum on the 9th or 10th day. After this the gonado— trophin content of the serum fell off rapidly, reaching 17 the level of zero after 14-18 days of treatment. Anti— gonadotrophic activity could be demonstrated in 90% of the rabbits at this time, and all of the remaining rab- bits by 25 days. It was concluded that antihormone forma- tion, beginning on the 9th to 10th day, neutralized the hormone. Similarly, regression of bovine thyrotrophin activ- ity has been observed in rabbits (Hertz and Kranes, 1934). Animals treated for periods of 2 to 7 days with daily injections of saline emulsions or acid and alka- line extracts of bovine pituitary glands showed thyroid hyperplasia. Animals treated 7 to 12 days showed early regression and involutional changes with remnants of hyperplasia in their thyroids. The thyroids of animals treated 12 to 28 days showed increasing degrees of involution and finally atrophy of the epithelium and marked colloid storage. Systematic assays of antihormone during the hormone treatment (Ostergaard, 1942; Chase, 1945) show that the antihormone levels rise rapidly to a stationary low lev- el. This level is considerably lower than some of the classical systems common in immunology, such as oval- bumin and bacterial polysaccharides. 18 Discontinuance of hormone injection generally results in an initial rapid decline in the antihormone content of the serum to a low level which may persist for several months. This persistence varies with different observers. A typical anamnestic response is observed if treat- ment is renewed after a period of rest. A second injec- tion series more readily induces antigonadotrophins which persist for a greater length of time (Chase, 1945; Leathem, 1949). A curious phenomenon, which is not too well under- stood, occasionally arises during the early stages of immunization with gonadotrophin. This phenomenon, first observed by Katzman g§_al. (1936), is the appearance of a gonadotrophin-augmenting substance in the serum instead of an antigonadotrophic substance. He observed this gonadotrophin-augmenting substance in the sera of rats in which rat pituitaries had been chronically implanted. The sera from these rats displayed this augmenting or progonadotrophic response only when injected in con- junction with rat hypophyses, and not with serum alone. Subsequently, Collip (1937) and Rowlands (1938c) repor- ted that the serum of sheep treated with sheep pituitary l9 extract exhibited an enhancing effect on the gonadotro- phic action of this extract in immature female rats. Thompson (1937b) obtained augmentary sera in the horse and dog following treatment with sheep pituitary indica- ting that the prodonadotrophic response could occur also with heterozoic gonadotrophin. Collip (1937) found that the presence of this activity in the serum was transi- tory, and replaced by inhibitory activity as injections were continued. Rowlands (1939a) found that serum from a goat which had been injected with hog pituitary extract augmented the action of this hormone in immature rats. However, the same serum also exerted an inhibi- tory effect on the ovulatory effects of the pig pitui- tary extract in estrous rabbits. In addition, the serum augmented the action on the ovaries of immature female rats of extracts of ox and sheep pituitary, but inhibi- ted that of horse and human pituitary, human chorionic gonadotrophin, and pregnant mare serum. Numerous theories have been advanced to explain these unusual results but it is generally conceded that the most logical explanation is that of Ely, §t_a1. (1950) and Deutsch §£_al. (1950). The observation that progon— adotrophin was usually present in the early stage of .I n.’ 20 immunization and gave way to antigonadotrophin led these authors to theorize that this was an immunological reaction resulting from antigen excess. In a typical precipitating antigen—antibody reaction, an insoluble complex or precipitate is formed when there is an excess of antibody; however, when there is an excess of antigen, a soluble complex is formed. Therefore, it is hypo- thesized that the progonadotrophic response results because the soluble complex dissociates and slowly releases the gonadotrophin to cause augmentation. The paucity of observations of progonadotrophic response with the placental gonadotrophins as compared with the pituitary gonadotrophins would seem to support this assumption. Human chorionic gonadotrophin and pregnant mare serum have a much greater half life than the pitui- tary gonadotrophins (Parlow, 1960). Thus, a mechanism which resulted in a slowed release of gonadotrophin would be less effective in promoting augmentation for a gonadotrophin with a longer half life (Cole gt_al., 1957). The presence in certain antisera of progonadotrophic activity toward one source of gonadotrophin, and anti— gonadotrophic activity toward another is probably the 21 consequence of differential cross reactions — that is, the consequence of the degree of similarity of the hor- mones as antigens. The foregoing framework of some of the generaliza- tions and peculiarities of the response to hormone injec— tion allows a simpler and more direct approach to the effect of the nature of the injected animal, the injec- tion method, and hormone antigenicity. The Effect of the Nature of the Injected Animal. The effect of the nature of the injected animal on antihor- mone response to antigenic stimuli has been recorded with regard to the effect of age, sex, pregnancy, hypo- physectomy, and species. Although the aim of Brandt and Goldhammer (1936) was to study the effect of the non-functional gonad on anti— hormone formation, they also gained information on the ability of the antihormone-forming system of the imma— ture animal. These authors observed that serum inhibi- tory to human chorionic gonadotrophin could be readily produced in rabbits when injections were started as ear- ly as 4 weeks of age. Starting with 12-week—old albino doe rabbits, Adams (1953) obtained antihormone by injec— tion of superovulating doses of horse pituitary extract. 22 Inhibition to pig pituitary extract was reached sooner in 80—day-old rats than in 21— to 30-day~old rats (Collip §£_31., 1938). A similar observation was made (Gordon, §t_§1,, 1939b) with pregnant mare serum and human chorionic gonadotrophin. Contrasting results were obtained by Hisaw, gt_al. (1936) in monkeys. Juvenile monkeys became refractory much more rapidly than adult monkeys.' Antigonadotrophin formed in males as readily as in females (Zondek and Sulman, 1937a; Zeldenrust, 1939). Castration has no effect on an animal's ability to form antihormone (Brandt and Goldhammer, 1936; Zondek and Sulman, 1937a; Guercio and Cazzola, 1939). Hypophysectomy in rats delayed development of inhibi- tion to pig pituitary extract in comparison to normal intact rats (Collip gt_§1,, 1938). Also, antihormones have been observed to disappear more rapidly from‘ immunized rats after hypophysectomy than from intact immunized rats (Freud and Uyldert, 1947). However, pregnant mare serum will cause development of antihor- mone in hypophysectomized monkeys within 20—25 days, and these inhibitory substances persist in very high titers for 7 months (Smith, 1942). 23 During pregnancy, rats and guinea pigs formed lower antihormonal titers than normal animals (Leathem, 1949). The response of different species of animals to hor— mone antigen is difficult to evaluate because of the small number of comparative studies which have been made. Injection of a foreign substance into an animal does not guarantee an antibody response, for substances antigenic for one species may not be so for another. In general, the rabbit has been the most commonly employed species for antigonadotrophin production. However, goats are thought to be better producers of antihormone to human chorionic gonadotrophin than rabbits (Rowlands, 1938a; Zondek and Sulman, 1942). Rabbits are considered to be superior to rats in ability to form rapid and higher ti- ters of antigonadotrophin (Haupstein and Otto, 1938; Gordon, 1941; Ostergaard, 1942). Although rats were the species in which antigonado— trophin was first demonstrated (Selye gt_a1., 1934), their response to hormone antigens has been reported to vary from one laboratory to another. Ostergaard (1942) and Leathem (1949) were of the opinion that there may be strain differences in rats with regard to antihormone response. Hoch-Legiti (1942) found that ovarian refrac— toriness to human chorionic gonadotrophin was clearly 24 delayed in hooded rats injected with methylcholanthrene. These results were not obtained in albino rats. Although no antihormone assays were carried out. strain differ- ences are clearly emphasized. According to Zondek and Sulman (1942), sheep and horses are good reactors to hormone antigen. Guinea pigs have been used only nominally for antihormone production. Dogs were successfully employed by Thompson (1939). In chickens, Zavodovsky gt_al. (1937) noted testicular atrophy following a period of excessive development when young cocks received chronic treatment with pregnant mare serum. Clinical observations on humans show that anti- gonadotrophins are unquestionably detectable after pro- longed treatment with pregnant mare serum (Rowlands and Spence, 1939; Jailer and Leathem, 1940; Ostergaard, 1942; Leathem and Abarbanel, 1943), sheep FSH (Maddock, 1949), and horse pituitary gonadotrophin (Rakoff and Leathem, 1946). In the bovine species the first observations on anti- gonadotrophins were made by Casida §;_a1. (1943). Cows which were injected with sheep pituitary gonadotrophin for 35 to 40 days demonstrated a slight hormone inhibi- tion in their serum, but the ovaries were 60 times normal 25 size. The most decisive demonstration of antigonado- trophin in the bovine was made by Cole et a1. (1957) using pregnant mare serum as the antigen. Cole's experi- ments demonstrated a definite relationship between the dosage and the amount of antihormone formed. He was of the opinion that cattle are relatively poor antigonado- trophin producers. This relatively poor ability of cat- tle to produce antigonadotrophin is borne out by a num- ber of reports. Injections of three cows three times a week with 2 gram equivalents of desiccated hog pitui— tary for a period of 5 months failed to cause antigonado- trophin production (Willett and McShan, 1955). Dziuk gt_§1. (1958) injected a cow daily for 59 days with 2,000-4,000 IU of pregnant mare serum and noted that the ovaries hypertrophied for 3 weeks and then regressed to normal. Despite this demonstration of refractoriness, Dziuk and co—workers were unable to detect any antihor- mone in the serum. The only other evidence available on the bovine con- cerning antihormones comes from the literature pertaining to superovulation. Superovulation is the induction of multiple ovulation by means of large doses of gonado- trophins. When studying the number of ovulations with 26 repeated superovulations, Willett gt_a1. (1953) observed refractoriness with various combinations of the follow- ing FSH and LH containing preparations: sheep pituitary FSH, unfractionated sheep gonadotrophin, pregnant mare serum, hog pituitary FSH, and human chorionic gonado- trophin. FSH preparations were injected subcutaneously and the LH preparation intravenously. The number of cor- pora lutea in four successive superovulations with nine cows averaged 12.9, 6.0, 5.9, and 3.1. This demonstra- tion of refractoriness could not be supported by rat assay of the serum of these cows for antihormone. There appeared to be some alleviation of the refractoriness when the dosage was increased on successive superovula— tions, and thus some indication that if antibodies were present they could be overwhelmed by increased antigen. However, the refractoriness was not altered when the source of the gonadotrophin was changed. A change of antigens should have nullified the effects of antihor- monal antibodies. The possibility of some other explanation for the above results is suggested by the inability of Dziuk §t_§l, (1958) to obtain inhibitory serum or a decrease in the number of corpora lutea on successive superovula— tions with pregnant mare serum. Also Nichols (1957) 27 reports that one cow was successively superovulated 11 times with hog pituitary FSH and slaughtered. Fifty-five corpora lutea were present on the ovaries from the eleventh superovulation at the time of slaughter. Although the foregoing factors concerning the nature of the injected animal and its response to hormone anti- gen were not always well documented, an even greater and inestimable factor is probably the variation of the indi- vidual animal. The Effect of the Method of Injection. The alliance of the nature of the injected animal and the method of injection of the hormone antigen is one of close inter- action. However, without more data to complete the puz— zle, a consideration of the individual main effects is in order. The injection method has been investigated in regard to the effect of the route of injection, dosage, frequency of injection, and the use of adjuvants. The subcutaneous route of injection is most commonly used. Occasionally the intramuscular route is used, but only very rarely the intravenous method, although the latter is generally preferred by immunologists. Anti- gonadotrophin formation against a combination of sheep anterior pituitary extract and human chorionic 28 gonadotrophin is more readily induced in the rabbit by the subcutaneous route than by the intravenous route of administration (Leathem and Rakoff, 1947). On the other hand, Bussard (1952) reports a more rapid development of antibodies to human chorionic gonadotrophin in rabbits treated by the intravenous method than by the subcutan- eous route. Pigon §t_§1, (1960) was not able to detect a difference between the subcutaneous and intravenous routes of administration of pregnant mare serum in sheep by means of the rate of antigonadotrophin formation. Chase (1949) found pregnant mare serum and ACTH to be more antigenic by the subcutaneous route than when intra- venous, intracutaneous, or intraperitoneal routes were used. The discrepancies in these results may be due to the rate of disappearance or biological half-life of these hormones. The biological half-life of some of the gonado- trophins has been reported: pregnant mare serum, 26 hours in the rat (Parlow, 1960), 24 hours (Hamburger, 1938) and 26 hours (Catchpole §£_§l,, 1935) in the rabbit, and 6 days in the gelding (Catchpole gt_§1., 1935); human chorionic gonadotrophin, 4.9 hours in the rat (Parlow, 1960); and bovine LH, 15 minutes in the rat (Parlow, 1960). 29 Pregnant mare serum does not pass the renal barrier in the horse and probably not in other species as readily as human chorionic gonadotrophin. Thus the route of injec- tion with pregnant mare serum would be expected to be of lesser consequence due to its greater half-life, whereas the intravenous injection of other hormones with a short half-life might be less desirable than the slower absorp— tion from a subcutaneous site. Although the route of administration did not influ- ence the rate of formation of antigonadotrophin in sheep in the studies of Pigon, gt_§l. (1960), the dosage of pregnant mare serum given twice a week did produce a change. Antigonadotrophin activity appeared by the sec- ond week with injections of 200 IU, by the third week with 500 IU, and by the fifth week with the highest dose of 1,000 IU.Cole.gE_al. (1957) obtained antigonadotro- phin more readily in cattle with 1,500 IU than with 10,000 IU weekly. Ostergaard (1942) observed a direct relationship at the end of 4 months treatment between the amount of pregnant mare serum injected daily (1.5- 160 IU) and the amount of antihormone. Daily injections of 1.6 IU resulted in no inhibition, while 3.2 IU gave partial inhibition. A dosage of 160 IU daily did not 30 increase the inhibition beyond that obtained with 80 IU. Thompson (1937b) made daily injections of a sheep pitui- tary extract into a dog and obtained a progonadotrophic response at 20 days which persisted at 130 days. A daily dosage 5 times greater in another dog resulted also in a progonadotrophic response at 20 days, but an antigonado- trophic response appeared at 90 days. Leathem (1949) obtained neutralizing activity in sera of rabbits after 21 days of daily injections of 2.5 rat units of equine pituitary, but not with 1.0 rat unit daily. Also, a combination of sheep pituitary gonadotrophin and human chorionic gonadotrophin did not elicit antihormone forma- tion at 1.5 rat unit dosages but was active at 4.5 rat units. It would appear from the foregoing that there is an optimum level of hormone administration which results in the maximum antibody production. This level probably has to be ascertained for individual systems or combinations of animals, hormones, and injection methods. Some comparisons on the frequency of injection of hor- mone and the resultant antihormone production are note— worthy. Zondek and Sulman (1942) were of the opinion that, if the dosage of human chorionic gonadotrophin given per 31 injection was 17-33 IU, the magnitude of the dose injected was of secondary importance to daily treatment. Treating rats daily for one year resulted in refractoriness, while two injections per week did not. In cattle, antihormone response of a greater magnitude was obtained by daily injection of 1,430 IU of pregnant mare serum compared to 10,000 IU and 1,500 IU weekly (Cole §t_§1,, 1957). Leathem (1949), in a study of the influence of spaced injections on antigonadotrophic formation, obtained inhi— bitory sera in three of five rabbits injected at monthly intervals with 400 IU of pregnant mare serum. A number of attempts have been made to increase the formation and persistence of antihormones. Numerous substances are known to immunologists which will enhance the immune response. One of the earliest attempts to increase the antigenicity of a hormone was that of Eichbaum and Kindermann in 1935. These investigators were able to increase the antigenicity of human chorion- ic gonadotrophin by absorption on collodion prior to injection. Gustus §E_al. (1935) tested the possibil- ity of a nonspecific stimulation of the antigonadotro— phin activity in monkeys by injection of typhoid vaccine. No increase in the inhibitory titer of the sera was found 32 although high typhoid agglutinin titers were induced. Chase (1945) repeated this work with pregnant mare serum in rabbits and also obtained negative results. However, Chase (1945) found that the simultaneous injection of a suspension of human erythrocytes and hormone resulted in a maximum titer being achieved more rapidly during the course of injection, and that this titer was maintained for a period twice as long as when hormone alone was used. A very effective procedure for enhancement of the immune response, which has recently come into widespread usage, is the adjuvant technique of Freund (Cohn, 1952; Carpenter, 1956). The Freund adjuvant technique of incorporating antigens in a mixture of paraffin oil and mannide mono-oleate as a water in oil emulsion generally induces strong and persistent antibody formation with several injections. The addition of killed M199- bacterium butyricum to the emulsion further enhances the response. The mode of action of adjuvants is discussed in reviews by Freund (1947 and 1951). Factors in promo- ting and sustaining immune response are: prolonged absorption as well as protection of antigens against destruction and elimination, favorable cellular reaction .. .- 33 about the antigen depots, and production of multiple foci of antibody formation in lymph nodes. Freund's adjuvant technique has been used in immuni- zation procedures with insulin (Lowell and Franklin, 1949; Moloney and Coval, 1955), growth hormone (Hayashida and Li, 1958), and anterior pituitary homogenates (Anigstein _e_t__a_1., 1960) . The Antigenicity of Hormones. It must be granted that a good measure of the antigenicity of a hormone is conferred upon it by the nature of the animal injected and the injection method. However, there are certain fac- tors inherent to the hormone which affect the degree of antigencity. Of these factors, it is pertinent to dis- cuss here the effect of homologous hormones, hetero- 1ogous hormones, denaturation and chemical treatment of hormones, and purity of hormones. Most antigens are substances foreign to the injected animal. However, well established exceptions are the organ-specific antigens - testes and lens. With the advance in knowledge on immunization techniques such as the use of adjuvants, more homologous substances are being added to the list of antigens. For example, the use of adjuvants permits the production of antibodies against . 34 brain tissue, which allows experimental production of conditions similar to the pathology of allergic encephalo- myelitis. The possibility of antibodies against homolo- gous hormones likewise has been considered from the stand- point of endocrine pathology. The early attempts to find antibodies for homologous hormones were dedicated more to the testing of Collip's concept of antihormones as inher- ent homeostatic substances. These attempts were carried out by means of various autoimmunity experiments and by parabiosis. Ehrlich's rule of, ”horror autotoxicus" states that an organism does not form antibodies against its own tissues. This immunological dogma tempers many of the conclusions drawn in regard to the investigations of antihormones. For exanple, Collip (1934a, 1935) concluded that the pres- ence of gonadotrophin-inhibitory substances in rats chroni- cally implanted with rat pituitary (Selye et_§l,, 1934) diminished the possibility that antihormones were anti- body in nature. However, in view of the preponderance of immunological evidence bearing on antihormones, it now appears that the above observations as well as many others should be reviewed in terms of autoimmune phenomena. 35 There is considerable contradiction in the literature concerning investigations in the area of autoimmunity. Antigonadotrophic activity in serum of untreated normal humans toward human chorionic gonadotrophin has been reported (Collip, 1935; Laroche and Simonnet, 1936). Other investigators (Haupstein and Otto, 1938; Fellows, 1940; Leathem, 1944) have been unable to confirm these observations. Antigonadotrophin has been reported in certain cases of human pathology. Laroche and Simonnet (1936) reported that antigonadotrophin was found in the blood of an ovariectomized woman and in an oliogomenor- rheic woman. Antigonadotrophin has been observed in the urine (Durupt, Lagarde, and Bregou, 1935) and sera of patients with amenorrhea. Eisenhardt and Thompson (1939) reported antigonadotrophin in the serum Of a patient with Cushing's Syndrome. Conversely, negative results were obtained in a search for antigonadotrophin in sera of patients with primary or secondary amenorrhea and steril- ity (Zondek and Sulman, 1942), eclampsia (Ostergaard, 1942), abortion and sterility (Leathem, 1949). Human pregnancy presents an optimal experimental situ- ation for the study of production of auto-inhibitory sub— stances for hormone. The continued high level production 36 of human chorionic gonadotrophin should afford an oppor- tunity for autoimmunization. The removal of the hormone source at parturition should provide an excellent oppor- tunity to test for antibodies. Antihormone assays of sera for inhibitory substances toward human chorionic gonadotrophin during and after delivery have with only one exception yielded negative results (Fluhmann, 1935a; Eichbaum et_gl., 1937; Sulman, 1937; Zondek and Sulman, 1939; Ostergaard, 1942; Leathem and Rakoff, 1946). The one exception was a single positive case reported by Bussard (1952). Also, no antihormones were evident against human pituitary hormones (Fluhmann, 1935a; Leathem and Rakoff, 1946). The possibility that the anti- body was tied up in an inactive complex with the hormone was also tested. Methods which were shown to be able to cleave known complexes of human chorionic gonadotrophin and its inactivating antibody were employed. Alteration of pH (Zondek and Sulman, 1942) and treatment with ethanol (Bussard, 1952) failed to demonstrate the presence of antigonadotrophin either in the blood of pregnant women or pregnant mares. 37 Another type of experiment in autoimmunity which is not as rigorous as the demonstration of antibodies against an organism's own tissue, is that of demonstra— tion of antibody against homologous tissue.) The demon— stration of antigonadotrophin in rat serum after contin- ued implantation of rat pituitary is of this nature (Selye et_al., 1934). Treatment of sheep with sheep hypophysis extract (Collip, 1937; Katzman et_al., 1947) and pigs with homologous pituitary extracts resulted in antigonadotrophin. Rowlands (1938c) obtained a progo- nadotrophin response in sheep serum following treatment with sheep pituitary extract. As discussed previously, the presence of a progonadotrophic response probably indicates the presence of antibodies at a very low titer. Extended treatment of humans with human chorionic gonadotrophin generally fails to produce antihormone (Twombly, 1936; Brandt and Goldhammer, 1936; Sulman, 1937; Spence e£_§1., 1938; Haupstein and Otto, 1938; Saphir g£_§1., 1939; Leathem, 1944). Dorff (1938) administered 112,300 rat units of hormone individually over a seven- teen month period without antihormone formation. Ostergaard (1942) observed no antihormone in a patient 38 after 45 daily treatments of 3,000 IU of human chorionic gonadotrophin. On the other hand, Brown and Bradbury (1947) treated a patient with 540,000 IU of hormone during the period of a month, followed 6 months later by another 220,000 IU in a month interval, and found antihormone on the 11th and Blst days after the last injection. Another experiment which sheds some light on the anti- genicity of hormones is that of parabiosis. A type of homologous immunization is achieved by joining an intact rat with a castrated rat possessing a hypersecretory hypo- physis. This procedure circumvents the problem of altera- tion of antigenicity through the extractions or chemical manipulations when homologous hormones are injected. These studies showed that the pituitary gonadotrophin from the gonadectomized parabion caused continued ovarian stimu- lation and constant estrus in the normal female partner (Martins, 1935; DuShane et_al., 1935; MacCahey et_al., 1936; Cutuly and Cutuly, 1938). Kupperman and Meyer (1945) joined parabiotic triplet rats composed of two females joined to a middle castrate male. When one of these females was injected with antigonadotrophin, which inactivated endogenous gonadotrophin, ovarian stimulation was prevented. a; 39 The uninjected female exhibited the symptoms of hyper- stimulation listed above. Bussard (1952) indicates that these "physiological" immunizations should be cautiously considered from three standpoints: first, parabiotic operations generally can not continue long enough for immunization to take place; second, the rat is a poor antibody producer; and third, the stress of the parabiotic condition may alter the hypophyseal—adrenal cortical equilbrium which in turn could alter the immune response. In summary, it can be stated that the autoimmuniza- tion to hormones has been only rarely observed, and that special techniques are probably needed for its detection because of the inherent neutralization of both antigen and antibody. The subtle and imperceptible alteration of hormones through extraction procedures has often been thought to be the reason for production of antibodies to homologous hor- mones. Although this is difficult to prove with homolo- gous hormones, the effect of denaturation has been inves- tigated with heterologous hormones. Twombly (1936) was able to produce antigonadotrophic activity in the serum of rabbits with human chorionic 4O gonadotrophin which had been heated until only 0.3% of the original hormonal activity was left. Similar results were Obtained with a sample of human chorionic gonadotrophin which was hormonally inactive due to prolonged storage in aqueous solution. Brandt and Goldhammer (1938) established that the inactivation of human chorionic gonadotrophin by heating, oxidation, or irradiation by ultraviolet does not destroy the antigenicity of this hormone. Destruction of approximately 99% of the hormone activity of human chori- onic gonadotrophin markedly reduced the antigenicity of the hormone in the investigations of Zondek e£_al,, (1938b). Similar experiments on the urine of children showed that heating destroyed the very low hormonal level and the antigenicity. Bischoff (1948) treated unfractionated sheep pitui- tary gonadotrophin with aqueous 40% urea at 37.50 C. After six hours' treatment only 8% of the original gonado- trophin activity remained. However, this treatment only reduced the antigenicity to 63% when compared with the untreated gonadotrophin. Singer (1941) is of the opinion that salting-out pro- cedures with ammonium sulfate alter the antigenicity of pitui- tary hormones in comparison to simple saline extraction. q. n - .av ..\. y . ..-. ...¢. 41 werner (1936, 1938) prepared thyrotrophic hormone from beef pituitary by sodium sulfate and by flavianic acid precipitation methods. The sodium sulfate preparation readily caused the refractory state in male guinea pigs and the presence of inhibitory substances in the serum, whereas the flavianate preparation did not, although equal potencies of both were administered. The refractory state induced by the sodium sulfate preparation was alleviated by the subsequent administration of the flavianate prep- aration. Another factor to be considered in the antigenicity of hormones is their purity. In general, injection of a pure antigen will elicit maximal antibody production. If one immunizes with a mixture of several antigens, the proba— bility of having antibody against any given one decreases. There exists the possibility that some of the minor compo— nents will have a greater antigenicity than the major component or hormone. This situation could tax the limi- ted ability of the antibody synthesizing mechanism, and thereby reduce or limit the synthesis of antibody to the major component. Gordon (1941) injected rabbits with hormonally equiva- lent amounts of four preparations of pregnant mare serum 42 of different degrees of purity. The antisera formed against the more purified hormone preparations were more inhibitory. Similar results were obtained with antisera produced in rats injected with crude and purified preg- nant mare serum preparations. In both cases the amount of antihormone was increased by removal of concomitant, non- hormonal material from the injected extract. Somewhat similar results were obtained by Ostergaard (1942). A highly purified pregnant mare serum containing 2,000 IU of activity per milligram was compared for antigenicity with a less pure preparation containing 600 IU mg. Equivalent hormonal activity was used for immunization in both cases. The resultant antisera were not greatly different in their ability to neutralize hormone. Rabbits immunized with unpurified pregnant mare serum did not form hormone- neutralizing antibodies in the same period of time. How— ever, the latter group of rabbits received only one— thirtieth the amount of hormone that the purified groups received in the same immunization period. A similar experiment on monkeys showed that unpurified pregnant mare serum was slower to induce the production of neu- tralizing antibodies than a purified preparation. When native human chorionic gonadotrophin from serum or urine ..,. ..,. .... 43 was compared with a hormonally equivalent amount of a purified preparation for antigenicity in rabbits, neutral- izing antibodies were formed for both preparations. Again, the titer was lower in serum from rabbits immunized with the unpurified preparation. There appears to be fairly good agreement between the results of the above two inves- tigators that the presence of non—hormonal concomitants in hormone preparations reduces the amount of antihormone formed. Epinephrine, thyroxin, and the steroid hormones have not demonstrated antigenicity (Bussard, 1952). Protrac- ted treatment with estrogen and progesterone has been observed to produce a refractory state, but inhibition was not transmissable in the serum (Selye, 1941). The octapeptides, vasopressin and oxytocin, also have not shown antigenicity (Zondek and Sulman, 1942). To summarize, the response to the antigenic stimuli of hormones is: greater in older than younger animals; unaffected by the sex of the animal or castration; slightly diminished by pregnancy and hypophysectomy and elicited in most of the domestic and laboratory animals and man by heterologous gonadotrophins. There is no widely accepted plan of immunization based on a detailed ‘a't I«"-' 44 injection procedure because of considerable interaction between the route and frequency of injection, dosage, animal, and hormone. An augmentation of antigenicity is afforded by the use of adjuvants in immunization. Hor- mones from a homologous source are only rarely antigenic and those which are non-protein are probably non— antigenic. Treatment of a hormone which destroys its biological action may not destroy its antigenicity. Purification of a hormone may increase its antigenicity by reducing the antigenic concomitants which tax a limited antibody-producing mechanism. Detection of Antihormones An antigen which exhibits biological activity permits the use of two methods for the detection of specific anti— bodies. The first method is the neutralization of the biological activity in vivo (in the case of hormones, an antihormone bioassay). The second method is the in vitro immunochemical reactions. Antigonadotrophin bioassay has been performed by a number of methods. The immature female rat has been the primary assay animal. However, female rabbits (Bachman, et a1., 1934) and female mice (Twombly, 1936; Ostergaard, ‘V ,.a 45 1942) have been used. Male mice have also been used (Laroche and Simonnet, 1936). Ostergaard (1942) and Bussard (1952) recommend the use of hypophysectomized animals for the bioassay of anti- gonadotrophins because of the possible interference by endogenous pituitary gonadotrophin. This recommenda— tion is probably based on the results of Noble et a1., (1939). These investigators reported that the differ- ential neutralization of the luteinizing activity of an extract of gelding pituitary gland can be effected by much smaller amounts of antisera to an ox pituitary gland in the hypophysectomized rat than in the normal rat. This difference was thought to be due to the production of luteinizing hormone by the pituitary gland of the intact test animal, stimulated by the follicular growth in the ovary. Also, rabbit antiserum to ox pituitary gland has no neutralizing action on the effect of an extract of human chorionic gonadotrophin on the ovaries of the hypophysectomized rat. However, this same anti— serum gave a strong, though incomplete, neutralization of human chorionic gonadotrophin in normal rats. These results were interpreted as being due to action of the antiserum on the hormones of the rat's own pituitary 46 gland and not on the gonadotrophic substance injected. Although the mechanisms involved in the above results are not too clear, these investigations point out the neces- sity of considering the possible interferenCe of the pituitary in the intact immature assay animal. Despite a general awareness of this possibility, only two inves- tigations following those of Noble and co-workers are known in which hypophysectomized rats served as the test animal (Chow, 1942; Anigstein et_al., 1960). _A point of controversy in the assay method has been the preliminary mixing of the hormone and antiserum prior to injection as compared to the separate injection of each. Zondek and Sulman (1937b) obtained a greater inhi- bitory activity in a given amount of serum when the hor- mone and antibody were combined and incubated prior to injection. Ostergaard (1942) obtained only a slight difference in favor of the separate method of injection, whereas McShan et_a1, (1943) observed a significant difference in favor of the separate method. Bussard (1952) concluded that the two methods were not in exact correspondence and favored the separate method because of the possibility of non-specific reactions when the hormone and serum are mixed in vitro. This non-specific 47 reaction is probably similar to that observed by McShan and Meyer (1945). They reported that when gonadotrophin is mixed with serum, heme compounds, or insoluble metal hydroxides, a non—specific augmentation of the ovarian response results. The augmentation by these compounds is thought to be due to delayed absorption of the hormone from the injection site. This hypothesis is supported by evidence that pregnant mare serum gonadotrophin can not be augmented in this manner (McShan and Meyer, 1941). Any augmentation which might be made possible by slower absorption is thought to be nullified by the slow renal clearance of pregnant mare serum (Hamburger and Pedersen- Bjergaard, 1938). Mixing of the hormone and antiserum in vivo must also be guarded against in the separate injection method to prevent non-specific reaction. Consequently, it has been advocated that the antiserum be injected subcutaneously on one side of the belly of the rat and the hormone on the other side of the belly (Bussard 1952). Katzman et_al. (1939) recommend injection of the hormone subcutaneously on the back and the antiserum intraperitoneally. Some authors are of the opinion that subcutaneous injections on opposite sides of the rat may even become mixed in the 48 sternal region due to sagging of the fluid down the trunk. Therefore, they recommended that the hormone be injected subcutaneously in a front leg and the antiserum subcu— taneously in the hind leg on the opposite side (Fluhmann, 1935; Gustus et_§1., 1935; Brandt and Goldhammer, 1936; Gegerson §t_al., 1936; Hamburger, 1938; Meyer and WOlfe, 1939; Zondek and Sulman, 1942). A problem which may also be important in the separate injection method is that of the Danysz—effect. The amount of antigen bound to a given quantity of antibody may be increased by adding the antigen in small aliquots instead of one portion. This is known as the Danysz-effect. This effect has not been shown in in vitro mixtures of gonadotrophin and antigonadotrophin (Zondek and Sulman, 1939; Chance, 1940; Bussard, 1952). It was concluded by Bussard (1952) from in vivo studies that the increased neutralization of hormone following injection of the antiserum intravenously and the hormone subcutaneously as compared to intravenous injection of hormone and subcu- taneous injection of antiserum was due to the Danysz effect. The subcutaneously injected hormone would be taken into the circulatory system slowly and would combine in small portions with the antibodies present there. The 49 Danysz effect would explain the increased neutralization of hormone taking place when hormone is injected subcu— taneously and the antibodies intravenously, as compared to the reversal of the sites of injection. Within the method of separate injection of hormone and serum many variations are found. A procedure employing a constant amount of antibody and a variable amount of hor- mone is less often employed than one of a constant amount of hormone and variable amount of antibody. Sufficient hormone is used to give a two- to three-fold ovarian weight increase over saline injected controls. The inhi- bitory activity of the serum is quantitated in terms of the dilution of serum necessary to neutralize the ovari- an stimulating activity of the hormone. Uterine weights are also used in conjunction with ovarian weights. Considerable variation in the number of injections of hormone and antihormone has been reported. One to five injections of hormone and antiserum have been giv- en in 48 to 73 hours followed by autopsy 48 hours after the last injection. McShan §t_a1, (1943) gave nine injections of hormone and antiserum in 96 hours followed by autopsy 48 hours later. 50 A factor which conditions the choice of the injection regimen is the biological half-life of both injected sub- stances. As was previously indicated, there is consider— able variation in the biological half—life of the gonado- trophins. Thus, pregnant mare serum with its longer half—life has been given with its total dosage in only one injection (Cole §§_§l,, 1957). In a study of the rate of disappearance of goat anti—human chorionic gonado- trophin from rats, Chance (1940) observed that 50% of the activity of the antigonadotrophic serum is lost on the first day, and that 14% remained on the 10th day after injection. Similar observations were made by Zondek and Sulman (1942). The rate of disappearance of antigonado- trophic serum may be less if the antiserum is from the same species. Simonnet (1938) observed that one injec- tion of rabbit antiserum to human chorionic gonadotro- phin protects rabbits from gonadotrophic action of this hormone for as long as 3 weeks. Thompson (1941) states that a single injection of antihormone in homologous ser— um may protect an animal from the effect of the antigenic hormone for from 8 to as many as 30 days. 51 It has been shown by Hamburger and Ostergaard (1949) that only a rough estimate of the activity of antigonado— trophic serum may be obtained even when the conditions of assay are strictly standardized. When a comparison was made between large and small doses of hormone and anti— serum mixed in constant proportions, the small doses of antiserum were found to be relatively more effective in neutralizing hormone than larger quantities. The anti— gonadotrophic, "titer" of the serum was calculated with equal justification as any value between 25 and 274. This type of discrepancy is often encountered in quanti- tative studies of toxin and antitoxin reactions, and lacks an adequate explanation. Inaddition to the limitations briefly indicated above, antihormone assays are hampered by the customary disadvantages of bioassays in general— variability of test animals, interactions between the endogenous endocrine system and the test hormone, and the extreme number of variables affecting the test animal. Consequently, there has been continued interest in immunochemical techniques for the detection of antihormones, which may minimize some of the foregoing disadvantages. 52 The primary advantage of the antigen—antibody reaction is its specificity. The immunochemical techniques based on this specificity of reaction also have some unique limitations. A major limitation is that in heterogeneous systems the total result is affected by each of the indi— 'vidual systems, which then must be sorted out. Thus it ‘behooves the experimenter to verify that a reaction is ibetween the desired antigen and its specific antibody. If an immune preparation contains more than one antigen, the resulting antiserum will contain a heterogeneous mixture of antibodies. Unless one is able to separate the anti- gens in the preparation and test each one individually in a given immunochemical technique, little information is obtained. The hormone—antihormone reaction has often been far from ideal for study by immunochemical techniques. Only a few hormones have been purified, and these in recent years. Consequently, most of the hormone immunization has been done with very crude preparations, and very heterogeneous antibody systems resulted. The antigenicity of the concom— itants in these crude hormone preparations was often great- er than that of the hormone. Thus the results of the early immunochemical experiments were seldom related to the antihormone activity. 53 Ehrlich in 1934 detected complement-fixing antibodies for human chorionic gonadotrophin. Other investigators concluded that these complement-fixing antibodies were not directed against the hormone itself but against non-specific urine antigens. Antiserum against human chorionic gonado— trophin, prepared from urine of pregnant women, also gave a positive complement fixation reaction with a preparation made in the same manner from human male urine that did not contain chorionic gonadotrophin (Bachman, 1935; Brandt and Goldhammer, 1936; Howell and Soskin, 1940). In addition it was observed that the levels of complement-fixing anti- bodies and gonadotrophin inhibition properties were not parallel in blood samples taken at different times during immunization. Brandt and Goldhammer (1936) detected anti- gonadotrophin formation after 2 weeks immunization with human chorionic gonadotrophin, but only a weak complement fixation reaction. After 4 weeks the antigonadotrophin potency of the serum had reached its maximum and the com- plement fixation reaction reached a plateau at 6—7 weeks and then disappeared, whereas the antigonadotrophin persis- ted. Bachman (1935) found that antigonadotrophin disap- peared from the blood sooner than the complement—binding antibody, which could be demonstrated in the serum for as 54 long as 4 months after discontinuance of injection. Contrary to the above results, Bussard (1952) established that complement-fixing antibodies which appeared to be hormone specific could be demonstrated after extreme absorption of the antiserum with normal urine extracts. The question of alteration of hormone antigenicity by purification or extraction procedures has frequently been raised in antihormone investigations. Ostergaard (1942) prepared a non—hormonal control preparation from normal horse serum by processing the serum in the same manner as pregnant mare serum. Antiserum from rabbits immunized with purified pregnant mare serum contained complement fixing antibodies in equivalent titer for the purified hor- mone andthe non-hormonal control preparation. Absorption of the antiserum with this same control preparation elimi- nated the complement-fixing antibodies but not the biolog- ical activity. The use of complement fixation, for quantitatively measuring growth hormone in bovine blood has recently been proposed (Trenkle et a1., 1960). After establishment of the specificity of the antiserum for bovine growth hormone, a standard curve was established for complement fixation with known amounts of growth hormone. This method gave 55 estimates of growth hormone similar to those obtained using the rat tibia test. Precipitin tests have also been extensively used for detection of antihormone. As with the results from comple- ment fixation experiments, there is considerable doubt that early precipitin experiments detected a specific hormone antibody. Positive precipitin reactions have been obtained between human chorionic gonadotrophin and its antiserum (Kindermann and Eichbaum, 1936; Gegerson et a1., 1936; Twombly, 1936). However, positive reactions were also obtained with the antiserum and normal human serum or muscle proteins (Guercio and Cazzola, 1939). It has like- wise been reported that human chorionic gonadotrophin gives rise to precipitins which have no relationship to the antigonadotrophin because precipitin reactions are brought about by urinary extracts devoid of hormone (Van den Ende, 1939a; Howell and Soskin, 1940; Miyamoto, 1954). Gegerson et a1., (1936) found that absorption of a bo- vine pituitary antiserum with bovine serum removed the precipitins but not the hormone inhibiting properties. Similar results have been established with antiserum to 56 pregnant mare serum gonadotrophin by absorption with nor— mal horse serum (Van den Ende, 1941), and with antiserum to human chorionic gonadotrophin absorbed with normal hu- man serum (Gordon et al., 1939b). Meyer and WOlfe (1939) observed that immunization of monkeys with a highly puri- fied pregnant mare serum gonadotrophin resulted in anti- hormone formation but not in precipitins, whereas both precipitins and antihormone resulted from a less pure preparation. Ostergaard (1942) absorbed antiserum to puri- fied pregnant mare serum gonadotrophin with a control preparation of normal horse serum carried through the same purification procedure as the pregnant mare serum. Absorption with this nonhormonal preparation removed the precipitins from the antiserum but not the antigonado- trophin. In contrast to the above observations for the involve- ment of non—specific horse serum proteins in the reaction of pregnant mare serum gonadotrophin with precipitins in neutralizing antiserum, Chase (1945) could not detect a reaction between horse serum and an antiserum to pregnant mare serum gonadotrophin. Gordon et_gl, (1939b) obtained a slight precipitin reaction between pregnant mare serum gonadotrophin and its antiserum after the antiserum had 57 been absorbed with normal horse serum. These results indicate that the hormone itself is antigenic. Horse serum carried through the purification procedure for pregnant mare serum gonadotrophin as well as normal horse serum fail to elicit antigonadotrophic activity when used for immunization (DeFremery and Scheygrond, 1937; Meyer and Wolfe, 1939; Van den Ende, 1941; Leathem, 1949). Likewise rabbit antiserum to human serum protein does not show antigonadotrophic activity (Bachman e§_al., 1934; Chen, 1937; Zondek and Sulman, 1942). The failure to obtain precipitating antibodies in anti- sera which were known to neutralize hormone has led some authors to conclude that these antibodies are of the "blocking" or non-precipitation type (Cole et_§1,, 1957). Bussard (1948b) was able to verify this hypothesis by cleavage of the hormone—antihormone complex in inactive supernatants by ethanol treatment. Sera of rabbits immu— nized with human chorionic gonadotrophin specifically precipitated the hormone and neutralized its gonadotro— phic activity. Reaction of the hormone with excess of antibody produced a gonadotrophin inactive supernatant. However, treatment of this inactive supernatant with ethanol yielded a slight gonadotrophin activity. Bussard (1948b) 58 was also able to recover hormone activity by ethanol treat— ment of the inactive precipitates. Thus it appears that there exist both precipitating and non-precipitating anti— bodies which neutralize human chorionic gonadotrophin. It is informative at this point to discuss some results of precipitin tests carried out with hormones which are more highly purified than the gonadotrophins. Hayashida and Li (1958) have utilized antisera against purified bovine growth hormone in precipitin ring tests. Although the growth hormone was considered to be highly purified, positive reactions were obtained with bovine gamma globu- lin and prolactin. Absorption of the antiserum with these proteins left a specific titer to the bovine growth hor- mone which was the same as before absorption. One micro- gram of growth hormone was detectable by these means. The quantitative precipitin reactions of Heidelberger and Kendall were also used by Li et_al. (1960) in the study of antibody reactions to a homogeneous human growth hormone. Attempts to show a parallel between precipitin titers of antiserum to hormones and its hormone neutralizing abil- ity have met with varied success. Gustus §£_al. (1935) observed that when monkeys were immunized with pregnant mare serum, precipitating antibodies appeared first and were 59 peculiar in that they did not neutralize the hormone in vivo. The biologically active portion appeared next, and, finally, the immunological activity of the serum disap— peared while the biological activity remained intact. These conclusions are tempered somewhat by the fact that the precipitin titers observed were extremely low and their significance questionable. Only four of a total of 25 observations have a recorded titer greater than 10. Van den Ende (1939a) was not able to show a parallel between biological inhibition and precipitins. The precipitin titer reached a plateau while the biological inhibitory power continued to rise. When the biological inhibitory power was at its maximum the amount of hormone inhibited in biological tests was almost six times as much as the amount at optimal precipitation proportions with the same amount of antiserum. Contrary to the above results, Twombly (1936) noticed a marked diminution in the strength of the precipitin reac- tion, which corresponded with the loss of hormone inhibi- tion following the cessation of injections of human chor— ionic hormone in rabbits. Meyer and Wolfe (1939) found- that immunization of monkeys with purified pregnant mare serum gonadotrophin did not result in the production of 6O precipitins, whereas whole serum from pregnant mares evoked precipitins as well as antihormones. In the latter case, however, the increase and decrease of precipitins and antihormones with repeated injection of serum from pregnant mares coincided. Wolfe et_al. (1945) injected sheep pituitary extract into rabbits until antigonadotro— hins and precipitins appeared. A drop in the titer of both could be incited by an intravenous injection of sheep pituitary extract. This negative phase reaction was seen only for the precipitin titer when sheep serum alone was injected intravenously. Although not as widely used as the precipitin tech- niques, agglutination procedures have been successfully employed in detection of antibodies to hormones. An early attempt by Zondek and Sulman (1942) to use human chorionic gonadotrophin-coated microorganisms (Serratia margesans) for detection of antibodies was unsuccessful. Chase (1945. 1949) developed an agglutination technique utiliz— ing collodion particles coated with pregnant mare serum gonadotrophin, and later crystalline adrenocorticotrophic hormone. Chase (1945) observed that the titer of antibodies determined in vitro followed the in vivo antihormonal titer of rabbit antiserum to pregnant mare serum gonadotrophin. 61 Although the titers obtained in the rat antihormone assay could not be directly correlated with those obtained in vitro, it was possible to compare the two sets of results when the sera from serial bleedings during the course of immunization were studied. An increase in serological antibodies accompanied an increase in the biological inhi— bitory activity. Roitt and Doniach (1958) used, as a method for detec— tion of thyroid auto—antibodies, agglutination of tanned sheep red blood cells coated with purified thyroglobulin. Arquilla and Stavitsky (1956) also used a hemagglutina- tion method for detection of antibodies to insulin. Read and Bryan (1960) reported successful employment of a hemag— glutination procedure for detection of antibodies to puri- fied human growth hormone. These authors in turn used this antiserum in a hemagglutination-inhibition technique to routinely detect quantities of growth hormone in the vicinity of 10-15 mug. A case in which the specificity of the antibody for a hormone is verified is that of Wide and Gemzell (1960). These investigators used a rabbit antiserum against human chorionic gonadotrophin in the hemagglutination—inhibition technique for detection of pregnancy in humans by virtue of 62 the presence of human chorionic gonadotrophin in the urine. Confirmation of this immunological diagnosis of pregnancy was verified by gynecological examination, Friedman test, or the Galli-Mainini test. Thus, an indirect correlation can be said to have been demonstrated between an in vivo and an in vitro test. The possibility of inducing anaphylaxis by hormone- antihormone reaction has been studied. Zondek and Sulman (1942) were unable to induce passive or active anaphylaxis with human chorionic gonadotrophin or pregnant mare serum gonadotrophin. In vitro anaphylaxis studies have been made on guinea pig uteri sensitized to human chorionic gonadotrophin (Van den Ende, 1939b) and pregnant mare ser- um gonadotrophin (Van den Ende, 1941). Van den Ende con— cluded from these studies that the antibodies causing anaphylactic response were directed against non-hormonal concomitants in both cases. Hayashida and Li (1958) recently used active anaphylaxis experiments to detect antibodies to purified bovine growth hormone. Arthus or cutireactions have been obtained against human chorionic gonadotrophin and dilute extracts of non-pregnant female and male urines in rabbits sensitized with human chorionic gonadotrophin (Zondek and Sulman, 1942). Similar 63 results have been reported by Twombly (1936), and Howell and Soskin (1940). This technique has also been used recently for the detection of autoantibodies produced in humans with thyroid disease (Buchanan et_al,, 1958). Electrophoretic analysis of the fractions of anti- serum shows that the gamma globulin to albumin ratio decreases in an inverse relationship to the serological antibody and antihormone titers (Chase, 1945). Similar results were obtained by Thompson and Melnick (1941). Berson and Yalow (1956) have demonstrated binding of 1131 labelled insulin to the gamma globulin fraction on paper electrophoresis and ultracentrifugation of serum from insulin-treated patients. It was suggested that the insulin-binding globulin was an antibody. Skom and‘ Talmadge (1956) reported that the binding of insulin I131 to serum globulins is demonstrable in all insulin-treated subjects by precipitation of the globulins with rabbit antihuman globulin. As indicated previously the injection of an impure antigen results in a heterogeneous or multiple system of antibodies. One must eliminate the undesired antigen- antibody reactions by means of absorption or additional fractionation of the antigen and antibody preparation. 64 These tedious and risky procedures are necessary to assure specificity of reaction in the above immunochemical meth- ods. However, the recent revival of gels asya medium for the diffusion of immunological reactants (Ouchterlony, 1958) circumvents some of the limitations outlined above for immunochemical procedures. Qualitative analysis of multiple systems is made possible by double diffusion of antigen and antibody toward each other through media sta- bilized by agar gel. Resolution of simple antigen-antibody reactions as precipitin lines is brought about by differ— ences in the diffusion coefficients of the individual reactants. Diffusion-in-gel methods have only recently been ap- plied to the hormone-antihormone reaction. Lunenfeld et a1. (1961) reported that human menopausal urine gonado- trophin contains more than one immunogenic component. However, only one of the components demonstrates biologi- cal activity when separated by solid matrix electro- phoresis. Growth hormone from several species has been studied in regard to its antigenic spectrum by the agar gel method (Anigstein et_al,, 1960; Li et_al., 1960; Read and Bryan, 1960). 65 In summary, the detection the antibodies to hormones has been carried out in vivo by means of neutralization of a standard test dose of gonadotrophin and in vitro by the various standard immunochemical techniques. Many varia— tions in these techniques have been utilized by different investigators. Much of the contradiction which exists in the literature arises from the immunization of animals with impure hormone preparations. The multiple system of antibodies, which results, makes the customary immuno- chemical tests difficult to interpret. Improvement in the detection of antibodies to hormones has been made possible by additional purification of some of the hormones and use of diffusion-in-gel methods of resolving multiple systems. Nature of Antihormones. Additional information which points toward recognition of the immunochemical nature of the antihormones is: their chemical nature, their effect in animals, and their specificity. 66 Chemical Properties and Preparation of Antihormones. The rather low titers of hormone antibodies which are usually formed in the injected animal are often difficult to differentiate from non—specific or non—hormonal phenom- ena affecting an immunochemical test. It is thus desira- ble to purify and concentrate the antibodies. Most of these methods are those pioneered in the fractionation of serum proteins and preparation of antibodies. The pres- ence of antihormone in the y—globulin fraction of serum has been taken as added reason for acceptance of anti- hormones as antibodies. Very little is known about the chemistry of the antihormones, as most investigations have placed more emphasis on the physiological effects. The principal methods of preparation have been preci— pitation with organic solvents or salting-out procedures. The solubility of antihormones in 40% acetone and insolu- bility in 50% at room temperature has been extensively used by Zondek and co-workers for recovery of approximate- ly 90% of the total inhibitory activity (Zondek and Sulman, 1942). These same investigators found ethanol to be less satisfactory. However, a cold ethanol method similar to the procedures of Cohn has been successfully used (Deutsch, 67 1952). Salting out procedures with ammonium sulfate preci— pitates the antihormonal activity in the globulin fraction of serum. Zondek and Sulman (1937c) and Thompson (1937b) found most of the activity in the pseudoglobulin fraction, whereas Harington and Rowlands (1937) found it distributed equally between the euglobulin and the pseudoglobulin fractions. On the other hand, Bussard (1952) detected the bulk of the antihormonal activity in the euglobulin frac- tion. A survey of other methods was made by Zondek et_al. (1938a). Precipitation with tannic acid, benzoic acid, flavianic acid, and KI plus NaCl were considered unsatis- factory. Absorption and elution, using kaolin or alumina, were also unsatisfactory. Chase (1945) recovered the antihormonal activity in the y-globulin fraction of serum following moving-boundary electrophoresis in a Tiselius cell. Li et_al, (1960) purified rabbit antiserum for growth hormone by means of fractionation with cations. The serum was first treated with a cationic substance, rivanol (2- ethoxy-6, 9-diaminoacridine lactate). Most of the serum proteins precipitated out, leaving the y—globulin in solu- tion. The supernatant was then passed through an Amberlite c... '1... 1. n- (1- . _-o‘ u. " v“ “'4. n ‘1 .- n N. “h- ‘hr 4 I , u (I. 68 cation resin (IRC—50) column. The final solution was found by electrophoresis to contain 73% y-globulin. The stability of the physiological response of anti- gonadotrophins has been studied with regard to the effects of heat, pH,enzymes, irradiation, and oxidation. Heating an aqueous solution of antigonadotrophin to 800C for an hour or boiling destroyed its activity, where- as heating to 700C for an hour did not destroy its activ— ity. HEating the acetone-dried powder to 1000C for one hour also did not destroy its activity (Zondek and Sulman, 1937c). Rowlands (1937) detected no loss of antihormonal activity in an aqueous preparation at 490C for 24 hours, whereas complete loss was observed at 600C for 24 hours. Haupstein and Otto (1938) incubated sera at 56°C for 2 hours without decrease in activity. Although physical factors other than temperature are probably involved, Chase (1945) detected a loss of antihormonal activity upon repeated freezing and thawing of antisera. Treatment of antihormone with N/lO HCl does not affect its activity, whereas N/10 NaOH destroys the activity (Zondek and Sulman, 1937c). Thus treatment of aliquots of neutralized mixtures of human chorionic gonadotrophin and its antiserum with acid and alkali have been used to recover 69 the gonadotrophic and antigonadotrophic activities (wunder, 1939; Ostergaard, 1942). Pepsin and trypsin di- gestion destroys the antihormonal activity (Zondek and Sulman, 1937c). Ultraviolet irradiation and oxidation with hydrogen peroxide are without effect (Zondek and Sulman, 1937c). Investigation of the physical—chemical behavior of the antihormones indicates that they are non—dialyzable (Harington and Rowlands, 1937; Zondek and Sulman, 1942). The electrophoretic mobility is similar to the y—globulins (Thompson and Melnick, 1941; Chase, 1945; Deutsch e§_al., 1950; Bussard, 1952). Bussard (1952) reported the mobili- ties for the antihormone active fractions for the serum of 5 rabbit, goat, and horse as 0.8 x 10— , 1.2 x 10_5, and 1.3 x lO-Scm 2v-ls_l, respectively. The albumin to globulin ratio decreases inversely in relationship to serological antibody and antihormone titers (Thompson and Melnick, 1941; Chase, 1945). 70 Effects of Antihormones in Animals. The mechanism of the inhibitory action of antihormonal sera has been interpreted for the most part from observa- tions of the effects of the antihormone in animals. The observations were in reality those of the secondary ef- fects of the presence of antihormone. They can be consi— dered secondary because they are the consequence of the primary effect, i.e., neutralization of the biological action of hormone. In the case of the gonadotrophin- antigonadotrophin reaction, the inhibition can be demon- strated as occurring within the framework of the recipro— cal axis of the hypophysis and the gonad. This conclu- sion comes from observations of all phases of the gonado- trophin reaction chain - the pituitary, the gonads, and the tissues dependent on gonadal hormones. A brief inroductory statement concerning antihormone specificity is necessary for a better understanding of the current area of review. The subject of specificity will be dealt with more fully in a subsequent section. During the course of immunization with some of the gonadotro— phins, an alteration in the specificity of the neutraliz- ing activity is noted. In the early stages of immuniza— tion, the serum will usually neutralize only the injected 71 hormone. However, as immunization progresses, this speci- ficity is lost, and additional gonadotrophins from other sources are neutralized. Eventually, the lack of speci— ficity may go so far as to neutralize the immunized ani- mal's endogenous gonadotrophin. Endogenous gonadotro- phin may be inhibited also by passive immunization with either an antihormone directed specifically against the animal's endogenous hormones or by an aspecific antihor- mone which has been developed by prolonged injection of some other source of hormone. The passive participation of the pituitary in the gonadotrophin—antigonadotrophin reaction comes from a num- ber of lines of evidence. It was shown in a previous sec- tion of this review that the immune response to hormones may be moderated but not abolished by hypophysectomy. Also, simultaneous injections of gonadotrophin and anti- gonadotrophin in hypophysectomized animals fails to stimu- late the gonads (Anigstein e§_a1., 1960). Marked changes in the histology of the hypophysis have been noted by a number of investigators following prolonged administration of gonadotrophins. After a moderate period of administration the changes are similar to those of the hypophysis in pregnancy. There is a marked degranulation b“ ‘54 n. a.‘ v.. "I o._ 72 and an increase in the chromophobe cells (Zondek and Sulman, 1942). However, if the treatment is continued for long periods the hypophysis begins to resemble that of the castrated animal, i.e., the pituitaries are heavier and larger and show marked basophilism with the characteristic presence of, "Signet-ring" cells. This increase in the number of basophil cells in the pituitary is often accom— panied by a decrease in the number of acidophiles and chromophobes (Koyano, 1923; Thompson and Cushing, 1934; Evans §t_al,, 1934; Collip et_al., 1934; Fluhmann, 1936; Collip §t_al., 1938; Fluhmann, 1939; Severinghaus and Thompson, 1939). The same histopathological picture can be duplicated by injection of antigonadotrophic sera which neutralize the endogenous gonadotrophin (Severinghaus and Thompson, 1939; Finerty et_al., 1940; Meyer et_§l,, 1942; Kupperman et_al,, 1944). When the antigonadotrophin injec- tions are discontinued, the percentage of basophiles gradu- ally returns to normal with a corresponding increase in the percentage of chromophobes (Finerty et_al., 1940). Ckmparable results are obtained by injection of estradiol (Finerty et a1., 1944). r 73 Along with the alteration of the histology of the pituitary, gonadotrophin is secreted in greater than nor- mal amounts. Hypersecretion of the gonadotrophic hormone by the pituitary of immature female rats follows treat— ment with antigonadotrophic serum. This hypersecretion results in a precocious development of the ovaries and premature occurrence of the cyclic variation in the vagi- nal smears after discontinuing the injections of anti— gonadotrophic serum (Meyer and Kupperman, 1939; Kupperman et_a1., 1942). Secretion of gonadotrophin has also been demonstrated by Kupperman and Meyer (1945) in parabiotic triplet rats composed of two females joined to a middle castrated male. Injection of an antigonadotrophin into one of the females of the parabiotic triplets prevented gonadal stimulation in the injected female only, whereas the other uninjected female exhibited ovarian hypertrophy. Direct evidence of the gonadotrophin secretion of the Irypophysis in the antigonadotrophin—injection rats was cflotained by assay of the pituitary glands. The gonado— t:xnophin content of these pituitary glands was found to eXCeed that of normal glands and to approximate the gonad Es‘tiimnulating activity of the hypophysis of castrated rats (Meller et a1. , 1942) . 74 Accompanying the above picture of pituitary basophilism (hypophysis of castration) is a condition of marked gonadal atrophy similar to that of hypophysectomy. 'Characteristic "wheel cells" appear in the theca of the ovary (Thompson and Cushing, 1934; Fluhmann, 1936; Collip §t_§l,, 1938; Fluhmann, 1939; Severinghaus and Thompson, 1939; Finerty et_al., 1940, 1944). The general picture, which has been pointed out repeatedly, of gonad refractoriness to continued gonadotrophic hormone treatment is also pertinent here. The ovary is usually regressed in size and weight and heav— ily'luteinized. Despite the marked atrophy of the ovary noted above, it can be shown that it still has not lost the ability to respond to gonadotrophin stimulation. Collip et_al., (1938) observed that rats, which were refractory to the immunizing pig pituitary gonadotrophin and endogenous ggonadotrophin, gave a marked ovarian response to an injec— txion of human chorionic gonadotrophin. It was shown by (Ikflcels (1937) that thyroid glands, from rabbits which were lféaiiractory to thyrotrophin, would respond to the thyro— troPhin in perfusion experiments. Iklthough only the ovary has been considered so far, in terms of gonadal atrophy in the presence of antigonadotrophin. 75 the effects on the testes have also been recorded. Anti- gonadotrophin in either active or passive immunization results in a regression of testicular weight and aspermia, or a morphological picture similar to that seen in hypo- physectomy (Korenchevsky, 1930; Rowlands, 1937a; Thompson and Cushing, 1937; Leathem, 1949; Maddock et_al., 1957; Trabucco et a1., 1958). Maddock et a1., (1957) observed that, upon discontinuance of active gonadotrophin immuni- zation in humans, the number of seminal fluid sperm increased and reached pretreatment levels within a few months. On the contrary, Trabucco §t_§1,, (1958) tended to find this aspermia to be irreversible after treatment of oligospermic patients with pregnant mare serum. It is the opinion of some investigators that the testis is more sensitive to the antigonadotrophic factor than the ovary (Thompson and Cushing, 1937; Zondek and Sulman, 1942). In agreement with the observations on the pituitary, anonsiderable alteration of the tissues dependent upon gycuaadal hormones occurs and reproductive functions such as pregnancy are markedly affected. The accessory repro— C311<3ttive organs are atrophic and resemble those of hypo— Physectomized castrated animals. The genitalia are also EitLITCDIphied (Korenchevsky, 1930; Rowlands, 1937a; Thompson 76 and Cushing, 1937). Thompson (1939) was able to inhibit development of the reproductive system of young rats by antigonadotrophin. Marvin and Meyer (1943) reported that injection of antigonadotrophin daily from birth was with— out effect on the differentiation of the seminiferous tubules for the first 11 days, but development after 11 days was almost completely inhibited by the antihormone. Attendant with the symptomology, which indicates the similarity between hypophysectomy, castration, and the presence of antigonadotrophins, is the effect on repro— ductive function. As would be expected, a diminution of elaboration of gonadal hormones prevents estrus and alters pregnancy. The efficacy of antigonadotrophin in preventing estrus was shown by the investigations of Marvin and Meyer (1941). About 5% of the rats in a colony of Sprague—Dawley rats eaxhibited estrous periods prolonged 10 days or more. Chraries containing numerous follicles, and vaginal smears sskuowing only cornified cells were altered by injection of aaIItLigonadotrophin to atrophic ovaries and the appearance of V’Eiglinal diestrus. Ovulation is suppressed in the pubertal rat and in the rabbit when they are injected with anti— gc>riacflotrophin after mating (Parkes and Rowlands, 1936) . 77 As in the case of hypophysectomy, injection of anti- gonadotrophin inhibited implantation of the blastocyst in the rabbit or caused resorption of the fetuses if injec- tions were begun later in pregnancy (Rowlands, 1937a). Thompson (1939) induced abortion in pregnant dogs by means of intravenous injection of an antigonadotrophin. Pregnancy was terminated without any apparent side effects on either the bitch or the fetuses. Two dogs were aborted a second time by this means. The fetuses were born alive. The possibility that the antiserum contained an oxytocic principle was ruled out by assay on guinea pig uterus. Similar results were obtained by Bachman et_al. (1934), who observed that rats immunized L with human chorionic gonadotrophin were unable to con— tinue gestation to term. In contrast to the above results, Leathem (1949) was unsuccessful in causing abor- tion of 3- to 40month pregnancies in humans with a goat :serum globulin capable of inhibiting human pituitary grenadotrophin. Van Wagenen and Cole (1938) reported that In<1nkeys which were refractory to pregnant mare serum be— CZEune pregnant. These latter two reports indicate a lack (335. involvement of the gonad and pituitary gland. The c‘l'cl‘l'lflict between these results and the foregoing reports, an! 4.»; .dv O ’1 b..\ ~9- .31 .Q4 .1- A. i.. 'I t!) ‘D ‘ 78 concerning antigonadotrophins and pregnancy, is probably due to differences in the specificity of the antisera. The duplication of the effects of hypophysectomy and castration simultaneously points to the inactivation of the endogenous gonadotrophin within the circulatory sys- tem. The foregoing evidence has helped to clarify the nature of the antihormonal antibody formed. Specificity. If antigen—antibody reactions depend on sterio- chemistry for their specificity, it can be seen that a consideration of the cross reactions in which an antigen or antibody participates may be of some aid in character- izing the reactants. It is in this vein that the litera— ture pertaining to the specificity of gonadotrophin- antigonadotrophin reactions will be reviewed. Considerable confusion surrounds the interpretation <>f observations on specificity of antihormones in the lJiterature. Bussard (1952) expresses the belief that £3Satica1 reaction of the ovary in immature or hypophysec— t:<:’Jnl'lized rats, or the ovulation response in rabbits, it 79 must be realized that certain differences exist in what is being analyzed in each case. For example, the various gonadotrophin hormones contain different ratios of FSH and LH, and if the antihormone being tested selectively neutralizes one and not the other, confusing results may occur depending upon the criterion of response. With due regard for the possible shortcomings in the data, it appears that certain general conclusions can be derived from the assembled information. Consequently, Table 1, adapted from a number of sources, is presented (Zondek and Sulman, 1942; Ostergaard, 1942; Leathem, 1949; Bussard, 1952). In studying Table 1 it is helpful to distinguish between several types of specificity as outlined by Thompson et_a1. (1941). 1. Species specificity; as shown by comparison of an antiserum against gonadotrophins from species other than 'the one used for the immunization. 2. Extract specificity, revealed by comparison of an aI’l‘tigonadotrophin against a hormone prepared by a given method against the same hormone prepared by other methods. loity of antigonadotrophic sera. if Table l. Spec Axearnird 60a Axeqrnqrd UBXDTQO Axeirnqrd 398 Kleqtnqrd QIquH Axeatnqrd eutms Klearnird daaqs Aleqrnird XO SWd Klearnqtd asaoH Source of neutralized hormone autxn p uemnH OOH Axeqtnatd uemnH C H S m QOOH O-IJUQ) H 230') JJ'U'U-H 0004-) 'OmHC‘. (USO-art! G O U SPOD SOUGIBJGH PMS 8O neutralization + = no neutralization 81 coflumuflamuuomc 0c coaumnaamuuowc u + + + u + + mmm sumuesufld moorm ma l + + + + + + : .. “H. + + + : : ON + + I. + + + + : : NH + + + + . sumuesuea mmmrm mg l + + + + : : N. + l l l : : HH + + l l + + + + + : .. m + + + + + + : : m + u unmeaspem xo ea l + + + l l .. v + I mzm m d G d 3 d d H d S d S 0 d d H 0.. d r.o r.u. r.% 1.9 r.M I.u. x W 1.0 m m r.m H 1.6 1.t. 3 4. 1.g. 1.1. 1.9 S 4 1 n m D 4 m e n n o n n a. n u n a d n s 1 e n e E n. ma n. nu. u... me n. n... nu uu e m I... m I... m I... n m . . 3......“ a T. .A .A .A .A .A .A .4 .A h moseow w m ou owns a A CH£m0Huonmcoo m ocofiuoa nouflamuusoc mo oousom W eggs .1 ea 82 COHUMNflHMHHDQS 0C GOHDMNHHmnuomc n + + I I : : FN + mumuesuad ham mm + l + + + : : .V + l : : ¢N + + : : NN + + + + + + + e = m + u mnmuesuam ocezm mm + + + + + + .. : ¢ + 2 .. HN + + + + + + + + : : mH + + I MHMDASDAQ moonm m d d D .d d S d S 0 d d H PH H d H Trm T.U.. T.%1m.w T.M T..U.. X W .L.O n D Trn mm. «46 1_¥ 141. 1.q .44: 1.3 S .471 n m 9 «4m 3 n n o n n q. n u n a .d n s 1 e n e e n a... n. .... n... n. n. a. u. a e e u e m 1 m m m m e 2 muomaucm u 1 I J . 1 o A A i a .A .A i .A .. 8...... . m ou now: a A Gaseouuonmcoo o be a mGOEHO£ Umnaamwuso: mo mousom eases, .L wag 83 COflflMNflHMHflgmg 0C coflumuflamuusmc n + + + 3 I + + .. am I + : .VN + I .— mm I I + I + + whom V I I + + + 2 : NI I + + mumuflsuflm Gmesm mm + + + : : : I + I + + mcflus mumuummu cmEsm AA + I mumuflsuflm cmxoflso 5H + mumufisuflm umm mm .a G Auqu d .d .a S Au“: A“ d d 1.0 .Inw I.W TIWm I.M T.q TIM "w I.W 94% mm w.m W 1,0 1.1. 1 1. 1.q. 1.1. 1 e 1 cu 1.1 n w my 1 m J m. m.m. m. m m” m w m.® n n a I a n e a 1 1 a 1 1 1 1 1 n” nu m.u uuu mummflucm m D. E U E E D. E P. D. a E m 5 HQ U 1 1 1 1 1 x 1 1 x o co 3 .A A A A .A .A .A .A .A 0p cows 9 cfismouuowwzow my MGOEHOS cmufiamuudwc mo munsom D a I “cwsaflpzoov .H mange Table 1. (Continued) Source of neutralized hormone Gonadotrophin Axeqrnqrd Boa Kleqrnnrd UBXDTQD Azeqrnqrd 198 Axenrnqrd 1IquH Axeqrnqrd aurms Axeqrnqrd deaqs Axenrnqrd X0 SWd £121In1td ason eurxn p uemnH 93H Axeqrnnrd uemnH m 00H #00) :30) 'U'U-r-I (DO-U ch: 504M 390:) 3311319393 + + + + HCG 31 8 + + + + + + + + + + 32 3O 33 + 34 11 + 84 + + + C1 0 -r-I C-IJ om VIN p-a IUr-l NM «484 '44-) (6:3 H0) : = 4J9 :5 (DO Cc: [\Ln IIII m +I Table 1. (Continued) Source of neutralized hormone Klenrnqrd 50a Axeqrnqtd UBXDTQD Axeqrnqrd 193 Axeqrnqrd 1IquH Kleqrnqrd eurms Axeqtnqrd daaqs Axeqrnqrd X0 SWd Aleqrnqrd ason aurln p uemnH 93H Axeqrnqrd uemnH Gonadotrophin used to produce antisera GPOD 80118193293 Horse pituitary 9 neutralization +: no neutralization 85 86 lawma Ammmav umsucmcamw Ammoav mcmxo cam momma» Ammmav magma» Ahmmav cmaasm Aommav umnmz .nmmav usoummmpum cam humamnmmo Ammmav umEEmscaoo cam uccmum inmmmav acmensam Ammmav cmEQUom cam cmfiasm Ammma .mmmav .AMIMM sznumM Aoomav aflMImw campmmflca lemmav awmlmw maamm Ammmav camfinmccflm cam Esmnnoflm Ammmav flawlmw mflaaoo Ammmav cemmfiona AhmmHV commfiona Amvmav .Amlmw cmrmoz .mm .vm .mm .mm .Hm .Om .mm .mm .hm .mm .mm .wm .mm .mm .HN .ON .mH Amvmac xuouomz Ama¢mav .Hm um cmEHmQQSM O O I\ CD H H lemmav maaaoo Ammmav .Hm um sownmmmo w 0 PI H Annmmav cmaaom cam xmcGON o m H AhmmHv mcflzmso cam GommEOSE O N H Ammmav Honoflz cam poacosflm . r-I I—I Ammmav mucmmm cam mwcmazom O O ,..I Anmmav mmxumm cam mommasom Ammmma .thmav mwcmazom Amwmav oummmumumo Ammmav mopmsw 0cm Homo: Amflmav mmoxmm 0cm Ewnummd Amfimav Ewflammq Aowmav Ewnummq 0cm HwHAMb NMV'mKDFCDO‘ Ammmav aflMIMw msumso AmmmHV maonumo wcm odoumso . Fa H magma How owoo mogouwmoq 87 3. Hormone specificity; the question posed is whether or not an antigonadotrophic serum obtained with follicle stimulating hormone can render animals resis— tent to luteinizing hormone and vice versa. referring to the possibility 4. Organ specificity, of cross reactions between antisera for hypophyseal gonadotrophins and the placental gonadotrophins, or vice versa. From a perusal of the table it can be seen that, in general, pregnant mare serum demonstrates an organ speci— its antiserum does that is, with few exceptions, ficity; Human rust react with horse pituitary gonadotrophin. cfliorionic gonadotrophin (HCG) presents a general picture as does human pituitary gonado- of species specificity, In contrast to the placental gonadotrophin, trophin. antisera to pituitary gonadotrophins of the various species represented show very little specificity, with The the exception of human pituitary as noted above. complete specificity shown by both fowl antiserum to mammalian gonadotrophin and mammalian antiserum to fowl gonadotrophin is also of interest. EKtract specificity has never been shown for gonado— ‘t1=’<2PhiJI Thormones. werner (1936) prepared two thyro- One trophic extracts from cattle pituitary glands. 88 extract was obtained by means of a sodium sulfate method and the other by a flavianic acid method. When guinea pigs were injected with hormonally equivalent amounts of each it was possible to demonstrate extract specificity. The thyroids of animals which had become refractory to the sodium sulfate preparation were still sensitive to the flavianate preparation. Hormone specificity has been demonstrated by a number of investigators. Rowlands (1938b) treated rabbits with ox pituitary extract which was primarily luteinizing hormone. When this rabbit antiserum was injected togeth— er with a horse pituitary extract containing both folli— czle stimulating and luteinizing hormone, the latter hor- nuone was selectively neutralized, leaving the FSH free -tc> act. Kupperman §t_§l. (1941b) immunized rabbits with FSH prepared from sheep pituitary glands by a tryptic digestion method. The resultant antiserum inactivated the gonad stimulating activity of sheep pituitary FSH, pregnant mare serum gonadotrophin, and sheep pituitary extracts in immature female rats. The absence of anti- LH Properties in the serum was shown by the serum's ‘j-IjaflflJlj:ty to prevent the increase in size of the seminal vesicles and prostate when injected simultaneously with (1' r) ‘3) ‘ti '- ’Ll 89 LH into immature male rats. The selective neutralization of FSH in unfractionated extracts of sheep pituitary and pregnant mare serum was demonstrated when tested with the antiserum. In the interval since these categories were formula— ted, some cross reactions between antisera to gonadotro- phins and substances outside the endocrine system have been discovered. Therefore a fifth area of specificity could be designated, namely, "system specificity." Some important cross reactions between the antigonadotrophins and nonhormonal substances have been reported. Eyquem and Bussard (1949) observed that sera of animals immu- nized with human chorionic gonadotrophin showed the property of agglutinating Diplo. pneumoniae Type XIV, and precipitated the type specific polysaccharide of these bacteria and C polysaccharide of Bacillus anthracig. Test for the antigonadotrophin properties of the antisera for the above substances indicated that these antisera possessed definite gonadotrophin—inhibiting properties. The antisera for these polysaccharide substances were less inhibitory than the antisera against human chorionic gonadotrophin, Antiserum against human type A blood - grOUP Substance was also shown to be slightly antigonadotrophic. In an earlier study (Bussard and 9O Eyquem, 1947), human blood group A antiserum, in contrast to anti-O serum, was shown to have an antigonadotrophic effect on the uteri of immature rats, while the action of anti-B serum was confined to inhibition of gonadotrophic stimulation of the ovaries. Tortora (1959) reported that injection of 20 women with human chorionic gonadotrophin stimulated a marked rise in A and B blood group antibody titers in 12 cases. In summary, the nature of antihormones can be arrived at from a number of types of evidence. Antihormones from sera can be purified by methods used to isolate the y- gglobulins. Electrophoretically, they behave as other inmnune proteins. From studies on the effect of the anti- hormones within animals we conclude that they react with- .ir1 the circulatory system to inactive hormones. This is verified by the in vivo reaction of antigonadotrophin to produce the simultaneous symptoms of hypophysectomy and castration in the intact snimal without affecting the inherent responsiveness of these organs. {H163 characteristics of the reactants in gonadotrophin- antigonadotrophin reactions can be adduced from the speci- ficitY Shown by the reactants. Thus, the non-hypophyseal 9° nadOtrophins demonstrate a greater specificity of 91 reaction than the hypophyseal gonadotrophins. The importance of the antigenicity of the carbohydrate com- ponents of gonadotrophins can be inferred from the cross reaction between human chorionic gonadotrophin and the polysaccharides of type XIV pneumococci. Mechanism of Inactivation of Hormone by Specific Antibody Inherent in any discussion of the mechanism of inacti- vation of hormone by specific antibody is the question of the relationship of the site of biological activity on the hormone molecule to the site which is responsible ftxr antigenicity. This relationship can be considered “Visthin one of three degrees of proximity: (1) Antibodies are formed against antigenic sites on impurities accom- I?Eilnying the hormone; (2) The hormonal site and the Eil‘lfitzigenic site are on the same molecule, but are distinct; ( 3 ) The hormonal site and the antigenic site are one and 1t1171ee same. Antibodies are indeed formed against concomitants ESL‘:='::ompanying the hormone as has been shown by the urine I:‘:>3=-‘<3tein specific antibodies elicited by human chorionic IEJ.<:>Iaadotrophin (Bachman, 1935; Eichbaum and Kindermann, 92 1935; Brandt and Goldhammer, 1936; Howell and Soskin, 1940), and antibodies against horse serum as a conse— quence of immunization with pregnant mare serum (Ostergaard, 1942). Absorption experiments with these non-specific proteins usually leaves the antigonado- trophin intact in the antiserum. The strongest argu- ment against neutralizing antibodies forming against impurities is the fact that in vitro gonadotrophin— antigonadotrophin precipitates are inactive. This argu- ment assumes that the concomitant is a separate entity from the molecule bearing the biological activity. The Lxresence of gonadotrophin in the precipitate has been demonstrated by cleaving the complex with ethanol (IBussard,l948). Data from the report of Gordon (1941) would appear t3<> reject the hypothesis that the neutralizing antibody 41-53 formed against an impurity in the hormone preparation, Ea--J_though the author did not interpret his data in terms ‘:>3f5 this relationship. In this study, the inhibitory est<:=‘tivity of antisera, from rabbits immunized with preg— nant mare serum gonadotrophin, was quantitated on hormone b3'1‘eparations of unequal purity. When equal amounts of ihtiserum (antibody) were tested against biologically 93 equivalent quantities of the pure and impure gonado— trophin, there was no significant difference between the ovarian weight response of the two groups. This would seem to indicate that a similar amount of antigenic material was reacted in the purified and impure prepa— ration. Because an equivalent amount of unneutralized hormone still remained in both preparations, the ovari- an weight response was the same. One can interpret this stoichiometry as evidence for the hormonal and antigenic sites being on the same molecule. Bussard (1952) ob— tained similar results with human chorionic gonado— ‘trophin. The number of units of hormone necessary to (flotain equivalence with a given antiserum was the same vwlth.diverse hormone preparations. The hypothesis that the antigenic site and the hor- nKnnal site are on the same molecule but are distinct, draws support from a number of observations. The unal- tered antigenicity of hormones in which the biological aactixrity has been destroyed supports the separate site hypothesis. Inactivation of hormonal activity by heat (Brandt and Goldhammer, 1938; Zondek et al. , 1938b) or age (Twombly, 1936) did not alter the antigenicity of gonadotrophin. Bischoff (1948) inactivated 92% of the 94 hormonal activity of gonadotrophin preparation by urea treatment, but it still retained 64% of the antigenicity. These results are explainable in terms of denaturation of a hormonal site on the molecule with very little altera— tion of the antigenic site. From these results one draws the conclusion that in the native hormone molecule neutralization takes place by steric hindrance, i.e., reaction at the antigenic site could block reaction at the hormonal site. The third hypothesis, that the hormonally active sites and antigenic sites are one and the same, may be supported by the similarity between blood group sub- stuances and gonadotrophins which are both glycoproteins cxnmtaining N—acetylglucosamine and hexoses. The impor— taruze of these groups to the hormonal activity of gonado- trophin is shown by the fact that gonadotrophin is inac- tixnated enzymatically by amylase (Evans and Hauschildt, 1942,- McShan and Meyer, 1938), receptor-destroying enzyme of Vibrio cholerae, a similar enzyme of influ- enza Virus (Whitten, 1948), and enzymes of Clostridium knelcflii:;u which destroy blood group O substance (Whitten, 1949) - Schiff and Akune (1931) found that amylase des- trxzyEeCi iblood group A substance, and Burnet et al.,(l947) 95 have shown that blood group substances are substrates for the receptor destroying enzyme of y, cholorae and a simi- lar enzyme of influenza virus. The destruction of sero— logical properties of blood group substances by the en— zymes of the culture filtrate of g, welchii was demon- strated by Morgan (1946). The involvement of the carbo— hydrate moiety in the hormonal activity is further shown by the observation of Raacke §t_al., (1957) that treat- ment of gonadotrophin with periodate under mild condi- tions for a period of 5 minutes destroys approximately 75% of the original potency. N-acetylglucosamine and lmexoses may not be the only requirements necessary for gnonadotrophic activity, because Bussard and Eyquem (1947) demonStrated that the blood group substances are. not gonadotrophic in action. The immunochemical evidence for the involvement of time carbohydrate moiety in the antigenicity of gonado— ‘trogflnins is shown by the cross reactions cited previously between gonadotrophin and blood group substances, and the Polysaccharides of type XIV pneumococci and I_3_. anthracis. -ITllrEBj.teration, these data show that antibodies against ti“? £><>lysaccharides of blood group A substance, Type XIV Pneumococci, and B. anthracis were antigonadotrophic. 96 It is concluded that the above evidence shows some involvement of the carbohydrate moiety in both the hor— monal and antigenic activity of gonadotrophin. That gonadotrophin activity depends on more than the carbo- hydrate moiety is shown by the fact that biological activity is lost when the hormone is degraded by proteo— 1ytic enzymes, ketene, formaldehyde, and reducing agents. Treatment with formaldehyde, nitrous acid, heat and urea also destroys the biological activity. Therefore, the importance of the protein structure of gonadotrophins :should not be discounted by the foregoing discussion of 'the carbohydrate moiety. Pregnant Mare Serum or Equine Gonadotrophin In 1931, Cole and Hart, and Zondek independently discovered the presence of high levels of gonadotrophin le‘the blood of pregnant mares. The gonadotrophin appears in the blood at about the 27th to 43rd day of Pregnancy, at which time implantation occurs. The gonadotrophin reaches a maximum concentration between 50 and 100 days of gestation and declines to a very low level ‘b}'-1<1C3 days (Cole and Saunders, 1935). Catchpole and Eycuass (1934) determined that the gonadotrophin content 97 was not detectable before the crown-rump length reached 2 cm, and that the maximal concentration appeared at 3.5—6 cm. Aylward and Ottaway (1945) observed that the levels of gonadotrophin in pregnant ponies at a peak con- centration of 73 to 443 IU/ml were usually higher than in larger breeds of horses. Similar observations were previously recorded by Cole §§_al., (1938). The site of origin of the gonadotrophin is thought to be in the endometrial cups. Catchpole and Lyons (1934) detected the hormone in the chorion before it was detec- table in the blood. The endometrium in contact with the <:horion was found to be richer in activity than the endo— nuetrium of the infertile horn. A secretion of the endo- nuatrial cups accumulates to push the allantochorion into a asac-like structure. This secretion is very high in gtniadotrophin activity, and stains with glycoprotein stains (Clegg M” 1954) . The hormone is thought to originate in enlarged epithelial or stromal cells on the surface of the endometrial cup (Day and Rowlands, 1947; Clegg et al. , 1954). 98 The high levels of gonadotrophin which persist in the blood of the pregnant mare are thought to be due to the failure of the gonadotrophin to pass the renal threshold. No activity is found in the urine of the mare (Catchpole g§_§l., 1935), in contrast to the high levels of gonado— trophin in the urine of pregnant women. It has been shown that pregnant mare serum remains in the blood stream not only in mares but also in animals into which it is injected (Evans §t_al., 1933b). The half—life of this hormone has been reported to be 6 days in a gelding (Catchpole §t_§l,, 1935); and 24 hours (Hamburger, 1938) and 26 hours (Catchpole ML, 1935; Parlow, 1960) in riats. Supporting evidence for long retention of this gcunadotrophin is shown by the observation of Saunders and (kale (1936) that substances which augment other gonado- trophins, such as zinc, sulfate, egg albumin, and casein were without effect on pregnant mare serum. Good retention is also implied by the fact that one injection <>f tilis hormone is usually as effective as multiple injections (Cole _e_t_a_l. , 1932; Hamburger and Pedersen- Bjergaard, 1938; Emmens, 1940; Meyer and McShan, 1941). 99 Pregnant mare serum possesses both follicle— stimulating and luteinizing activity. When the hormone is given to hypophysectomized female rats in small doses, it has a predominantly follicle-stimulating effect. How— ever, when the dosage is increased, the LH effect asserts itself and produces ovulation and luteinization (Nalbandov, 1958). Evans et al. (1936) and Hellbaum (1937) claimed an actual separation of the follicle- stimulating and luteinizing fractions. Frahm and Schneider (1957) fractionated pregnant mare serum by paper electrophoresis and obtained separate components ‘witfli follicle-stimulating and luteinizing activity. kawever, Raacke et al. (1957) found only one compo- nerfl: by starch electrophoresis, and this component con- tainmed both follicle-stimulating and luteinizing activi- tyn Cole et al. (1940) also concluded that there was cnaly (one gonadotrophin responsible for both activities. flflie gonadotrophic activity in pregnant mare serum has been purified by a number of methods: salting out with sodium sulfate and ammonium sulfate (G055 and C016, .1931; Evans et al., 1936; R-imington and RonhLariciss, 1941), reduction of dielectric constant by organic solvents, such as acetone (Cartland 100 and Nelson, 1937; G033 and Cole, 1940) and alcohol (Cartland and Nelson, 1937; Rimington and Rowlands, 1941); adsorption on aluminum hydroxide (Evans gt_§1., 1933a; Gustus §t_§1., 1936), and ultracentrifugation (Severinghaus gt_§1., 1938). By solid matrix electro- phoresis on starch, Raacke §t_§l., (1957) achieved a purity of 30,000 IU per mg of protein. This is the highest biological activity achieved so far. The reported observations on elementary analysis and physical—chemical properties of pregnant mare serum gonadotrophin are reported in Table 2. The effect of the alteration of various chemical groups on the gonadotrophic activity of pregnant mare serum has been studied. Ketene (HZC:CO:) is considered to be a mild and specific acetylating agent for aqueous protein solutions. Three groups in proteins are known to react with the agent: the amino, phenolic hydroxyl, and sulfhydryl groups (Li et_a1., 1940). Li and co- workers observed that gonadotrophic activity is des- troyed by treatment of pregnant mare serum with ketene. Bischoff (1942) reported that nitrous acid also destroys gcniadotrophic activity by reaction with essential free antino groups. Cartland and Nelson (1937) destroyed the Table 2. 101 Physical—chemical characteristics and elementary analysis of pregnant mare serum gonadotrophin. Isoelectric point Sedimentation coef. Diffusion coef. Partial specific volume Extinction coef. Molecular weight Frictional ratio UV absorption max. Mobility (du/deo) Protein N (%) Amino N (%) (%) (%) Tyrosine Tryptophan Lysine (%Q (%) Arginine (%) Histidine Carbohydrate (%) Hexose (%) Hexosamine (%) Sulfur (%) Biological activity IU/mg a Bourillon b Bourillon C Bourillon d Evans and and Got (1957) (1959) and Got (1960) Hauschildt (1942) and Got U‘U‘U‘ 2.41 28,000b, 68,500C, 30,000 1.06b, 1.9C 279 mub 4.0 x 10' 10.6f .46 .89b, 3.54f .53b. 1.37f .8d .25d .1d 14.1f 15.6—17.6 (galactose)e, 16 8.3-8.46, 8h 0.50-0.85d 30,0009, 12,500 f 5f H) NmeI—‘O h Gurin (1942) (1940) (1957) Li et al. Raacke gt_§1. Rimington and Rowlands h (1944) 102 gonadotrophic activity by treatment of pregnant mare ser- um with formaldehyde, but Bischoff (1941) was unable to corroborate this observation. In general, these reac- tions appear to demonstrate the necessity of free amino groups for gonadotrophic activity. Treatment with reducing agents also appears to des- troy the gonadotrophic activity. Prolonged treatment with an excess of cysteine destroys the biological activity of pregnant mare serum, while sodium cyanide treatment results in only partial inactivation (Evans et al., 1942). On the contrary, Fraenkel—Conrat gt_§l., (1939) were not able to destroy the hormone activity of pregnant mare serum with cysteine. These observations indicate that disulfide linkages may be necessary for biological activ— ity. Enzymatic degradation indicates the necessity of the integrity of both protein and carbohydrate portions of pregnant mare serum gonadotrophin for biological activity. Trypsin and pepsin destroy the gonadotrophic activity. There is the possibility that the low pH necessary for .Pepsin action may also have been a destructive agent (Evans et al., 1933; Cartland and Nelson, 1937; Evans and HEiuschildt, 1942). Destruction of biological activity is 103 also caused by papain, carboxypeptidase, and chymotrypsin (Evans and Hauschildt, 1942). The latter investigators also reported rapid degradation by ptyalin, takadiastase, and emulsin, which indicates the necessity for the carbo— hydrate moiety. Bischoff (1944, 1946) demonstrated by means of the action of urea and heat on gonadotrophin activity that the loss of biological activity agrees with classical protein denaturation. 104 CHAPTER III MATERIALS AND METHODS Immunization Immunization of cows and rabbits with pregnant mare serum gonadotrophin was carried out with the commercial preparation, "Gonadogen“ (The Upjohn Company). This gonadotrophin was prepared by acetone extraction simi- lar to the method reported by Cartland and Nelson (1937). Antisera against normal horse serum was produced by injection of horse serum (Bacto Horse Serum, Difco Labs.). This serum was collected from horses at a slaughter house and filter-sterilized, and is normally supplied for enrichment of bacteriological culture media. When an adjuvant was employed in immunization, Freund's complete adjuvant and incomplete adjuvant were used (Bacto—Adjuvant, Difco Labs.). The complete adju- vant contains: Arlacel A (mannide monooleate), 1.5 ml; Bayol F (paraffin oil), 8.5 m1; and Mycobacterium INJtyricum, 5 mg. The incomplete adjuvant contains the saline quantities of mannide monooleate and paraffin oil, 105 but no bacteria. A water-in-oil emulsion of the aqueous solution of antigen and adjuvant was prepared at various times by one of three methods: forcible ejection of the mixture through a small gauge needle with a syringe; mixture in a tissue homoginizer; or mixture in a Servall Omnimixer blender and homogenizer. The emulsion was considered satisfactory in all three methods when a drop placed on water did not spread. Four cows were employed in the immunization for this investigation. Cow No. 565, a multiparous Holstein six years of age, was culled from the MSU herd because of chronic mastitis. This cow was injected with 1,500 IU pregnant mare serum daily, six days a week, for four weeks. Cow No. 3039, a parous Brown Swiss three years of age, was culled from the MSU because of low production. This cow was injected with 5,000 IU in 10 m1 saline at approximately weekly intervals over three and one-half months. After a five month lapse, this animal was fur— ther injected 13 times with 500 IU of gonadotrophin at two dayintervals. Two months later, injections of 5,000 IU fo gonadotrophin emulsified in complete adjuvant were 106 started, and continued at irregular intervals (Table 4) for a period of approximately one year. Cow No. 3044, a multiparous Brown Swiss four years of age, was culled from the MSU herd because of infertility. This cow was injected at weekly intervals for 12 weeks with 5,000 IU of pregnant mare serum in 5 m1 of saline emulsified in 5 ml of complete adjuvant. Subsequent injec- tions were performed at irregular intervals (Table 5) with pregnant mare serum gonadotrophin emulsified in either complete or incomplete adjuvant, for approximately two years. Cow No. 3053, a parous Brown Swiss two and one-half years of age, was culled from the MSU herd because of low production. This cow was injected weekly for 12 weeks with an emulsion of 5 ml of complete adjuvant and 5 ml of saline as a control. Subsequent injections were made with an emulsion of gonadotrophin and complete or incomplete adjuvant at irregular intervals (Table 6) for approxi— mately one year. The injections were given subcutaneously or intra— Ifluscularly in divided doses in several sites within the Ileek, crop, or thurl regions of the cows. 107 Blood was collected by vena puncture from the jugular vein, and allowed to clot. The serum was removed and centrifuged. One percent of 1:1,000 solution of merthi- olate was added and the serum stored at -20°C or lyophil- ized. The individual serum samples were identified in subsequent operations by the cow number and collection date. This same identification scheme will be used herein. Cows 3039 and 3035 were slaughtered. The blood was collected and clotted to separate the serum. The thy- roid, adrenal and pituitary glands as well as the ova— ries and uterus were collected for gross observation and histological examination. Hematocrit determinations were made on the weekly blood samples to assess the effect of repeated blood collection. These hematocrit determinations were made by sedimenta- tion in heparinized micro-hematocrit tubes. Determina- tion of total protein in the serum was also made on the weekly samples by a modification of the Folin—Ciocalteu procedure (Lowry §E_al., 1951). New Zealand White and Dutch Belted rabbits were immu- nized with pregnant mare serum by injection of an emulsion om o o ooo.m mks mmIoHIm o o ooo.m mks omImIm ooomso oz 6 o ooo.m omm omImmIe oozmso oz 6 o ooo.m mks omIHNIe suoosm 50 m I .smao o o ooo.m mes omImIe o o ooo.m mks omIan o o ooo.m mks omImmIm so m I .amflo ooo.m mks omImHIm o o ooo.m mam omIHHIm moaoaaaou oonmq o o ooo.m ooo.H omIeIm 6 mm omImmIm mownm>o mo osofiuos sasmouu aMNmmm Eamon ADHV AHEV coausoooo Iauoa Ioomooo .s omuom mzm mam .floa oon 6066 nosed cauflmflooum unmasmmua mo mGOHuummH ossfl> .mnmmflusm UGMUHSmmu on» as com o>H> CH map can mmom 300 mo cmfifimmu soaumua:SEEa 0:8 127 as o I.H.o mama EU N.Hxaxm.m Ia so N.me.axm Im omImmINH oom mmIoHImH oom omIeaImH oom omIHHINH oom mmIoImH oom mmIsImH oom mmIvaH oom omImINH oom mmIOmIHH oom omInmIHH oom omImmIHH oom omImmIHH oom mmIHmIHH EU H .EMHUlofloU m 56 m.xaxm.m In so m.axm.mxm.m Im o 0 com omv omImHIHH wwfium>o mo mGOEHOS cflnmoup amfimmm EDHmm ADHV AHEV coaoaoooo Iauc< Ioomcoo as mmuom mam mam .nom swam mono mmsHH sfluflmflomum ucwEumeB Acmscfiucooo .w magma 128 ooo.m .osoo ooIoaIm ooo.m .osoo ooImIo o o ooIHmIs ooo.m .osoo ooIeaIs ooo.m .osoo ooIoIs oom ooImIo o o ooo.ma.osoo ooIoHIm + oom ooIeIm so me.ax~ In so Nxmxm Im ooo.ma.oaoo ooIoHIv o a ooo.a ooIeIe ooo.m .osoo ooIomIm ooo.m .oaoo ooIomIm ooo.m .osoo ooIomIm ooo.m .msoo ooImHIm I o 0 com omIHmINH mosum>o mo wachon swnmouu mMNmmm Esnmm ADHV AHEV cofluaocoo Iauc< Ioomooo .m mmuom mam mam .no< swam mums moswa cfiufimwomnm usmEumwna Aomsaaucoov .v magma 129 .Hom Hamsm I o H.m In so k.H I.H.o so a. .e.a I.Hou m o m.ma Im + o oouounosmam ooo.om ooIsImH ooo.oa.o5oo ooIHHIHH ooo.m .osoo ooIoanH ooo.m .osoo ooIeIoH mmflhm>o mo maoEHo: CHSQOHH .HNuSQ Eamon ADHV AHEV ooapaocoo Iauom Ioomooo as omuom mam mam .flom swam mums mocfla sfluflmflomum psmEumoue Aomscaucooo .6 magma 130 H H H ooo.H mmIHmIs so m.xme.H H N m ooo.m .msoo ooo.H omImHIo + H H m e ooo.m .osoo mew omIomIm mucosa oz H m a ooo.m .osoo mas omImHIm 26 H .Hom m IH H H m a ooo.m .msoo mks omImHIm H Am a ooo.m .osoo oom mmImIm spoosm H o H H ooo.m .msoo omm omImmIe so m.xm.me o H o ooo.m .osoo mks mmIHmIv oNHm Hmsuoz o o ooo.m .osoo mks omIHIe o o ooo.m .osoo mes omImmIm wNH m HGEHOZ Hooosm uoz o o ooo.m .oeoo mas omImHIm o o ooo.m .msoo oms mmIHHIm moHoHHHou ooHMH o o ooo.m .oeoo omm omIqu I o o ooo.m .oaoo ooo.H omImmIm mwfium>o mo mmcofiuos aflnmouu aMNmmm Enumm ADHV HHEV coHuHoooo IHH:< Ioomooo as omuom mam mam .hoa oon moon mocfia caufimfiumum usoaumwue .mumwausm usmuadmmu 0:» mo mCOHuommH OHUH> CH psm O>H> as mfiu paw voom 300 mo :mEHme nofiumuflcsfifiw m:& .m magma 131 ooo.mH .osoo ooIoHIm 80 H I.H.o m Hxme + o H o ooo.H oololo ooo.m .UCH ooIomIm ooo.m .ocH ooIomIm ooo.m .UsH ooIoNIm ooo.m .osH ooImHIm m.me.me.m H m m ooo.H mmIHMImH ooo.m .ocH mmIoHINH oHoHHHou moumH Im ooo.m .ocH omImImH ooo.m .UCH mmINImH oom.m .usH mmImNIHH EU H I.H.o N EU HxN.me.m H m m ooo.m .osH 0mm mmImHIHH .H.0 can .Hom H H N m mes mmIHmIm Eu m.HxHxN.N IH EU m.HXm.me Im ooo.m .osH mmIOHIm mmHHm>o mo msoEHon cHnmouu .MNmmm Edumm ADHV AHEV coHuHocoo IHuc< Ioomcoo as mmuom mam mam .flom omHm moms mosHH :HuHmHomHm usmfiumwua HomscHucooc .m oHnms 132 H a m ooo.m .ocH oos.m HoIoHIm H a m omm.m HoIoIm ooo.m .ooH HoIoNIH noun .mouumm CH H o m ooo.H HoIHHIH ooo.m .ooH HoImIH H , m m ooIoHImH ooo.m ooIMHImH ooo.oH .msoo ooIHHIHH ooo.m .osoo ooIoHIoH ooo.m .osoo ooInIoH ooo.m .osoo ooIoHIo ooo.m .msoo ooImIm H m m ooIHmIn ooo.m .osoo ooIvHIs ooo.m .osoo ooIoIs + H m m oom ooImIo mmHHm>o mo msoEHo: CHQQOHH aMNmmm Esuwm ADHV AHEV ooHuHoooo IHuc< Ioomooo as omuom mam mam .noa oon mums mmcHH GHUHmHume ucmEummHB HomscHucooo .m mHnms 133 mDHumw CH Holmlv + H a m ooo.m .ooH ooo.m HoIomIm + H a m ooo.m .ooH ooo.m HoIoIm H v m ooo.m HoImIm H a m oom.m HoImmIm mmHHm>o mo mGOEHOS anmonv .HNusn Essen HDHV AHEV ooHuHocoo IHuca Ioomcoo as mayor mzm mam .Hoa oon mums mmsHH cHuHmHomHm usmEummHB HoosoHuoooc .m oHnme 134 Homooouo o o ooo.m .ooH ome omImHIHH so m.xme.H IH 50 N.me.me.m Im o o ooo.H omIoHIo H o o .osoo mks omIomIm o o .osoo mse omImHIm o o .osoo mes omImHIm o o .msoo oom mmImIm moHoHHHom m IH o o .osoo omm mmImmIe EU N.Hxme .QEOU mew mmIHmIv mHoHHHou IH .osoo mes mmIHIa o o .osoo mam mmImmIm oNHm Hmsuoz .osoo mks omImHIm o o .osoo oom mmIHHIm oHoHHHom IH .meoo oom mmIoIm o o o .osoo ooo.H mmImmIm mmHHm>o mo wCOEHOS CHCQOHH aMNmmm Eamon ADHV HHEV ooHuHoaoo IHucs Ioomooo «a omuom mam mam .Ho< oon mums meHH CHuHmHomHm quEumwue .mumwdqu quuHCmmn 05¢ mo mCoHuom0H OHHH> CH pCm o>H> CH wnu pCm mmom 300 m0 CmEHme COHHMNHCCEEH one .o wHQMB 135 ooo.mH .QEOU owlmHlm oom ooIva ooo.mH .msoo ooImHIv anon uHmo Hmsuoz ooIvHIv + o o ooo.H ooIer ooo.m .osoo ooIomIm ooo.m .osoo ooIomIm o o ome ooIeIm ooo.m .ooH ooImmIm ooo.m .ocH ooImHIm + o o ooo.H mmIHmImH ooo.m .ooH omIoHImH ooo.m .ooH mmIoImH ooo.m .ocH mmINImH ooo.m .ooH omImmIHH mmHHm>o mo mCOEHOC CHCQOHH aMNmmm ECme HDHV HHEV coHuHocoo IHuc< Ioomcoo as omuom mam mam .Hoa oon moms mMCHH CHuHmHomHm quEummue HmeCHuCOUo .o mHnt 136 .H.o..o m.0H IH .Hou .o o.m Im + H oouousoomHm ooo.e~ ooIVIHH ooo.m .osoo ooImHIOH ooo.m .ogoo ooIHIOH ooo.m .osoo ooIoHIo ooo.m .oEoo ooImIo H m H ooIHmIs ooo.m .osoo ooIeHIH ooo.m .osoo ooIoIe + m H oom ooImIo mmHHm>o wo mCOEHOC CHCQOHH .HNHDQ Eamon ADHV AHEV coHuHocoo IHuc< Ioomooo «a omuom mam mam .Ho< omHm moms mmCHH CHuHmHomum ucmfiumwue HooooHucooc .o mHnme 137 Table 7. Assay for hormones neutralizing antibodies in sera of cow 3039. PMS Serum Sample Ovarian weights Avg. .D. %I (IU) (mg) Saline 18.9 19.0 17.9 18.6 i 0.2 10 48.5 69.0 34.9 50.8 i 17.1 10 3039-5-26-59 40.9 71.2 36.4 49.5 t 18.9 4 10 3039—12-31-59 37.8 53.8 56.8 49.5 i 10.2 4 10 3039-6-2-60 39.5 28.8 17.6 28.6 i 11.0 69 Saline Colony average 13.8 1 2.4 15 Colony average 42.2 I 17.4 15 3039-12-7-60 H20 soluble globulin 16.2 16.2 16.2 i 0.0 Saline Colony average 13.8 t 2.4 15 26.7 25.8 26.2 i 0.1 15 3039-5-4-60 22.8 20.6 21.7 i 1.6 27 138 Table 8. Assay of sera of cow 3044 for hormone neutralizing antibodies. PMS Serum sample Ovarian weights. Avg. 1 S.D. ‘%I (IU) (mg) Saline 18.9 19.0 17.9 18.6 i 0.1 10 48.5 69.0 34.9 50.8 i 17.1 10 3044-4-4-60 28.6 38.8 31.0 32.8 i 5.3 56 3044-6-2-60 17.6 18.7 14.0 16.8 i 2.3 106 Saline 13.5 11.8 12.6 i 1.2 10 21.7 29.5 25.6 i 5.4 10 3044—3-30-61 11.0 12.8 11.9 i 1.3 105 Saline Colony average 13.8 i 2.4 10 " " 29.9 i 8.3 15 " " 42.2 i 17.4 15 3044-4-28-59 22.9 34.0 28.4 i 7.8 15 3044-4-1-59 17.8 23.8 20.8 t 4.2 15 3044-4-21-59 25.5 25.5 15 3044-4-28-59 20.8 20.5 20.6 i 0.0 Table 9. 139 Assay of sera of cow 3053 for hormone neutralizing antibodies. PMS Serum sample Ovarian weights Avg. 1 S.D. %I (IU) (mg) Saline 18.9 19.0 17.9 18.6 i 0.9 10 48.5 69.0 34.9 50.8 1 24.3 10 3053-12-31-59 23.2 23.2 28.4 24.9 i 4.2 80 10 3053—4-4-60 32.3 32.5 34.5 33.1 i 1.7 55 10 3053-6-2-60 18.5 41.5 30.8 30.3 i 16.3 64 Saline Colony average 13.8 t 2.4 10 " " 29.9 i 8.3 15 " " 42.2 i 17.4 15 3053-5-26-59 28.3 28.6 28.4 i 0.1 15 3053-12-31-59 44.1 39.4 41.8 + 3.3 140 Cow 3039 did not form precipitating or neutralizing antibodies against pregnant mare serum during a lZ-week- immunization regimen of weekly injections of 5,000 IU of gonadotrophin. The failure to detect hormone—neutrali- zing antibodies is shown by serum sample 3039—5-26-59 in Table 7. Similar results were obtained when injec- tions of 500 IU were given every other day for a month as shown by serum sample 3039-12-31-59 in Table 7. Both precipitating and neutralizing antibodies were obtained later when Freund's complete adjuvant was used in immunization. (serum sample 3039—6-2-60) Despite the failure of cow 3039 to show precipita- ting or neutralizing antibodies during the first immun- ization period, the ovaries demonstrated typical symptoms of refractoriness to the gonadotrophin. The ovaries hypertrophied to a diameter of 5 cm after two injec- tions and subsequently regressed in size, although high levels of gonadotrOphin were still being injected. Cow 3044 possessed both precipitating and neutrali- zing antibodies after six weekly injections of an emul- sion of Freund's complete adjuvant and 5,000 IU of gonadotrophin. Both types of antibodies were readily demonstrated in all subsequent serum samples. This cow's 141 ovaries appeared to be showing some stimulation from the gonadotrophin through much of the early immunization per- iod. There appears to have been no marked hypertrophy of the ovaries. Immunization of cow 3053 was without consequence on pregnancy as a normal calf was born during this interval. Conception took place during the period that this cow was receiving only complete adjuvant. Immunization with preg- nant mare serum was carried on only during the last five months of gestation. Neutralizing antibodies for preg— nant mare serum could be demonstrated during this gesta- ‘don period but not precipitating antibodies. Both types of antibodies could be detected later when complete adju- vant was used for immunization. Examination of cow 3039 at the time of slaughter yielded the following observations: The tissue weights and gross characteristics: thyroid gland 60.3 9 left ovary 5.1 g, several small follicles, 1 to 3 mm in diam. right ovary 12.5 g, two follicles, 1.7 and 0.7 cm in diam.;one c.l., 1.7 cm in diam. adrenal glands (2) 28.7 g pituitary 5.3 g bodyweight 1505 lb 142 Histological examination of these tissues revealed that in the ovaries there were several small atretic follicles forming gland-like structures within the capsule. In two different areas these adenomatous structures had proli— ferated and formed what appeared to be small granulosa cell tumors. The pituitary gland appeared to have decreased numbers of both basophils and acidophils and there was some edema in the pituitary stalk. In the uterus, there was a marked increase in capillaries sur- rounding the uterine glands and some edema of the endo- metrial stroma. The tissue weight and gross observations obtained at the time of slaughter of cow 3053 were as follows: thyroid gland 45.3 g adrenal glands (2) 18.0 g left ovary 10.8 g, corpus luteum right ovary 8.6 g, follicle pituitary no weight taken bodyweight 1222 lb Histological examination of the ovaries revealed that the follicle on the right ovary contained degenerating granu- losa cells, and an area adjacent to the follicle contained an area of proliferating cells which were adenomatous in 143 nature. The cells making up the adenoma appeared to be of the granulosa type. In the pituitary gland there ap- peared to be a decrease in the PAS positive, small baso- philic cells without a decrease in the large cells. There was a slight increase in the amount of collagenous fibers. The adrenal glands showed no detectable changes. The endometrial epithelium of the uterus was composed of tall columnar cells. The uterine glands showed no secre- tory material present in their lumina. Antibody Spectrum to Pregnant Mare Serum The earliest samples of serum from cow 3044 to show precipitating antibodies demonstrated heterogeneity on agar gel plates. Three precipitin lines were seen with these early samples whereas with later samples five to six lines could be resolved. A comparison of antibody spectra of bovine and rab- bit antisera showed some of the lines to be common antigen- antibody reactions (Figure 2, Plate A7, Figure 3, Plate V9) . 144 Figure 2, Plate A . Comparison of rabbit anti-PMS serum with bovine anti- MS sera. Center well (lower) well 1 (left) well 2 (upper) well 3 (right) PMS, 500 IU/ml Rabbit anti-PMS serum 3044-5—26-59 3044-1-11-61 Figure 2, Plate T . Precipitating antibody spectrum of bovine anti-PMS serum. Center well (lower) well 1 (left) well 2 (upper) well 3 (right) PMS 3044-7-21-60, absorbed with horse serum. 3044-7-21-60, absorbed with EL butyricum. 3044-7-21-60, unabsorbed. 145 Figure 2, Plate A7 Figure 2, Plate T5 146 When normal horse serum was reacted with the anti- serum, a number of lines appeared in agar gel diffusion plates. Two precipitin lines were evident with horse serum reacted with serum samples taken in the early sta- ges immunization of cows. In later samples, this number increased to four and sometimes five precipitin lines. Rabbit sera usually formed three to five precipitin lines with horse serum even with samples taken in the early stages of immunization. The identity of the horse serum specific lines was demonstrated in plates employing the Bjorklund inhibi- tion method. Plate T5 (Figure 2) indicates the horse serum specific antibodies. The horse serum applied ini- tially to well 1 absorbed the horse serum specific anti- bodies of the bovine serum within the well. Excess horse serum diffused from well 1 toward well 2 and reacted in the intervening space with the specific antibodies from well 2. These lines extend downward from between well 2 and the center well to between well 3 and the center well. Thus the horse serum specific antigens diffusing from the pregnant mare serum placed in the center well are identi- fied. A single line remains between well 1 and the center well and identifies with a line extending around the 147 center well by wells 2 and 3. This line indicates an antigenic difference between normal horse serum and preg- nant mare serum Further identification of the horse serum specific antigens which are contaminants in pregnant mare serum was determined by use of horse serum fractions obtained by ammonium sulfate fractionation. When bovine antiserum was reacted with horse serum fractions it was apparent that one of the contaminating antigens in pregnant mare serum was found predominantly in the water soluble euglobulin fraction and in lesser quantities in the albu- min fraction precipitated with two thirds to three-fourths saturation of ammonium sulfate. Another contaminant was more widely distributed in the water soluble euglobulin, water insoluble pseudoglobulin, and in both water solu- ble albumin fractions. Rabbit antiserum reacted with two horse serum antigens distributed throughout all fractions. (See plates C and D 11 11' one of these antigens has been concentrated during the Figure 3). It is apparent that purification for pregnant mare serum. The innermost preci- pitin line is more dense between well #6 and the center well than elsewhere. 148 Figure 3, Plate V9 Comparison of rabbit anti—PMS sera with bovine anti-PMS sera Center well PMS, 500 IU/ml well 1* Rabbit anti-PMS serum 028-3—30—61 well 2 Rabbit anti-PMS serum 038-3-30—61 well 3 Rabbit anti—PMS serum MS-15—2-16-6l well 4 3044-3-30-61 well 5 3044-3—9-61 well 6 3044-3-9—61, water-soluble globulin, 100 mg/ml Figure 3, Plate Cll Precipitin reaction of rabbit anti—PMS with fractions of horse serum Center well Rabbit anti-PMS serum 038-3-30-61 well 1* Horse serum, unfractionated, 1/50 well 2 Horse serum, HZO—insoluble euglobulin, 1/50 well 3 Horse serum, H20 soluble euglobulin, 1/50 well 4 Horse serum, H20 insoluble pseudoglobulin 1/50 well 5 Horse serum, H20 soluble pseudoglobulin 1/50 well 6 PMS, 500 IU/ml Figure 3, Plate D11 Precipitin reaction of rabbit anti-PMS with fractions of horse serum Center well Rabbit anti-PMS serum 038-3-30-61 Well 1* Horse serum, unfractionated, 1/50 well 2 Horse serum, H20 insoluble 1/2 to 2/3 sat'd. (NH4)ZSO4 fraction, l/50 well 3 Horse serum, H20 soluble 1/2 to 2/3 sat'd. (NH4)ZSO4 fraction, 1/50 well 4 Horse serum, H20 insoluble 2/3 to 3/4 sat'd. (NH4)ZSO4 fraction, 1/50 Well 5 Horse serum, H20 soluble 2/3 to 3/4 sat'd.' (NH4)2SOQ fraction, 1/50 Well 6 PMS 500 IU/ml *Wells numbered clockwise from well 1 at one o'clock. Figure 3, Plate D11 150 The presence of antibodies against M, butyricum was also demonstrated on agar gel diffusion plates. (Plate 0 ,Figure 4 and Plate T 3 Figure 2). In this plate T 5' 5 the homogenate of M, butyricum was placed in the upper or #2 well before addition of the antiserum to specifi- cally inhibit the M. butyricum antibodies in the anti— serum. These antigens also diffused toward wells 1 and 3 and reacted with antibodies near the upper border of these wells. These lines show no relationship to any other lines in the plate. M. butyricum cells extracted with saline and water failed to elicit a reaction on agar gel plates. However, breakage of the cells by sonic oscillation or homogeni- zation with the aid of glass beads in the Omnimixer freed one antigen from the cells. (Plates w and Y, Figure 4). 151 Figure 4, Plate 0 . Antibody spectrum of bovine anti-PMS serum. 3 Center well 3044-5-26-59. well 1* Horse serum, 1/64. Well 2 PMS, 1,000 IU/ml well 3 M. butyricum, concentrated homogenate. Well 4 Horse serum, 1/128. well 5 PMS, 500 IU/ml. well 6 M. butyricum, unconcentrated homogenate. * wells numbered clockwise from well 1 at one o'clock. Figure 4, Plate W. Effect of several methods of disruption of EL butyricum on the reaction with antiserum. Center well (lower) 3044-5-26-59. well 1 (left) M, butyricum, untreated. well 2 (upper) M, butyricum, ground with levigated alumina. Well 3 (right) M, butyricum, homogenized in blendor with glass beads. Figure 4, Plate Y. Effect of several methods of disruption of M, butyricum on the reaction with antiserum. Center well (lower) 3044-5-26-59. well 1 (left) M. butyricum, ground with levigated alumina. well 2 (upper) M, butyricum, lysed in water. well 3 (right) .M. butyricum, sonic treated. 152 Figure 4, Plate W Figure 4, Plate Y 153 Additional evidence that the M, butyricum antigen- antibody reaction is independent of the reaction of anti- serum with pregnant mare serum is shown in plate Fla (Figure 5). The antiserum in well 3 was collected before cow 3053 was injected with pregnant mare serum. The identity of this line with the line between antiserum 3044-5—26-59 and the bacterial antigen indicates the spec— ifity of the reaction. A single precipitin line which could not be attri- buted to either horse serum antigen or M, butyricum formed on agar gel plates when pregnant mare serum and its rabbit or bovine antiserum were reacted. This line shown on plate T (Figure 2) is continous between all three 5 wells and the center well. The inhibition of the bovine antiserum with horse serum in well 1 removes all but one precipitin line, which indicates the existence of an anti— gen in pregnant mare serum not found in normal horse ser- um. Additional evidence for this antigen was found by absorption of pregnant mare serum with rabbit anti—horse serum. This reaction in turn identified with the single reaction between unabsorbed pregnant mare serum and anti- serum absorbed with horse serum. The precipitin line show— ing this identification lies between well 1 and the center 154 Figure 5, Plate F . Reaction of serum of cows 3044 and 3053 with M- butyrIcum. Center well (lower) well 1 (left) Well 2 (upper) Well 3 (right) M, butyricum. sonic treated. 3053—2-25-59. 3053—5—26-59. 3044-5-26-59. Figure 5, Plate U . Demonstration of a specific antigen in PMS. 14 Center well (lower) Well 1 (left) Well 2 (upper) Well 3 (right) PMS, 500 IU/ml. Rabbit anti-PMS serum absorbed in the well with horse serum. Rabbit anti-PMS serum, unabsorbed. PMS, 500 IU/ml, absorbed in the well with rabbit anti-horse serum. 155 Figure 5, Plate Fla Figure 5, Plate U14 156 well and connects to a line between well 2 and the center well and finally identifies with the line between well 2 and well 3 on plate U14(Figure 5). The other lines in this plate are due to reaction of excess horse serum and rabbit anti-horse serum used in the absorption procedure. When antiserum against pregnant mare serum was absorbed with an excess of normal horse serum, gonadotro— phin neutralizing antibodies were still present as demon- strated by rat antihormone assays. (Table 10). Likewise, concurrent administration of a standard dose of pregnant mare serum and rabbit anti-horse serum resulted in gonadal stimulation. Thus absorption of either the non-specific horse serum antibodies or antigen failed to affect the gonadotrophin or antigonadotrophin reaction. Antiserum Fractionation When tested on agar gel diffusion plates, the frac— tions of antiserum obtained with ammonium sulfate-salting out procedures showed the pregnant mare serum precipita- ting antibodies to be located primarily in the water solu- ible portions of euglobulin and pseudoglobulin. A lesser annount was found in the water insoluble pseudoglobulin. 1RD precipitating antibodies were found in the albumin or Table 10. 157 Assay of hormone-neutralizing antibodies in the water—soluble globulin fraction of serum sample 3044—3-9-61 following absorption with horse serum. PMS Serum sample Ovarian weights Avg.ii S.D. ‘%I (IU) (mg) (mg) Saline 12.9, 14.2, 14.0 13.7 i 0.2 15 63.1, 51.2, 26.8 47.0 i 18.5 Horse serum 13.2, 15.3, 9.6 12.7 i 2.4 15 3044-3-9-61 11.7, 16.7, 14.8 14.4 i 2.5 98 Saline Colony average 13.8 I 2.4 15 " " 42.2 i 17.4 15 Rabbit anti- horse serum 37.8, 36.2, 44.2 39.5 i 3.6 158 water insoluble euglobulin fractions. Fractionation of antiserum with rivanol was less suc- cessful than the ammonium sulfate salting out procedures. Although quantitative results were not obtained regarding the loss of precipitating antibodies by rivanol purifica- tion, it was observed with several samples that precipi- tin lines failed to appear in agar gel plates after rivanol treatment of antiserum. Serum sample 3053—11-4-60 failed to show precipitin lines when it was used untreated on agar gel plates. Gamma globulin extracted from this serum sample with rivanol also failed to form precipitin lines. HOwever, a water soluble globulin fraction obtained by salting—out procedures with ammonium sulfate formed lines against pregnant mare serum. A major disadvantage with the rivanol technique was the loss of gamma globulin which occurred during the remov- al of rivanol from the supernatant with charcoal. By means of biuret protein determinations it was ascertained that approximately one third of the protein in the super- atant was lost during charcoal treatment. Anion exchange cellulose chromatography of water solu- ble globulin from bovine antiserum did not yield clear cut fractionation of precipitating antibodies for pregnant mare 159 serum. Precipitating antibodies for the non—specific horse serum proteins were also present in the fractions showing a reaction with pregnant mare serum. Precipitating anti- bodies could be detected in two effluent protein peaks oc- curring at ionic strengths of 0.096 and 0.108. The trail— ing side of the latter peak was considerably skewed. At an effluent ionic strength of 0.2, precipitating anti— bodies for pregnant mare serum were not detectable. Approximately 70% of the total protein initially applied to the column was eluted by the time the effluent ionic strength gradient reached 0.2. Twelve to 14% of the total protein initially applied to the column failed to be adsorbed on the column and was washed directly through the column. A sample of this fraction (fraction 10) failed to show precipitating antibodies to pregnant mare serum on agar-gel plates (Figure 6). Table 11 presents the results of the rat assay for hormone-neutralizing antibodies in the chromatographed fractions. It is evident that fraction 10 did not contain any inhibitory substance whereas fractions 48 and 53 did contain hormone neutralizing activity. 160 Figure 6. Reaction of PMS with the water-soluble globulin of 3044-3-9-61 fractionated by anion exchange cellulose chromatography. Plate X10 Center well Well Well Well well well well Plate Y Center well well Well well well well well Plate Z Center well well Well Well Well Well Well 1* 0101wa 1* 0301-wa 1* PMS, 500 IU/ml Water-soluble globulin of 3044-3-9-61, unfractionated chromatography run 4—13—61, u=0.01 chromatography run 4—13-61, chromatography run 4-18-61, u=0.01 chromatography run 4-18-61, chromatography run 4-18-61, Fraction Fraction Fraction Fraction Fraction 5, 22. 9. 79, 85, PMS, 500 IU/ml Water-soluble globulin of 3044-3-9-61, unfractionated Fraction 23, chromatography Fraction 77, chromatography Fraction 80, chromatography Fraction 85, chromatography Fraction 47, chromatography PMS, 500 IU/ml Water-soluble globulin of 3044-3-9-61, unfractionated chromatography run 5-2-61, u=0.ll Fraction Fraction 62 and 63, u=0.13 Fractions 72 and 74, u=0.l6 Fractions 81 to 91, u=0.20. 3044-3-9- 53, 61, unfractionated run run run run run 4-13-61. 4-18-61. 4-18-61. 4-18-61, 5-2-61. *Wells numbered clockwise from well 1 at one o'clock. u=0.12 u=0.l u=0.l4 u=0.l4 u=0.09 u=0.10 u=0.14 u=0.09 chromatography run 5-2-61, chromatography run 5-2-61, chromatography run 5-2-61, Figure 6, Plate 210 162 Hm m.H H H.om m.mm .m.mH .o.oH m.mH mOH.o mm oH mo m.HH H «.mm m.oH .a.mm .m.eH m.mH ooo.o ma mH mmI m.mH H H.mm m.oo .H.ma .m.ve m.eH moo.o oH mH m.mH H o.Ha m.om .N.Hm .H.mo _ mH m.mH H ~.vm m.ve .o.em .H.om OH m.o H e.mH o.vH .~.aH .o.mH mnHHmm 35 H95 3.5 S: CHmuoum d .02 Ha .o.m H.m>< nusoHos nmanmao Hmuoe HomaHmmm noHuomnm mam .HCanmoumeouso mmoHSHHmoImmCmsoxmlCoHCm Ha oomeOHuomum HoImImlevom Eamon m0 CHHDQon 0HQCH0mIHmum3 mnu CH mchonHqu mCHNHHmHusoCImCOEHOC Mom wound .HH mHnma 163 Alligon-Humphrey-Diffusion-in-Ge1 Method The tangent of the angle between the antigen well Plate X 4) was calculated and precipitin line (Figure 7, 1 as 1.0489 from several observations of the two sides. A diffusion coefficient of 4.0 Ficks was calculated from the value for the tangent and a diffusion coefficient (of 3.65 Ficks for bovine gamma—globulin. Immunoelectrophoresis in an Agar Matrix Immunoelectrophoresis of pregnant mare serum resulted iri only one precipitin arc, when this hormone preparation was initially subjected to electrophoresis and followed ‘byr reaction with bovine antiserum to pregnant mare ser- Inn in a lateral trough. High concentrations of hormone were necessary to elicit this precipitin arc. Concentra— tixDr1 of 2,500 IU/ml resulted in an arc while 1,000 IU/ml gave negative results. This anode—migrating component was not identified in relation to the non—specific horse sezqun antigens or the pregnant mare serum specific line. ZImmunoelectrophoresis of normal horse serum produced eVidenCe of two antigenic components with bovine anti- serI-u‘n to pregnant mare serum (Figure 7, Plate IE2). 164 Figure 7, Figure 7, Plate X14. Determination of the diffusion coefficient of the PMS-specific antigen. Horizontal well— antigen, PMS, 500 IU/ml. Vertical well- antibody, bovine anti-PMS water—soluble globulin of 3044-3-9-61, absorbed with horse serum. Immunoelectrophoretic analysis of the reaction of horse serum and bovine anti-PMS. Horse serum was applied to the circular wells. Migration toward the anode by two horse serum antigens is detected by placing bovine anti-PMS in the trough lateral to the electrophor- etically separated horse serum. Figure 7, Plate Xl4 a; ~~~~ Figure 7, Plate IE 2 166 Two antigenic components in horse serum had also been demonstrated on agar—gel diffusion plates with this particular antiserum. The close proximity of both arcs to each other indicates that their electrophoretic mobil- ities do not differ greatly. Demonstration by immunoelectrophoresis of the two antigenic components common to both normal horse serum and pregnant mare serum was possible with a variety of conditions. Veronal buffers at pH 7.65, 8.0 and 8.5 were used for both the electrophoretic and immunological portions of these trials with equal success. Separation of the antigenic components was as readily demonstrated on micro-plates (26 x 76 mm with a 2 mm layer of agar) as on macro-plates (130 x 180 mm with a 4 mm layer of agar). Immunoelectrophoresis on CelluloseiAcetate Although good electrophoretic separation of proteins was possible on cellulose acetate, precipitin lines were very seldom formed between the separated components and the antiserum. Attempts to achieve optimal conditions for immunodiffusion reactions were unsuccessful. I. 1 Veronal or phosphate buffers at pH 8.0 were used without success. Employment of pH 7.4 phosphate buffered saline customarily used for agar-gel immunodiffusion did not improve results on cellulose acetate. Complement Fixation Tests The antiserum control tubes in preliminary complement fixation tests revealed that antiserum from cow 565 was anticomplementary at dilutions of 1/10 and 1/20. This sample of antiserum was subsequently shown to be anti- complementary in dilution as high as 1/160. Samples of serum from.cow 3044 which were known to contain precipi- tating antibodies in agar-gel diffusion plates were also tested in complement fixation tests. Serum sample 3044- 6-12—59 was not anticomplementary at a dilution of 1/10. When this amount of serum was reacted with a solution of pregnant mare serum containing 2,000 IU/ml, no comple- ment fixation was detected. This serum was further tes- ted undiluted with pregnant mare serum at a level of 3,000 IU/ml. At these higher concentrations approxi- Inately 25% of the erythrocytes remained unlysed, indica- ‘ting that there was a slight fixation of complement. An Eittempt to increase the sensitivity of the test at these 168 higher concentrations of reactants by reducing the amount of complement from two-100% units to one 100% unit was unsuccessful. The undiluted antiserum proved anticomple— mentary at this level of complement. It was concluded from these results that insufficient complement was fixed by the reaction of pregnant mare serum and its bovine antiserum to warrant the use of complement fixation for in vitro quantitation of the reaction. Bentonite Particle Fixation Tests Five serum samples of cow 3044, and 3053 which were shown on agar gel diffusion plates to possess precipita- ting antibodies against Mycobacterium butyricium, were tested in a bentonite particle fixation test. All five serum samples in dilutions to 1/16 agglutinated the sensitized bentonite particles. It was concluded that the coincidental titers on the serum samples must have been due to some non—specific reaction which was not detectable with the controls used because these five ser— um samples were collected at different times and from different cows. 1:... ~ ‘ 169 Because of the possibility of an interferring non- specific reaction and disadvantage of differential centri— fugation procedures necessary to obtain the proper size of bentonite particles, this test was discontinued. Latex Fixation Test Non-specific agglutination of unsensitized latex particles in control tubes by bovine antiserum to preg— nant mare serum was invariably encountered in latex fixa- tion tests. Dialysis of the antiserum prior to use in the test reduced this non—specific autoagglutination but did not eliminate the problem. Varying the ionic concen- tration of the Tris buffer from 0.15 to 0.05 also did not eliminate autoagglutination of unsensitized latex parti- cles by bovine antiserum. Altering the level of Tween- 80 (polyoxyethylene sorbiteui monooleate) from 0.7% to 0.04%.was equally unsuccessful. Results are given in Table 12 of a 2 x 2 x 2 x 8 fac- torial experiment in which the effect of pH, ionic strength and levels of Tween—80 were tested on auto- agglutination of latex particles by both saline and bovine serum. The bovine serum was dialyzed with saline prior to the test. It is apparent that none of the 170 COHHMCHHsHmmm mumHQEoo u ++++ COHHMCHusHmmm 0C H I +++ +++ ++ ++++ ++ + + + ¢o\H +++ +++ +++ ++++ ++ ++ + + mm\H +++ +++ ++ ++++ + + + + oH\H +++ ++++ ++ ++++ ++ + + + m\H +++ ++++ +++ ++++ + + + + ¢\H ++++ ++++ +++ ++++ + I + + m\H ++++ +++ ++++ +++ I ++++ I +++ H\H +++ ++++ +++ ++++ ++++ ++++ ++++ ++++ o a oInIHHm Hololmm 3 on me on me o.o mm on so H.o n a mo.o u a H.o u a No.0 ",3 om noose an no ooHusHHo Essen wCH>om mCHHmm omummmanD .mmHoHpHmm waMH owNHuHmComCC mo COHHMCHusHmmmousm C0 Eamon 0CH>0Q oCm mCHHmm omummmoQCs CH om C0039 mo me>mH oCm Adv CumCmHum UHCoH .mm H0 HomMHm .Nfl manna 171 factorial combinations were satisfactory for use with bovine serum in latex fixation test. Rabbit antiserum to pregnant mare serum exhibited less tendency to cause auto- agglutination of unsensitized latex particles than bovine serum. The difficulty in establishment of optimal condi- tions for latex fixation tests appeared to outweigh the ultimate value of this test. Rat Assays for Hormone-Neutralizing Antibodies. The results of the test in rats of antisera for inter- fering substances and the effects of the storage method are presented in Table 13. It is evident that there were no gonad-stimulating substances present in the sera tes— ted. Also the method of storage did not affect the ova- rian weight of these rats. The results of an attempt to quantitate the amount of inhibitory substance in antiserum 3044-5-26-59 are given in Table 14. This table shows the results of two separate trials on the same sample of antiserum. From these data, one can estimate that the titer lies in the range of l to 8 or that the antiserum contains 10 to 80 antigonadotrophin units per milliter. 172 Table 13. Assay of bovine antisera for the effect of serum storage and presence of possible interfering ' substances. Serum sample Method of Ovarian weights Average storage (mg) (mg) 3039-3—4-59 Frozen 15.8, 21.9 18.8 3039-3-25-59 Frozen 16.8, 19.4 18.1 3044-2-25-59 Frozen 18.5, 18.4 18.4 3044-2-25-59 Lyophilized 17.1, 16.6 16.8 3044-3-11-59 Frozen 20.4, 17.0 18.7 3044-3-11-59 Lyophilized 17.8, 18.1 17.4 3044-5—26—59 Frozen 16.7, 16.1 16.4 3044-5-26-59 Lyophilized 15.0, 18.2 16.6 Saline 18.3, 16.5, 17.7 17.5 173 mo O.~ H O.Om m.om .H.O~ .o.Om Oe O.O H.v.am m.ma .m.mm eo\H OH 66 6.6 H H.4m ~.Hm .m.em .o.Om ma\H OH on e.~ H O.O~ H.mm .O.om .m.Om me a.m H O.mm m.em .m.mm .O.mm mmxH OH mH m.m H H.ov m.me .m.Hm .O.vm em\H OH me o.m H «.mm m.o~ .O.em .H.HN OO O.m H O.mH O.Hm .O.mH .O.HH OH\H OH om m.m H H.om O.mm .m.mm .O.H~ mH\H OH mm O.e H O.OH O.HH .o.a~ .m.mH OH H.» H e.mm m.mm .m.am .m.aH m\H OH on O.m H H.oH m.eH .O.mH .m.eH o\H OH mm a.m H O.HH O.eH .m.eH .~.Hm 6O H.m H O.eH O.Hm .e.OH .o.mH «\H OH on H.m H ~.eH O.OH .O.¢H .O.HH «\H OH OOH m.m H H.mH O.OH .e.eH .e.mH H\H OH m.Hm H H.om H.me .O.mm .e.me m.OH H O.Oe H.H¢ .e.¢m .m.mo OH H.om H ~.HO O.HOH .H.Oa .H.Nm esnon HMEHOG OH O.m H H.6H m.mH .m.eH .m.OH H\H H.o H m.mH o.mH .a.OH .a.mH a.H H O.mH m.HH .a.mH .m.eH onHHmm Hose Hose Hose Hose HOHO Ha. .o.m H .m>< nusmHoz cmHHmao .wa .o.m H .mam nusmHoz cmHnmSO coHusHHo HH HmHHe H HmHHs esnmnHucm mam .mmIoNImlooom mHmEom ECHmm Ho mCOHuCHHo mo ammmm GCOEHOCHHC< .VH oHnt 174 Calculation of the regression equations for the percent inhibition and the log of the antiserum dose yielded the following: Trial I. y = 36.46 + 40.03 (log x + 2). Trial II. 9 31.49 + 49.31 (log x + 2). These data are graphically shown in Figure 8. The antiserum dose corresponding to 50% inhibi- tion was calculated from these equations. The dilution of serum 3044—5—26-59 corresponding to 50% inhibition was 1/42 and 1/46 for trials 1 and 2, respectively. These dilutions are equivalent to an inhibition of 210 and 230 IU of gonadotrophin per milliliter of anti— serum. Quantitation of the antigonadotrophic potency of antiserum by means of in vitro reaction of antiserum and dilutions of gonadotrophin followed by in vivo determination of the excess of hormone is shown in Table 15. It can be seen that some loss of gonadotro- phin activity was incurred during the incubation of the pregnant mare serum. Sufficient gonadotrophin activ— ity remained after reaction with antiserum and incuba— tion to stimulate the ovaries in the 300, 350 and 400 IU levels. The lack of activity at the 250 IU level 175 on“ .m wHCmHm uoooongguoa noH DHHI sMH N.H @mw own 1 I 1 i H... . u 8:2“ . H.~ .. H An uovaonm o ouH u H 88:. 43d 821 e6 380 8 . a 83:33 . 3.: I u n Hanna 3 . x 858.3 . 3.6m . H H 3...:- okfiéiio HQm MCOHOU mCHHmm as as 8: .Q.m H mommm>¢ mquHmB CMHHm>O HCmEummua mzm .ECHmm mono: Ho mmmoxm Cm CHHB omQHOQO Coon own CUHCB AECHmm.mzmIHHCm 0CH>0QV HoImIMIvvom Ho COHHUMHH CHHCHon mHnCHOthmpms 03p CHHB mam Ho mHm>mH mCHHHm> mo CoHuommH OHHH> CH Hmumm CHCQOHuoomCom mmwoxm How mmmmm .mH mHQma 177 probably indicates the combined effects of inactivation by antibody and incubation. The results of correction of ovarian weights to an equalized body weight of 100 grams are shown in Table 16. Individual variation was not reduced as indicated by the failure to reduce the standard deviation or the coefficients of variation. The correlation value (r) and the coefficient of determinationIrZ. lOOIalso indicate very little relationship between body weight and ovarian weight and very little common variance. 178 Table 16. Effect of correction of ovarian weights to an equalized bodyweight of 100 grams. Saline injected rats PMS(10 IU) injected rats Uncorrected Corrected Uncorrected Corrected No. of animals 21 21 21 21 Avg. ovarian wt. 17.0 26.9 42.1 69.6 Standard deviation 2.0 3.9 13.1 25.5 Coef. of variation 11.7 14.6 31.1 36.7 r €33¥¥F° ’ 0.12 -0.27 r2.100 0.24 7.29 179 CHAPTER V DISCUSSION OF RESULTS Immunization of Cows The data obtained in this study agree with those reported by Cole §£_al., (1957). Daily injections of 1,500 IU of PMS resulted in the presence of neutrali- zing antibodies in the serum after 3 to 4 weeks. Thus, the total amount of hormone necessary for the production of gonadotrophin-neutralizing antibodies was 30,000 to 35,000 IU. When the gonadotrophin was injec- ted as an emulsion with adjuvant in cows 3039, 3044, and 3053, the total amount of gonadotrophin necessary to elicit hormone-neutralizing antibodies ranged from 25,000 to 35,000 IU. The failure of cow 3039 to form antihormones with a comparable amount of gonadotrophin, without adjuvant, would appear to indicate a difference due to the use of adjuvant. Cole and coworkers failed to obtain inhibi— tory substances with weekly injections of 10,000 IU of gonadotrophin in cows. Dziuk et al., (1958) observed that injection of 5,000 IU daily for 60 days did not ‘1 180 result in the presence of antihormone in the serum of cows. Apparently, large doses, especially at longer interVals, are less apt to produce hormone-neutralizing activity in the serum than small repeated doses or injection of adjuvant emulsion to give a slow sustained release. Thus it would appear that the exceptionally long half—life of pregnant mare serum gonadotrophin found in other animals may be of less immunological importance in the bovine than a low sustained antigenic stimulation by pregnant mare serum. The observations of the ovaries of cows 3039 and 3044 deserve some comment. It is difficult to ascer- tain whether or not the lessened response of the ova- ries of cow 3044 in the early stages of immunization was due to the low levels of gonadotrophin being released from the injected adjuvant-gonadotrophin emul— sion. Alternatively, very low inactivating levels of antihormone may have been present in the serum very early in the immunization and were not detectable by rat assay. The latter possibility may be supported by the failure to detect antihormone in the serum of cow 3039 despite the presence of ovarian refractoriness to injected gonadotrophin. Similarly, Dziuk et al.,(l958) 181 also failed to detect antihormone by rat assay in the presence of ovarian refractoriness to pregnant mare ser- um in cows. The post-mortem observations on cows 3039 and 3053 reveal a number of interesting aspects of the antigonado- trophin phenomenon. The presence of follicles and cor- pora lutea in the ovaries and a lack of basophilism in the pituitary reveals that there was not a direct immunochemical involvement of the cow‘s endogeneous gonadotrophin. This is in agreement with the literature reviewed on the specificity of the antibodies to preg- nant mare serum gonadotrophin. Anti—PMS sera has only rarely been observed to react with other sources of gonadotrophin. 3 The lack of excessive gonadotrophic stimulation of the ovaries in turn reveals that the PMS was not exer- ting its influence on the ovaries. Thus it was probably being immunochemically inactivated as indicated in the laboratory immunochemical studies. The presence of two fairly large follicles (1.7 cm and 0.7 cm in diameter) on the right ovary of cow 3039 reveals that there was more than normal endogenous gonadotrophic stimulation present, but not as much as would be normally expected 182 from 5,000 IU of PMS. This is in agreement with the observation that the hormone-neutralizing ability of the serum collected from this cow at slaughter could be demon- strated in a concentrated water soluble globulin frac- tion and not in the unconcentrated antiserum. Thus, this cow may have had only a very low level of circulating antibodies to PMS. The presence of granulomas in the ovaries of both cows is probably in agreement with the reports of the effects of continued excessive levels of gonadotrophin on tumorigenesis (Ely, 1957). Antibody Spectrum to Pregnant Mare Serum The heterogeneity of the antigen spectrum of preg- nant mare serum is not unexpected in light of the starch electrophoresis studies of highly purified pregnant mare serum preparations (Raacke §t_g1., 1957). Starch electro— phoresis separated numerous protein components which show no biological activity. Rimington and Rowlands (1941) detected gonadotrophin activity spread through euglobu— lin and pseudoglobulin fractions as well as an albumin fraction of pregnant mare serum precipitated with 1/2 to 183 2/3 saturation of ammonium sulfate. The acetone- precipitated gonadotrophin used in the immunization of the cows reported herein resulted in antibodies that reacted with several fractions of horse serum in the diffusion studies on agar. A question of major interest is raised by the results of this investigation. Is the single precipitin line, which is found on agar diffusion plates and attributed solely to pregnant mare serum, the same antibody which neutralizes the gonadotrophin activity in vivo? Four types of evidence bear on this question: 1). Absorption of pregnant mare serum with rabbit anti-horse serum does not remove this particular line. In addition, this antiserum does not diminish the gona- dotrophic activity of PMS in vivo. 2). Absorption of anti-PMS sera with horse serum results in a single line on agar-gel diffusion plates and retention of the antigonadotrophic activity. 3). Results of agar diffusion studies by the meth- od of Allison and Humphrey (1960) yields a diffusion coefficient for the antigen (4.0 Ficks) which is com— parable to that published in the literature for an electrophoretically homogeneous fraction of PMS (4.2 Ficks) 184 LJ 4). The presence and absence of antigonadotrophic activity and precipitin reactions for pregnant mare ser- um was directly correlated in the fractions separated by anion-exchange chromatography. While these four points are affirmative, they are only indirect evidence that the precipitin line contains the gonadotrophin—antigonadotrophin complex. The possi- bility that a non-hormonal horse serum antigen has been altered in specificity during the acetone extraction of the gonadotrophin from the native serum still exists. If a horse serum antigen were so altered, it could not be removed by absorption with horse serum. The results obtained in points 1 and 2 above could have occurred with such an altered horse serum antigen. The last two points tend to refute this possibility, but they are based on observations of such imprecision that the possi— bility of an altered horse serum antigen is not ruled out. How can the question of altered non-specific horse serum antigen be resolved? Samples of unpurified preg- nant mare serum and purified hormone could be absorbed with rabbit anti-horse serum and then compared in agar diffusion studies and rat antihormone assays. A rather involved procedure would be necessary for direct proof. 185 The inactive precipitate and supernatant, following in vitro reaction of PMS and anti-PMS, could be treated so as to cleave the gonadotrophin-antigonadotrophin complex. The cleaved products could then be tested in vivo and in vitro for manifestation of precipitating and biological activities. Quantitation of the Antigonadotrophic Activity of Antisera. Quantitation of the antigonadotrophic activity of antisera yielded widely varying results on the same ser- um sample. The sigmoid nature of the antiserum dose response curve probably makes the use of criteria, based on complete inhibition, an insensitive method. This may account for the wide range of dilutions of antiserum necessary for expression of the titer or number of anti- units of antigonadotrophin. The use of the 50% inhibi- tion method would appear to circumvent the difficulties mentioned above. There was close agreement of the dos- ages of antiserum at the 50% inhibition point for both trials on 3044-5-26-59. To test the 50% inhibition method still further, the data presented in the report of Hamburger and Ostergaard (1949) was treated by this method. These 186 authors had assayed mixtures of pregnant mare serum gonadotrophin and rabbit antiserum in various combina— tions in immature rats. They concluded from their data that the expression of the titer on this particular sample of antiserum could be in the range of 25 to 274. Therefore, these data seemed ideal for testing the repeatability of the 50% inhibition method. For the calculation of the percent inhibition of each level of antiserum, values for the ovarian weights of the saline control and hormone control were estimated from an accompanying standard curve. The average ovarian weights for four of the hormone levels are listed in Table 17 with the calculated figures for percent inhibition. A regression equation was calculated for the log of the antiserum dose plus 2 and the percent inhibition at each hormone level. A comparison of the regression lines for these data were made with those from the two trials on serum 3044-5—26—59 (Figure 9). A test of homogeneity of the regression coefficients (Steel and Torrie, 1960) indicated that the regression coefficients for Trials I and II, the data of Hamburger and Ostergaard, (1949) and the data of Pigon and coworkers (1960) were not signifi— cantly different (F = 0.44). 187 we OH HHOH.O eo\H mm oH mm em mama.o mm\H mm mH Om mm mm no omOH.O OH\H me aH mm Hm am so OOmO.H m\H NO NH no Hm mm mm on me Oemm.H e\H mm mm Heme.H m.m\H OH mm mmom.H H.N\H so NH Ne mm me am OOOO.H mxH OO OH HmeO.H «\m no mH OOOO.N H HH mm He no em O Hose Hose Hose Hose Hose neoHoz uroHoz panama usmHmz onoHoz CmHHm>O HR CmHHm>O HR CmHHm>O HR CMHHM>O HR CMHHm>O + mmoo mmoo O NH.m ooH eonmn IHonm ADHV mam Ho mmoo onoCmum .ooeoos noHoHnHeaH Rom as“ an MUCmuom UHCQOHHOUMComHqu Eamon mo COHHMHCUHMU now owns mCOHHMCHQEOU mCOHHm> CH ECmeHqu HHQQmH oCo CHCmouuooMCom ECHmm meE HCMCmmHm Ho mmHCHxHE CHHB omumwuu msmu wHCHmEEH mo mquHmB CMHHm>o mmmum>m Co AmomHo oummmumpmo oCm Hmmusnemm EOHH mama .RH mHame 188 (\l r\ a\ r (\J 0.] Q 4 o x HOHOmH.eH . ~4.m~ a nOHVHH.eO . HO.H~I a meHvHe.~n . eO.e~I x mOvao.so o smonnI o.H 1‘ H .mza B NHAo H .wza DH m.~H o H H .o mHCmHm w 4 coon laps-Hv:< non :.H . ~.H o.H m.o 0.0 H.o ~.o 4 d .mE OH OH 0 .nxa OH mm 0 HH DH NH.n HES 6.3.338 65 63355 9m .8 323 wash...» a. 69:5... Sufism QEIHHS p.389» 5%: pounds-co mMIONImlgm 5.59 .3 Roan... :2 6.3638 35. 5:8 Co 88 I 33.3 "Om 9; .8 832.33 OH OH OH OH Om O1 0» uorqpqrqux quaoaag 189 The antiserum dose at the 50% inhibition point for the data of Hamburger and Ostergaard was obtained from the regression equation. This antiserum dose was in turn multiplied by its respective hormone level to equalize the antigonadotrophic activity in terms of 1 ml of anti- serum. The calculated amount of hormone inactivated per milliliter of antiserum, at the assay levels of hormone of 25, 19, 12.5 and 3.12 IU were 62, 52, 46 and 54 IU respectively. These figures are directly comparable to the expressions of titer given in the Hamburger and Ostergaard paper as ranging from 25 to 274. Thus, there appears to be a considerable reduction of variability by use of the method of Pigon and coworkers which is based on the midpoint of the antiserum dose response curve. Immunodiffusion It has been pointed out in the literature review that numerous investigators were aware that antigenic horse serum concomitants were present in the gonadotrophin extracted from pregnant mare serum. However, these in- vestigators did not consistently find precipitins for the gonadotrophin after absorption with normal horse ser— um even though antigonadotrophin could be demonstrated in vivo with the absorbed antiserum. The lack of any 190 precipitin reaction was taken, by these investigators, as evidence for rejection of the hypothesis that the antigonadotrophin activity was due to antibodies. On the other hand, if the level of antibodies were quite low there is a good possibility that the precipitin reaction was not of an observable magnitude by the tech- nique used. For example, based on the results reported in this study, one ml of antiserum inactivated approxi- mately 220 IU of PMS in one case and 250 to 300 IU of PMS in another case. If the biological activity of 30,000 IU/mg of protein reported by Raacke et_§l,, (1957) is accepted, the amount of PMS inactivated in the above example is contained in approximately 7-10 ug of protein. While this amount of protein is readily detectable by its hormonal action, it is possible that its detection as an antigen in immunochemical reactions may have been difficult by the techniques used in earli— er investigations. A considerable amount of gonadotrophin was required for the demonstration of lines on agar—diffusion plates. Approximately 0.6 ml of gonadotrophin solution contain- ing 500 IU/ml or 300 IU of total hormone was usually necessary. Agar-gel plates with smaller wells and 191 shorter inter-well distances could be used, but there usually is a loss of resolution of the lines on plates of miniaturized design. Therefore, in order to reduce the amount of hormone, separation or absorption of the anti— bodies or antigens to reduce the neterogeneity of the system, is required to circumvent the reduced resolution of miniaturized agar—gel diffusion patterns. Immunodif- fusion on a cellulose acetate matrix has been shown by Consdon and Kohn (1959) to retain the resolution of larger agar—gel diffusion plates, while requiring only micro quantities of reactants. However, it appears from the investigations reported herein that an inordinate amount of skill is required for utilization of cellulose acetate in comparison to agar—gel diffusion methods. Relationship of Antigenic Site to Hormonal Site The results of this study add no apparent clarifica- tion to the relationship between the antigenic site and the hormonal site on the gonadotrophin molecule. However, the methods developed in this study suggest several avenues for the further investigation of this relationship. 192 The most direct approach would be to purify preg- nant mare serum first by absorption with rabbit anti- horse serum and then by anion-exchange chromatography. The resulting purified material could be subjected to enzymatic cleavage and the products of varying degrees of such cleavage could subsequently be compared by both precipitin and biological reactions with the antiserum for PMS. An additional approach would be to study the effects on the precipitin and biological activities by reactions which affect specific chemical groups. For example, the effect of periodate, and p—chloromercuribenzoate could be studied. 193 SUMMARY AND CONCLUSIONS Immunization of cows with pregnant mare serum gonado- trophin (PMS) can be performed as readily with weekly injections of 5,000 IU of hormone emulsified with Freund's adjuvant as with daily injections of 1,500 IU of hormone. In either case, a total of 35,000 IU of PMS results in hormone-neutralizing antibodies. Immuniza- tion of rabbits with an emulsion of PMS and adjuvant also results in antibodies toward the hormone. The antibody spectrum as demonstrated by agar- diffusion techniques indicates that some of the anti— bodies produced in the cow were identical to those pro- duced in rabbits. Horse serum absorption of the antisera in the well of the agar—diffusion plates demonstrated that two to five antibodies resulted from horse serum concomitants in the hormone used for immunization. Fractionation of horse serum by salting—out methods with ammonium sulfate failed to separate the horse serum con- comitants into any discrete fractions of horse serum when tested against rabbit anti—PMS. However, when these horse serum fractions were tested against bovine anti-PMS one of the contaminating antigens was found predominantly in the water-soluble euglobulin fraction with lesser 194 quantities in an albumin fraction precipitated with two- thirds to three-fourths saturation of ammonium sulfate. A second concomitant horse serum antigen was more widely distributed in the water—soluble euglobulin, water- insoluble pseudoglobulin and in two albumin fractions precipitated between one-half and three-fourths satura— tion of ammonium sulfate. Immunoelectrophoretic analysis of precipitin reac- tion between horse serum and bovine anti-PMS revealed that two of the horse serum concomitants in PMS possess electrophoretic mobilities which are not greatly differ— ent from each other. The anti-PMS sera were found to contain an antibody against homogenized, desiccated Mycobacterium butyricum. The presence of this antigen did not contraindicate the use of this organism in the adjuvant during immunization because no cross reaction could be demonstrated with PMS° A single precipitin reaction was observed between horse serum absorbed rabbit anti—PMS sera and PMS. This reaction was identical with one between unabsorbed rab- bit anti-PMS sera and PMS which had been absorbed with rabbit anti-horse serum. The antigen participating in this reaction was shown by an agar-diffusion technique 195 to have a diffusion coefficient of 4.0 Ficks. A value of 4.2 Ficks has been reported in the literature for the diffusion coefficient of a hormonally active fraction of PMS prepared by starch electrophoresis. Concurrent administration of anti—PMS serum and PMS into immature female rats prevented the gonadotrophin activity of the hormone. Absorption of the antisera with horse serum did not prevent the antigonadotrophin activ— ity. Simultaneous administration of normal horse serum and PMS into immature female rats did not prevent the gonadotrophin activity of the PMS. Rabbit anti-horse serum also did not affect the gonadotrophin activity. Quantitation of the antigonadotrophic potency of antisera was found to be less variable when the determina- tion of the 50% inhibiting dose of antiserum for a stan- dard dose of gonadotrophin was used. Correction of the ovarian weights of the rats, used in the antihormone assays, on the basis of bodyweight did not reduce individual variability. Attempts to ascertain the optimum conditions for the use of several quantitative immunochemical methods on PMS— anti-PMS reaction (complement fixation, bentonite and la— tex fixation) were unsuccessful. 196 Likewise, the use of cellulose acetate as a matrix for immunodiffusion met with little success in these studies. 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Quantitative aspects of the prolan-antiprolan reaction. Proc. Soc. Exptl. Biol. Med. , 33: 96. ‘3 3005.3. USE C33. .91 .'; 9 fl ,{Wf‘ri :13? f"."‘!V’ I’ ‘33,, 2'. \ K. ‘3 .4 V. , . « . .- ‘ ‘ - " .4" '. “f . J ”'1111111111[[1]]11111111111“ 169 2126