L 3:. 2:52er 1....;.:..: . I 13. I... x: . » it: .3... ‘ .34; X1 uiidu 1. 3 p: 1.; 427.1. .t. .. \uv...1_r1.‘uam§c..o!.~wm2 ~ . inn: .3: LEE. :nC. rm £5.17. - huiigveenbwa . .13... -..: » P335» 5.55.? .... .1 at 3.5. .2 :7: Lav: 5.x. ,. ,_ £2? . \. A. .L,.9M..u.-.l.,.» tau. . 5? .6, ‘ ‘ ‘ ‘ .a u, Cut..1.0.n.¢’...(s¢:t its», L I?» " " Michigan $3 University , « . "f _ This is to certify that the thesis entitled Senescent Alterations of LH and Prolactin Regulation ~ in the Female Rat presented by Barry Ernes t Watkins has been accepted towards fulfillment of the requirements for Ph.D. degree in Physiology WM 19 Wage, Major professor Date 6/4/74 800K BINDERY Ell": may II No as SPIIIIFIIN. maul“. ABSTRACT SENESCENT ALTERATIONS OF LH AND PROLACTIN REGULATION IN THE FEMALE RAT By Barry Ernest Watkins Effects of L-dopa and LRH on serum LH and prolactin were determined in young (4 mo) proestrous, estrous and second day diestrous, as well as in aged (23-33 mo) constant estrous and constant diestrous female Long-Evans rats. For all experiments a pretreatment control sub-orbital sinus blood sample was obtained about 10 minutes before drug treatment. In the L-dopa studies, intraperitoneal administration of 0.5 ml saline or a saline suspension of 3 or 30 mg L-dopa was made at 1:00 p.m. Serial bleedings were then performed at 15, 60 and 120 minutes after drug injection. In other experiments, 0.5 ml of saline or LRH doses of 5, 50 or 500 ng were intravenously injected at about l0:00 a.m., followed by bleedings at 15, 30 and 60 minutes. Further experiments were conducted to analyze effects of altered steroid feed- back on prolactin and LH responses following L-dopa or LRH treatment. TWO hour Progesterone pretreatment (5 mg s.c.) was used for animals in all reproductive states of concern. A similar 2 hour priming regine using 20 ug estradiol benzoate was used for all groups except the young diestroUs and aged constant diestrous rats, which received 24 hours of' EStTOQE" pretreatment. Serum prolactin and LH were then measured r.-- buy" I 32%. in"! Barry Ernest Watkins following acute administration of saline, 30 mg L-dopa or 50 mg LRH in the same schedules as previously described. Basal serum prolactin was elevated during proestrus and estrus compared to diestrus. Aged rats had prolactin levels which were inter»- mediate between those of young proestrous or estrous, and those of diestrous rats. The 30 mg dose of L-dopa maximally suppressed serum prolactin levels in both young and old rats by 15 minutes, with the suppression lasting for at least two hours. Conversely, 3 mg of L-dopa caused only transient depression of serum prolactin, which was of longew‘ duration in young than in senescent rats. These results suggest that perhaps old rats are less capable of releasing prolactin release- inhibiting factor (PIF) or that pituitary responsiveness to PIF de- creases with age. The possibility of age related changes in control of prolactin releasing factor secretion also exists. Although neither estradiol benzoate nor progesterone priming affected the ability of 30 mg L-dopa to depress serum prolactin, the duration of prolactin suppression was generally more transient in both young and old rats than that occurring in those not receiving steroid priming. These changes induced by ovarian steroid pretreatment may be due to decreased L-dopa availability within the central nervous systeni (CNS) following impaired drug absorption or accelerated metabolism. These steroids may also suppress pituitary responsiveness to endo- genously released PIF following L-dopa administration. Basal serum LH was greatly elevated only on the afternoon of proestrus. 0f the low levels representative of all other reproductive Barry Ernest Watkins states, LH values of old constant estrous rats were slightly greater than those of either estrus or diestrus, which in turn exceeded those found in aged constant diestrous animals. Young diestrous rats had a smaller LH increase following 30 mg L-dopa than did those in either proestrus or estrus. LH levels in old constant estrous rats increased more slowly than did those of younger ones, while aged constant diestrous rats were completely unresponsive to 30 mg of the drug. The delayed LH enhance- ment in old constant estrous rats and the lack of response in the aged constant diestrous group could indicate the senescent development of a variable degree of latency for either endogenous LRH release or of delayed pituitary response to LRH. Fifty and 500 ng LRH elevated serum LH in all groups by 15 minutes in a dose related manner. The magnitude of LH response was greater in young proestrous and estrous rats than in the diestrous animals. Both groups of aged rats showed a smaller LH response to LRH induction than did any of the younger ones. The induced LH elevation was also delayed in both groups of aged rats. These results indicate that with senescence, the pituitary becomes less capable of secreting LH in response to a given LRH stimulus, and that the response generated occurs after a longer latency period. Pretreatment with either estradiol benzoate or progesterone did not affect basal serum LH concentrations. Although progesterone pretreatment did not greatly alter the ability of L-dopa to increase serum LH levels, estradiol benzoate therapy abolished the L-dopa effect in all groups other than that of young proestrous rats. On the other Barry Ernest Watkins hand, estrogen pretreatment increased pituitary responsiveness to LRH in estrous, diestrous and old constant estrous animals. Progesterone pretreatment preferentially enhanced pituitary responsiveness in all young rats without significantly affecting that of senescent animals. These results show that while estradiol benzoate therapy can restore pituitary responsiveness of constant estrous rats to levels correspond- ing to young cycling rats, increasing the steroid's availability is incapable of restoring functional pituitary response capacity of aged constant diestrous rats. It was also shown that pituitaries of aged rats were refractory to the progesterone effect of enhancing pituitary responsiveness. Thus estradiol benzoate plays a dual role in providing feedback information for control of LH secretion by augmenting the ability of the pituitary to secrete LH while under LRH stimulation, while simultaneously blunting L-dopa's effect to elevate serum LH through presumed dopaminergic activation of hypothalamic LRH release. SENESCENT ALTERATIONS OF LH AND PROLACTIN REGULATION IN THE FEMALE RAT By Barry Ernest Watkins A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1974 ACKNOWLEDGMENTS I would like to express thanks to Dr. G. D. Riegle, the chairman of my guidance committee, for counseling me throughout my graduate program. I am also grateful to Dr. G. D. Riegle and Dr. W. R. Dukelow for their patience and encouragement, which alleviated much of the anxiety associated with completing requirements for the Ph.D. degree. Appreciation is also extended to the remaining members of my guidance committee, Dr. W. D. Collings, Dr. L. F. Wolterink and Dr. R. A. Merkel, for the significant roles they played in guiding me through the final stages of my degree program. The friendship and helpfulness of everyone at the Endocrine Research Unit have been a constant source of encouragement to me. I am also appreciative that the faculty and graduate students of the Department of Physiology made my experiences here exciting and fruitful. ii TABLE OF CONTENTS Page LIST OF TABLES ......................... iv LIST OF FIGURES ........................ v INTRODUCTION .......................... 1 LITERATURE REVIEW ....................... 2 Ageing ........................... 2 Prolactin Control ..................... 6 General Considerations ................. 6 Hypothalamic Monoamines and Prolactin Control ..... 7 LH Control ......................... 16 General Considerations ................. l6 Hypothalamic Monoamines and LH Control ......... 22 Effects of LRH on Pituitary LH Secretion ........ 27 METHODS ............................ 31 Experimental Animals .................... 31 Hormone and Drug Treatments ................ 32 Blood Collection ...................... 33 Radioimmuno-Assay for LH and Prolactin ........... 33 Statistical Analysis .................... 37 RESULTS ............................ 38 Vaginal Cytology Data ................... 38 Effects of L-Dopa on Serum Prolactin ............ 41 Effects of L—Dopa on Serum LH ............... 59 Effects of LRH on Serum LH ................. 77 DISCUSSION ........................... 93 LIST OF REFERENCES ....................... 110 iii TABLE l0. LIST OF TABLES Effects of acute L-dopa or LRH therapy, and of pretreatment with 20 pg estradiol benzoate or 5 mg progesterone on patterns of vaginal cytology ...... Effects of L-dopa treatment on serum prolactin levels of young and senescent female rats . . . ........ Effects of L-dopa therapy on serum prolactin levels of young and senescent female rats receiving 20 pg estradiol benzoate pretreatment ............. Effects of L-dopa therapy on serum prolactin of young and senescent female rats receiving two hour pre- treatment with 5 mg progesterone ............ Effects of L-dopa treatment on serum LH levels of young and senescent female rats ............. Effects of L-dopa therapy on serum LH levels of young and senescent female rats receiving 20 ug estradiol benzoate pretreatment .................. Effects of L-dopa therapy on serum LH of young and senescent female rats receiving two hour pretreatment with 5 mg progesterone ................. Effects of LRH therapy on serum LH levels of young and senescent female rats .................. Effects of LRH therapy on serum LH levels of young and senescent female rats receiving 20 ug estradiol benzoate pretreatment .................. Effects of LRH therapy on serum LH levels of young and senescent female rats receiving two hour pretreatment with 5 mg progesterone ................. iv Page 40 44 45 46 62 63 64 BO Bl 82 FIGURE l. 10. ll. l2. LIST OF FIGURES Serum prolactin levels in female rats after administration of various doses of L-dopa, without regard to reproductive status ......... Serum prolactin concentrations in young and aged female rats after administration of various doses of L-dopa ....................... Effects of L-dopa therapy on serum prolactin concentrations in young cycling female rats ...... Effects of L-dopa therapy on serum prolactin concentrations in aged female rats ........... Effect of steroid pretreatment on basal serum pro- lactin concentration in young and aged female rats . Effect of steroid pretreatment on the ability of L-dopa therapy to suppress serum prolactin concentration in female rats, without regard to reproductive status ................. Resting serum LH levels in young and aged female rats Serum LH levels in female rats after administration of various doses of L-dopa, without regard to reproductive status . ................. Effects of L-dopa therapy on serum LH concentrations in young cycling female rats .............. Effects of L-dopa therapy on serum LH concentrations in aged female rats .................. Effect of steroid pretreatment on basal serum LH concentration in young and aged female rats ...... Effect of steroid pretreatment on the ability of L-dopa therapy to increase serum LH concentration in young and aged female rats . . . . ........ Page 50 52 54 56 58 66 68 7O 72 74 76 FIGURE Page 13. Serum LH levels in female rats after administration of various doses of LRH, without regard to reproductive status ................... 84 14. Effects of LRH therapy on serum LH concentrations in young cycling female rats ............... 86 15. Effects of LRH therapy on serum LH concentrations in aged female rats ................... 88 16. Effect of steroid pretreatment on the ability of LRH therapy to elevate serum LH concentrations in female rats, without regard to reproductive status ....... 90 17. Effect of steroid pretreatment on the ability of LRH therapy to elevate serum LH concentrations in young and aged female rats ................... 92 vi M‘- .1 ,- INTRODUCTION The field of experimental gerontology has been less extensively investigated than most other areas of bio-medical research concern. The current status of knowledge regarding the physiology of ageing has been aptly summarized by the Russian scientist Vladimir Frolkis (1968) who stated that "the essence of ageing is the sum total of unequal changes in regulatory processes at the molecular, cellular and systemic levels of the organism." In other words, debilitations of ageing appear to be related to impaired functioning of homeostatic control mechanisms. The endocrine and nervous systems are recognized to be major mediators of long and short term regulation of homeostasis, respectively. Mainte- nance of homeostasis through time is thus to a large part dependent on functional patency of endocrine control systems as they are influenced by neural and neuroendocrine regulation. Reproduction appears to be an ideal system to investigate senescent alteration of regulatory processes because mammals typically exhibit impaired reproductive capacity with ageing. The rat was chosen as an experimental model for this research because of its short life span. Also normal neuroendocrine control of gonadotropin and prolactin secretion, as well as ovulation, has already been rather fully characterized in this species. The research described here was primarily conducted to determine the influence of senescence on LH and prolactin control in the female rat. LITERATURE REVIEW Ageing The rat has been extensively used as a convenient experimental model to study the impairment of reproductive performance that occurs during senescence. Cyclic patterns of vaginal cytology are altered with advancing age. Aged rats have irregular cycles of vaginal cytology which show unpredictable patterns, long periods of vaginal cornifica- tion, or persistent leucocytic smears characteristic of pseudopregnancy (Ingram, 1959; Clemens and Meites, 1971; Peng and Huang, 1972). Some aged rats can show continuous leucocytic smears accompanied with long term ovarian atrophy, suggesting that loss of gonadotropin secretion may be responsible for onset of anestrus (Peng and Huang, 1972). Sexual acceptance of the male was also shown to be less strongly associated with periods of vaginal "estrous" cornification in old than in young adult rats (Ingram, 1959). Mandl and Shelton (1959) further reported that reduced fertility precedes the exhausting of ovarian oocyte supply in the rat. This decreased reproductive capacity could be related to the generalized ovarian atrOphy which was reported to occur in old constant estrous rats by Clemens and Meites in 1971. However it has been demonstrated that ovaries of old constant estrous animals do not appear overtly abnormal, typically having well developed follicles with no evidence of luteal tissue (Clemens and Meites, 1971). In addition, it was shown by Aschheim in 1965 that intravenous administration of purified LH to aged constant estrous rats caused resumption of estrous cycling, presumably through the induction of ovulation. Further, transplantation of ovaries from young adult or immature female rats into old constant estrous or pseudopregnant recipients did not restore normal estrous cycles (Aschheim, 1964; Peng and Huang, 1972). On the other hand, young adult female rats receiving transplants of ovaries from aged donor rats can continue to show normal estrous cycling (Aschheim, 1964; Peng and Huang, 1972). These observations suggest that ovarian dysfunction is not the primary cause of reproductive decline in the rat. Age related changes are known to develop at the hyp0physial level in the rat. Pecile et a1. (1966) reported that pituitaries of old rats have proportionally higher numbers of acidophils than do young counterparts, a finding which is compatible with the observation that old constant estrous rats have elevated pituitary prolactin content (Clemens and Meites, 1971). Clemens and Meites (1971) further indicated that such senescent rats have higher pituitary FSH and lower LH content than do those of young cycling rats on the day of estrus. Age related changes in the integrity of pitutary function was evaluated by Pecile et al. in 1966. Using young hypophysectomized female rats they found that gonadal function, as assessed by vaginal estrous cyclicity and by uterine and ovarian weight, could be restored by transplanting pituitary tissue from 30 day old donor rats into the evacuated pituitary capsule of the hypophysectomized assay animals. However, hypophysial ‘0 Iii i 5 . 1 Flu l“ a We transplantation from 8 or 24 month old donors did not restore gonadotropic function. In a similar series of experiments Peng and Huang (1972) transplanted pituitaries from rats over 16 months old under the hypothalamic median eminence of hypophysectomized young adults. Following such surgical manipulation estrous cycling was restored in 10 of 30 rats treated, and fertility was demonstrated. They also found that 3 out of 4 young rats which received both pituitary and ovarian transplants from old donors showed continued estrous cycling. These pituitary transplant studies indicate that some aged rat pituitaries can release gonadotropins in cyclic patterns when transplanted to the median eminence of young recipients. Although functional alterations at the hypophysial level are implicated, they may not totally be responsible for senescent impairment of reproduction. There is evidence that central nervous system parameters, particularly hypothalamic structures involved with neuroendocrine feedback control systems, are functionally altered during the course of ageing. A recent series of experiments has assessed age related changes within the hyp0physial-adrenocortical axis (Hess and Riegle, 1972; Riegle and Hess, 1972; Riegle, 1973). These studies have shown that experimental procedures entailing elevation of endogenous blood plasma corticosteroid levels following depo ACTH treatment, direct enhancement of glucocorticoid titers with dexamethasone therapy, or chronic stress-induced corticosteroid increases; all result in atten- uation of acute stress-related plasma corticosteroid elevation to a greater degree in young than in old rats. These results indicate that adrenocortical feedback control system sensitivity is depressed in the senescent rat. It has also been suggested that the threshold for hypo- thalamic feedback control of gonadotropin secretion elevates with age (Dilman, 1971), possibly due to impaired neurotransmitter synthesis (Frolkis, 1966). In support of this concept, Clemens and Meites (1971) reported elevated hypothalamic follicle stimulating hormone-releasing factor (FRF) activity in aged constant estrous rats compared to that of young ones on the day of estrus. Attempts have recently been made to experimentally restore reproductive capacity of senescent rats. It has been shown that intravenous LH administration (Aschheim, 1965) causes resumption of estrous cycling. Similarly, systemic progesterone treat- ment of several days duration to aged rats in constant estrus is capable of inducing ovulation in association with at least one apparently normal estrous cycle (Clemens at aZ., 1969). Clemens et a1. (1969) also demon- strated that direct electrical stimulation of the hypothalamic preoptic area of old constant estrous rats can cause ovulation. Their report further indicated that prolonged systemic epinepherine administration could induce ovulation in old rats; the effect possibly mediated through alteration of certain central nervous system neuroendocrine functions. Similarly, chronic administration of L-8-3-4-dihydroxypheny1alanine (L-dOpa) or iproniazid, drugs assumed to increase hypothalamic cate- cholamine availability, restored regular cycling patterns in old constant estrous rats (Quadry et aZ., 1973). ~ Prolactin Control General Considerations In vitro work by Meites et al. in 1961 suggested that pituitary prolactin production is twice as great in mature female rats as in pre- puberal ones. By incubating pituitaries with 1"C leucine, Ieire et a2. (1972) reported that in vitro prolactin synthesis rates in mature female rats is highest during proestrus and estrus, and that prolactin release is lowest on the day of diestrus. In the normal cycling rat, serum prolactin levels have been found to surge on the day of proestrus (Amenomori et aZ., 1970; Wuttke and Meites, 1970; Neill et aZ., 1971; > Uchida et al., 1972; Taya and Igarashi, 1973). The precise duration a of serum prolactin elevation has been variously reported by different groups of investigators. Neill et al. (1971) indicated that serum prolactin returns to baseline Oevels by midnight on the day of proestrus. However other investigators have demonstrated elevated serum prolactin levels which lasted Trom the afternoon of proestrus through the day of estrus (Niswender et aZ., 1969; Amenomori et aZ., 1970; Taya and Igarashi, 1973). It was further shown by Uchida et al. in 1972 that about two-thirds of their rats exhibited prolactin surges which ended during the night of proestrus while the remaining animals had prolactin elevations which were still evident on the day of estrus. It has recently been demonstrated that serum prolactin levels can also show two separate elevations which occur on the afternoon of proestrus and on the day of estrus (Riegle, unpublished). The exact cause of these nonuniform patterns of prolactin surging is not yet known. These findings could be a reflection of variability in normal secretion patterns between animals or between strains of rats. It is also possible that on the day of estrus some rats have exaggerated sensitivity to nonspecific stressors associated with experimental manipulation which are thought to increase serum prolactin concen- trations (Neill, 1970; Valverde-R. et al., 1973). Hypothalamic Monoamines and Prolactin Control Considerable investigative effort has recently been devoted to attempt to elucidate the role that the central nervous system (CNS) plays in the control of hypophysial prolactin secretion. It is gen- erally accepted that the major central regulation of prolactin secretion is of suppressive nature mediated through a hypothalamic prolactin inhib- iting factor (PIF) (Meites et al., 1961; Talwalker et aZ., 1963; Kamberi et aZ., 1971c; Kanematsu and Sawyer, 1973). In addition, recent evi- dence suggests the presence of a hypothalamic prolactin releasing factor (PRF) which appears to be elaborated during conditions such as those associated with ether stress (Valverde-R. et aZ., 1973). It has recently been shown that the synthetic tripeptide pGlu-His-ProNHz, known as thyrotropin releasing hormone or TRH, can cause prolactin to be released from the pituitary (Bowers et aZ., 1973). In addition the findings of Bowers et al. (1973) suggest that since TRH is equally capable of increasing secretion of prolactin as well as of thyroid stimulating hormone (TSH), TRH might be a physiological PRF. In support of this concept, it has been reported that intravenous TRH injection results in elevation of serum prolactin concentrations (Mueller et aZ., 1974), even in rats with median eminence lesions (Porteus and Malven, 1974). As a consequence of the discovery of hypothalamic regulating factors, many investigations were carried out to evaluate possible involvement of various CNS neuro-transmitters in the control of PIF release. Coppola et al. (1965) found an inverse relationship between CNS catecholamine availability and the ease of pseudopregnancy induc- tion in the rat. In these experiments, the use of reserpine, a-methyl- dopa or tetrabenazine to deplete brain catecholamine levels stimulated the onset of pseudopregnancy, presumably due to drug-evoked impairment of PIF release. They also found that the effect could be reversed fol- lowing restoration of monoamine stores with systemic treatment of L-dopa, the metabolic precursor of dopamine, norepinepherine, and epinepherine. The assumption that these effects occurred within the brain were con- firmed in 1968 by van Maanen and Smelik, who demonstrated the induction of pseudopregnancy associated with monoamine depletion in the hypo- thalamic median eminence following implantation of reserpine within the basal hypothalamus. They also showed that simultaneous enhancement of hypothalamic catecholamine levels with the monoamine oxidase inhib- itor iproniazid could overcome the reserpine effect. With the advent of radioimmuno-assay techniques for assessment of rat prolactin (Niswender et aZ., 1969),an attempt was made by Lu et a2. (1970) to verify and expand these findings concerning the neuro-humoral control of prolactin secretion. Intraperitoneal administration of drugs known to inhibit catecholamine activity (reserpine, chlorpromazine, a-methyl-para-tyrosine, or a-methyl-meta-tyrosine) markedly elevated serum prolactin levels in proestrous rats by 30 minutes after injection and generally decreased pituitary prolactin content when measured at 4 hours after drug treatment. However direct systemic treatment of sus- pected neuro-transmitters such as dopamine, epinepherine, norepineph- erine or serotonin, did not affect serum prolactin, presumably due to their inability to diffuse across the blood-brain barrier (Lu et al., 1970). It has further been shown by Kleinberg et al. (1971) that the ability of systemic chlorpromazine therapy to elevate serum prolactin concentrations is strikingly inhibited when coupled with L-dopa pre- treatment. Intraperitoneal L-dopa administration has been reported to effectively suppress serum prolactin levels in both intact female rats and in hypophysectomized rats bearing anterior pituitary grafts (Lu and Meites, 1971; Lu and Meites, 1972). Using an in vitro assay protocol, Lu and Meites (1972) also showed that L-dopa treatment elevated PIF activity both in peripheral serum and within the hypo- thalamus. In addition, the drug was effective in elevating hypothalamic and blood serum PIF activity in hypophysectomized rats (Lu and Meites, 1972). These results present strong evidence that systemic administra- tion of L-dopa can suppress serum prolactin concentration by acting within the CNS to increase dopamine availability which in turn enhances hypothalamic synthesis and/or secretion of PIF. Using in vitro incuba- tion methods, Kamberi et al. (1970a) demonstrated that injection of dopamine directly into the third ventricle of the brain caused oh- ! . pk- . .7. 10 enhancement of PIF activity in hypophysial stalk portal blood plasma of adult male rats. These results can be interpreted to mean that dopamine acted within the hypothalamus or associated CNS areas to stimulate secretion of hypothalamic PIF into the hypophysial portal system. Alternatively, it is possible that dopamine diffused into the pituitary stalk portal circulation where it directly acted on the pituitary as a prolactin inhibiting factor in a manner which had been earlier hypothe- sized by van Maanen and Smelik (1968). In an attempt to clarify the role of hypothalamic neuro-transmitters on prolactin secretion, Kamberi et al. (1971a) found that intraventricular administration of dopamine hydrochloride to male rats markedly decreased serum prolactin levels while similar injection of epinepherine or norepinepherine was without effect. However very high nonphysiological doses of epinepherine and norepinepherine did result in significant serum prolactin suppression. In addition, none of the monoamines altered prolactin concentration either when perfused to the anterior pituitary by way of a cannulated hypophysial portal vessel or when injected into infundibular or peduncular arteries (Kamberi et aZ., 1971a). Kamberi et a1. (1970c) also found that simultaneous intraventricular injection of dopamine and pronethalol (a B-adrenergic blocker) caused typical prolactin suppression, while dopamine in association with an a-adrenergic blocker such as phentolamine or phenoxybenzamine, prevented the prolactin response seen with dopamine alone. These observations indicate that dopamine, rather than epinepherine or norepinepherine, is the monoamine which exerts greatest inhibition of pituitary prolactin secretion. It 11 is also apparent that the dopamine effect is mediated through activation of certain hypothalamic a-adrenergic receptors to induce the release of a hypothalamic PIF since dopamine itself is not able to suppress pituitary prolactin when directly perfused into the pituitary or when delivered via general cephalic arteries. However evidence is accumulating which suggests that cate- cholamines may directly inhibit prolactin release at the pituitary level. There have been reports that in vitro incubation of pituitaries with dopamine, epinepherine or norepinepherine causes decreased prolactin release accompanied by accumulation of the hormone within the adeno- hypophysis (MacLeod, 1969; Birge et aZ., 1970; Schally et aZ., 1974; Shaar and Clemens, 1974). In in vitro experiments Shaar and Clemens (1974) have also shown that the PIF activity of hypothalamic extracts can be abolished with pre-incubation with monoamine oxidase or with aluminum oxide to absorb available catecholamines. Other in viva evidence presented by Donoso et al. (1973) indicates that L-dopa treatment can suppress serum prolactin in castrate rats with complete median eminence lesions, suggesting that high catecholamine levels may directly inhibit pituitary prolactin release. Perfusion of hypophysial portal vessels with glucose solutions containing dopamine or norepineph- erine also has been shown to decrease serum prolactin, implying that these monoamines may be prolactin release-inhibiting factors (Takahara et aZ., I974). Techniques employing histochemical fluorescence of hypothalamic catecholamines have allowed qualitative assessment of catecholamine 12 content within the predominately dopaminergic tubero-infundibular neurons of the median eminence. Such procedures, accompanied by treat- ment with an inhibitor of catecholamine synthesis (a-methyltyrosine- methylester), makes possible general determination of catecholamine turnover as reflected by the relative depletion of histochemical flu- orescence (Ahrén et aZ., 1971; kufelt and Fuxe, 1972; Fuxe et aZ., 1973). Absolute levels of hypothalamic dopamine content as gauged by fluorescent intensity in tubero—infundibular neurons are not altered by hypophysectomy, castration, or treatment with exogeneous pituitary hormones (Hfikfelt and Fuxe, 1972). However tubero-infundibular catecholamine turnover varies as a function of the estrous cycle, being lowest during proestrus and early estrus (Fuxe et aZ., 1969; Ahrén et aZ., 1971). Intravenous pro- lactin pretreatment in normal and hypophysectomized rats of both sexes generally enhanced hypothalamic dopamine turnover rate to that charac- teristic of cycling female rats during diestrus (kufelt and Fuxe, 1972). These findings provide further evidence linking deopminergic activity within the hypothalamus with PIF release since an inverse relationship is demonstrated between previously reported prolactin levels during the estrous cycle (Amenomori et aZ., 1970; Taya and Igarashi, 1973), and corresponding alterations in tubero-infundibular dopamine turnover rates. These results support the concept that prolactin treatment acts to inhib- it the endogenous secretion of prolactin by increasing hypothalamic dopamine turnover, which presumably causes enhancement of PIF release (Hdkfelt and Fuxe, 1972). lull- n\u 13 Investigations have also been carried out to evaluate possible involvement of other suspected CNS neuro-transmitters on prolactin control. Intraperitoneal administration of serotonin and its immediate metabolic precursor, 5-hydroxy-L-tryptophan, had no effect on serum prolactin concentrations in rats (Lu et aZ., 1970; Smythe and Lazarus, 1973). However a recent report by Lu and Meites (1973) indicated that high intravenous doses of S-hydroxy-L-tryptophan markedly increased serum prolactin by 30 minutes after injection, while serotonin itself did not alter peripheral prolactin levels. They theorized that the reason serotonin was without effect on serum prolactin was likely due to its inability to pass the blood-brain barrier. Using intraventric- ular administration of serotonin and melatonin, Kamberi et al. (1971b) showed that these indolamines could elevate serum prolactin. They also discounted the possibility that serotonin or melatonin directly promotes hypophysial prolactin release when they found that perfusion of the pituitary by way of a cannulated hypophysial stalk portal vessel did not affect serum prolactin levels. These observations suggest the additional existence of a serotonergic system in the hypothalamus which may act in antagonism to the dopaminergic inhibitory system by facili- tating pituitary prolactin secretion. In addition, cholinergic pathways have recently been implicated in the control of pituitary prolactin release. The blocking of cholinergic synapses with atropine has been shown to depress serum prolactin in rats on the day of estrus (Gala at aZ., 1972) as well as to block the proestrous preovulatory prolactin surge (Libertun and McCann, 1973). Similarly systemic treatment with l4 nicotine, known to be a cholinergic pre-ganglionic blocking agent, has been reported to delay or totally block the proestrous prolactin elevation in the rat (Blake et aZ., 1973; Blake, 1974). Therefore involvement of certain CNS cholinergic neuron systems may also promote preovulatory prolactin surging in the rat. Electrophysiologic studies have also been performed to try to anatomically define hypothalamic structures involved in the control of prolactin release. Although acute electrochemical stimulation of the ventral medial hypothalamus and medial preoptic area of the hypothalamus did not alter serum prolactin (Wuttke and Meites, 1972; Gala at aZ., 1973), stimulation of the hypothalamic medial preoptic area with chron- ically implanted electrodes was shown to evoke marked elevations of serum prolactin (Wuttke and Meites, 1972). It is well established that estrogen therapy to ovariectomized rats elevates serum and pituitary prolactin levels (Amenomori et aZ., 1970; Lu and Meites, 1971; Bishop et aZ., 1972; Kalra et aZ., 1973). In vitro experiments have demonstrated that this effect is at least partially mediated by a direct stimulatory action of the hormone on pituitary prolactin release (Nicoll and Meites, 1964; Lu et aZ., 1971). In 1972 Bishop et al. reported that suprachiasmic lesions cannot affect estrogen's ability to elevate serum prolactin, while lesions in the nedian eminence prevent the estrogen effect. The authors concluded both that estrogen's stimulatory effect on prolactin release is mediated at the median eminence, and that the direct enhancement of estrogen on pituitary prolactin secretion may not be physiologically important since 15 the steroid was ineffective in ovariectomized rats with medium eminence lesions. By injecting normally cycling rats with an antiserum to estradiol on the second day of diestrus, Neill et a1. (1971) were able to block the expected proestrous surge of prolactin. In 1972 Neill also demonstrated that estrogen treatment of ovariectomized adult rats resulted in initiation of periodic prolactin surges which could be overcome by disrupting anterior neural input to the hypothalamus. These results indicate that estrogen is essential for induction of the pre- ovulatory prolactin surge, and that it likely acts within CNS centers rostral of the hypothalamus to promote prolactin secretion. In intact female rats, systemic estradiol benzoate given on the morning of estrus resulted in significant plasma prolactin elevation by the afternoon of the following day, while if the steroid was aministered on the first day of diestrus, serum prolactin remained unchanged when measured on the second day of diestrus (Kalra et aZ., 1973). However Ying and Greep (1972) showed that estradiol benzoate given on day one of diestrus invariably induced the onset of pseudopregnancy, presumably associated with elevated serum prolactin levels. These data suggest that the sensitivity of feedback mechanisms by which estradiol benzoate affects prolactin secretion alters during various stages of the estrous cycle. In vitro experiments by Nicoll and Meites (1964) indicated that progesterone does not directly affect hypophysial prolactin release. 0n the other hand, systemic injection of high doses of progesterone elevated serum prolactin in castrate female rats (Kalra et aZ., 1973). In intact rats, progesterone administration is capable of both increasing 16 and decreasing estrous cycle length, depending on time of injection (Paup, 1973). It has also been reported that progesterone therapy on the morning of proestrus in intact rats can advance the onset time of the preovulatory prolactin surge by about 3 hours, as well as enhance the magnitude of response (Uchida et aZ., 1972; Kalra et aZ., 1973). LH Control General Considerations Using the ovarian ascorbic acid depletion technique, Ramirez and McCann (1964) determined normal LH levels during the estrous cycle of the rat. They found that plasma LH began to rise on the morning of proestrus, peaked that afternoon, and had not yet returned to basal levels by the morning of estrus. With the development of a sensitive radioimmuno-assay specific for rat LH (Monroe et aZ., 1968), it became possible to more precisely evaluate normal patterns of LH secretion as reflected by peripheral hormone concentrations. Subsequent investiga- tions have shown that the preovulatory LH surge occurs in its entirety during the afternoon or evening of proestrus, with measurable serum LH elevation between 3 and 9 p.m. in most rats (Goldman et aZ., 1969; Monroe et aZ., 1969; Wuttke and Meites, 1970; Neill et aZ., 1971; Taya and Igarashi, 1973). However in any given rat, the duration of the LH surge is thought to last for only 1 to 3 hours (Monroe et aZ., 1969). It is believed that control of gonadotropin secretion in cycling female rats involves tonic CNS negative feedback regulation by ovarian steroids and by LH itself. Both in vitro and in vivo experiments have 17 shown that estrogen can act within the hypothalamus to presumably decrease LRH-induced pituitary LH secretion, thereby depressing serum LH levels (Kalra et aZ., 1973; Legan et aZ., 1973; Saksena et aZ., 1973). In addition, the existence of short loop negative feedback of LH on gonadotropin control was shown by the report that implantation of LH into the medial basal hypothalamus can inhibit ovarian function (Ojeda and Ramirez, 1969). Activation of hypothalamic preoptic mechanisms which induce LH release is periodically superimposed on this tonic inhibition of gona- dotropin secretion. It has been reported that lesions within dorsal hypothalamic areas have no effect on elevated serum luteinizing hormone- releasing factor (LRF) activity in chronically hypophysectomized rats, while median eminence lesions depress LRF activity (Naller and McCann, 1965). These results can be interpreted to mean either that the median eminence is itself the site of LRF synthesis or that the median eminence acts as an area for storage of LRF which had been synthetized in more anterior hypothalamic regions. The combined findings that suprachias- matic lesions decreased hypothalamic LRF activity and that marked LRH activity was present in the supra-optic area of the hypothalamus indi- cate that this region may constitute a major site of LRH synthesis (Crighton and Schneider, 1969). The normal cyclic changes in serum LH levels of mature female rats can be altered by appropriately timed administration of gonadal steroids or of certain centrally acting pharmacologic agents, notably barbiturates (Wuttke and Meites, 1970; Krey and Everett, 1971; Ying and 18 Greep, 1972; Beattie and Schwartz, 1973). Pentobarbital is capable of blocking both the expected preovulatory LH surge and subsequent ovulation when given between 1:30 p.m. of the last day of diestrus and 1:30 p.m. of proestrus (Redmond, 1968; Wuttke and Meites, 1970; Beattie and Schwartz, 1973). The report by Beattie and Schwartz (1973) demonstrating pentobarbital's ability to inhibit LH surging 24 hours after administration, suggests that the short acting drug may not have direct adenohypophysial action but rather that it interferes with a neural clock mechanism involved with preovulatory gonadotropin elab- oration. In accord with this hypothesis, it has been found that administration of pentobarbital in the early afternoon on any day in four day cyclic rats could delay the next ovulation by 24 hours (Domingues and Smith, 1971). Wuttke and Meites (1972) reported that the pentobarbital induced suppression of preovulatory gonadotropin release could be overcome by electrochemical stimulation of electrodes which were chronically implanted in the hypothalamic medial preoptic area or within the arcuate nucleus region. It was also concluded from these studies that the neural triggering mechanisms causing LH release are at least partially located within the medial pre0ptic area. Estro— gen can also influence the onset time of LH surging. Injections of estradiol benzoate given on the morning of the first day of diestrus in 4 day cycling rats advanced ovulation time by 24 hours (Ying and Greep, 1972; Krey et aZ., 1973). Ying and Greep (1972) also showed that this estrogen effect can be overcome by pentobarbital treatment during the critical period before the expected precocious gonadotropin l9 surge. Recent work has suggested that the stimulatory feedback effects of exogenous estrogen on gonadotropin release depended on progesterone synergism (Krey et aZ., 1973; Mann and Barraclough, 1973). Further evidence suggests that subcutaneous progesterone injections can par- tially overcome the inhibitory influence of minimally effective pento- barbital doses on ovulation (Kabayashi et aZ., 1973). In addition, administration of progesterone on the second day of diestrus or during proestrus increased serum LH within 6 hours after therapy (Naller et aZ., 1966). It was also shown by Redmond (1968) that progesterone adminis- tration on the morning of proestrus could induce premature gonadotropin release as indicated by the time of ovulation. More recently, Uchida et al. (1972) extended the work of Redmond with the demonstration that progesterone given during the morning of proestrus advanced the pre- ovulatory gonadotropin surge by about 3 hours in intact cycling rats. Although the mechanism of action for these progesterone effects is unknown, it is quite possible that the steroid acts within the CNS both to counteract pentobarbital induced suppression of LH release and to alter normal clock mechanisms that control the timing of preovulatory gonadotropin secretion. Surgical ovariectomy is another approach which has been employed as an experimental tool to determine requirements for initiation of pulsatile gonadotropin surging. Ovariectomy has often been shown to remove negative feedback of ovarian steroids on gonadotropin secretion, thereby resulting in elevated serum gonadotropin levels (Ramirez and McCann, 1963 and 1965; Blake et aZ., 1972; Kawakami et aZ., 1973) as 20 well as enhanced serum and hypophysial portal vessel LRF activity (Schneider and McCann, 1970a; Ajika et aZ., 1972; Ben-Jonathan et aZ., 1973). Ajika et a1. (1972) also demonstrated that 24 or 48 hours of estradiol benzoate therapy decreased both pituitary LH content and hypothalamic LRF activity. Likewise, replacement therapy with estradiol benzoate and progesterone in long term ovariectomized rats can suppress the elevated serum LH concentrations (Naller et aZ., 1966; Blake et aZ., 1972; Kalra et aZ., 1973). There is also evidence that estrogen alone is able to markedly decrease serum LH levels in ovariectomized rats (Saksena et aZ., 1972; Kalra et aZ., 1973). Estrogen availability may be the only requirement to induce pulsatile gonadotropin surging. Treatment of ovariectomized rats solely with estrogen can result in resumption of LH surging on the second day after injection (Neill, 1972; Legan et aZ., 1973). Further, estrogen is capable of inducing daily afternoon LH surges in long term ovariectomized rats which are of com- parable duration to normal proestrus gonadotropin surging in intact cycling rats (Legan and Midgely, personal communication). However lesions in the median eminence of ovariectomized rats prevented this estrogen effect (Bishop et aZ., 1972b). Collectively, these observa- tions suggest that positive feedback may act as a stimulus to implement the triggering of neural clock mechanisms responsible for pulsatile LH release. Support for this hypothesis was found in intact rats, where treatment with antiserum to estradiol at 1 p.m. of the second day of diestrus abolished the expected preovulatory gonadotropin surge (Neill et aZ., 1971). 21 Progesterone may interact with estrogen to control gonadotropin secretion. Increased serum LH concentrations were induced within 4 hours after progesterone administration in chronically ovariectomized rats pretreated with testosterone propionate to depress serum LH levels (Jackson, 1973). Using ovariectomized rats, Kalra and co-workers (1972) indicated that systemic progesterone given 48 hours after a priming dose of estradiol benzoate caused elevation of serum LH evidenced at 12 hours after injection. Activation of a-adrenergic norepinepherine pathways within the CNS was thought to be responsible for this effect since selective inhibition of hypothalamic norepinepherine content with diethyldithiocarbamate, or treatment with the a-adrenergic blocking agent phenoxybenzamine inhibited the gonadotropin elevation. Thus use of the ovariectomized rat as a model for assessment of the influence of gonadal steroids on CNS triggering mechanisms for pulsatile LH release shows the existence of complex relationships between steroid availability and subsequent timing of neuroendocrine events. Precise anatomical location of the sites of steroid action on control of gonadotropin secretion is not yet well understood. In addi- tion to proposed hypothalamic areas within the arcuate nucleus and me- dian eminence area for negative steroid feedback interaction (Schneider and McCann, 1970; Bishop et aZ., 1972; Piva et aZ., 1973), there is evi- dence that the limbic system may affect control of gonadotropin secre- tion (Ellendorf et aZ., 1972; Kawakami et aZ., 1973; Piva et aZ., 1973). Ellendorf et a2. (1972) reported that micro-electrode stimulation of electrodes implanted in the amygdala during the afternoon of proestrus blocked ovulation. They also demonstrated that amygdalar stimulation 22 in chronically ovariectomized rats caused transient decrease in serum LH concentration. 0n the other hand, Kawakami et a1. (1973) found that stimulation of either medial amygdaloid regions or of the dorsal hippo- campus increased serum gonadotropin levels. There is evidence that tritiated progestins are concentrated in both the hypothalamus and hippocampus in the mouse (Luttge et aZ., 1973). It has also recently been shown that implantation of progesterone into the amygdala causes decreased hypothalamic LRH content, and presumably depressed levels of serum LH (Piva et aZ., 1973). Although these results are somewhat con- flicting, they nonetheless provide strong evidence for involvement of the limbic system in gonadotropin control. Hypothalamic Monoamines and LH Control Much experimentation has been done in an attempt to clarify that part played by the CNS in control of ovulation. In 1969 Kordon and Glowinski investigated the effect of altered CNS catecholamine avail- ability on gonadotropin induced ovulation in prepuberal female rats. They found that inhibition of catecholamine synthesis with a-methyl tyrosine administration on the afternoon of the day preceding expected ovulation blocked its occurrence. They also made the observation that L-dopa treatment to restore catecholamines in these blocked rats par- tially restored ovulation, while L-threodihydroxyphenlyserine therapy to selectively restore brain norepinepherine levels was ineffective. These results suggest that functional patency of CNS dopaminergic synapses was necessary for successful ovulation induction. In a 23 similar series of experiments, Rubinstein and Sawyer (1970) examined the effects of depressed CNS catecholamine availability on ovulation in cycling rats. They found that depletion of brain catecholamines with reserpine treatment in the morning of proestrus inhibited ovulation and decreased the ability of electrochemical stimulation of the hypothalamic medial preoptic area to induce ovulation. Intraventricular injection of epinepherine was better able to overcome the pentobarbital block to ovulation than was norepinepherine, dopamine or serotonin, suggesting that hypothalamic epinepherine releasing synapses could be important in bringing about ovulation (Rubinstein and Sawyer, 1970). Kamberi et a1. (1969) utilized an in vitro assay technique in an attempt to link brain catecholamine availability to associated LRH activity and LH release. They found that co-incubation of norepinepherine or dopamine with rat pituitaries generally did not affect the rate of LH release as measured by the ovarian ascorbic acid depletion assay. When stalk median emi- nence fragments were included in the incubations, dopamine rather than norepinepherine or serotonin caused enhanced pituitary LH release. These results indicated that dopamine stimulated pituitary LH release by promoting LRF secretion from hypothalamic stalk median eminence tissue. The development of a sensitive radioimmuno-assay for rat LH (Monroe et aZ., 1968) made possible a more critical examination of CNS catecholamine involvement in the control of gonadotropin secretion and of subsequent ovulation. Using radioimmuno-assay for LH determination, further in vitro studies were performed showing that co-incubation of pituitaries with dopamine, norepinepherine or serotonin did not alter 24 basal LH release, while epinepherine caused slight elevation of LH release (Schneider and McCann, 1969). It was also demonstrated by Schneider and McCann (1969) that with stalk median eminence added to incubations, only dopamine was able to facilitate pituitary LH release. Further experiments employing a- and B-adrenergic blocking agents demon- strated that dopamine's action to enhance LRH secretion was mediated through a-adrenergic mechanisms (Kamberi at aZ., 1969; Schneider and McCann, 1969; Schneider and McCann, 1970b). Additional evidence that the dopamine effect to enhance pituitary LH elaboration is mediated through promotion of hypothalamic LRH release comes from the report that LRF activity is increased in hypophysial portal stalk plasma of rats treated with intraventricular dopamine (Kamberi et aZ., 1969). In in viva experiments it has been shown that while intraventricular dopamine therapy increased LH levels, norepinepherine or serotonin was without effect (Schneider and McCann, 1970b; Kamberi at aZ., 1970b). The observation that dopamine was ineffective in promoting pituitary LH release when perfused directly into the pituitary through portal vessels or when injected into the basilar artery (Kamberi at aZ., 1970b) also indicates that the monoamine induces its effect through mediation of hypothalamic LRH secretion. Schneider and McCann (1970b) reported that dopamine is more effective to induce serum LH elevation on the second day of diestrus and on proestrus than during estrus or day one of diestrus. They also indicated that the catecholamine could elevate serum LH in ovariectomized rats which had been primed with estrogen and progesterone pretreatment. 25 Studies have also been conducted to assess possible involvement of other CNS neuro-transmitters on the control of gonadotropin release. Intraventricular administration of the indolamines serotonin and mela- tonin are known to decrease serum LH levels in ovariectomized female rats (Schneider and McCann, 1970b) and in intact male rats (Kamberi at aZ., 1970b). These findings indicate that, in addition to catecholamine mechanisms promoting LRH release, there is an opposing serotonergic pathway in the hypothalamus that acts in some manner to inhibit LRH secretion. Evidence has recently been published that implicates the presence of CNS cholinergic pathways which facilitate gonadotropin secretion. Both systemic and intraventricular treatment with atr0pine to block cholinergic receptors were able to block the preovulatory pro- estrous gonadotropin surge and to suppress serum LH levels in ovariec- tomized female rats (Libertun and McCann, 1973). Since exogenous LRH still increased serum LH in rats which had been treated with atropine, the major site of the drug's action was assumed to be not at the hypo- physial level, but rather at higher CNS centers. Feedback characteristics of estrogen on neuroendocrine control of LRH secretion have likewise been examined. Schneider and McCann (1970c) showed that co-incubation of estradiol with pituitaries directly increased basal LH release, while the response to the addition of stalk median eminence fragments or purified LRF was not affected. They also demonstrated that inclusion of estrogen with incubates containing pituitaries, stalk median eminence fragments and dopamine inhibited the catecholamine's ability to enhance LH release through LRH mediation. 26 It was assumed that estrogen's suppressive effect on dopamine induced LRH release was brought about through synthesis of an inhibitory peptide since inhibition of protein synthesis with puromycin or cycloheximide abolished the effect. By using steroid blocked ovariectomized rats as assay animals for LRH activity, Schneider and McCann (1970a) extended the study of estrogen effects on dopaminergic control of LRH secretion. They reported that intraventricular dopamine injections increased LRF activity in serum of hypophysectomized rats. However pretreatment with intraventricular estrogen at 2 hours before dopamine administration prevented the monoamine's ability to increase serum LRF. These results indicate that although estrogen acts directly at the adenohypophysis to enhance LH release, it interferes with hypothalamic dopaminergic mechan- isms which promote LRH secretion. The recognized ability of estrogen to exert negative feedback on gonadotropin control may therefore be imple- mented by inhibiting dopaminergic LRH release. However estrogen's action to facilitate pulsatile gonadotropin surging appears to involve different neural mechanisms. Fluorescent histochemical examination of catecholamine content within infundibular d0paminergic neurons has been conducted in order to relate hypothalamic dopamine turnover rates with conditions known to alter endogenous LH secretion. After treating rats with a-methyltyrosine- methylester to inhibit catecholamine synthesis, a qualitative assessment of monoamine turnover is possible by measuring the relative degree of fluorescence attenuation through time. Fuxe et a2. (1969 and 1972) reported that the low dopamine turnover rates characteristic of 27 ovariectomized or immature female rats is elevated after treatment with low doses of exogenous estrogen or pregnant mare gonadotropin. Simi- larly, in intact female rats, it was shown that dopamine turnover was lowest during proestrus and early estrus, times of depleted pituitary LH content and presumed elevation of serum LH (Ahrén at aZ., 1971). Finally, intravenous LH therapy did not alter the-low dopamine turnover of hypophysectomized rats. These results provide evidence that dopa- minergic neural systems within the tubero-infundibular area of the hypothalamus may act to suppress LRH secretion. The conclusions from histochemical experiments concerning neuroendocrine involvement in control of LRH and gonadotropin secretion are in complete opposition to those concepts of gonadotropin control generated from direct alter- ation of hypothalamic neuro-transmitter availability. Future studies involving simultaneous use of both of these experimental techniques will be required to fully reconcile these currently contradictory hypotheses. Effects of LRH on Pituitary LH Secretion In 1966, Antunes-Rodrigues and co-workers attempted to gauge pituitary responsiveness to purified LH-releasing factor throughout the estrous cycle of normally cycling rats. Using bioassay techniques for serum LH determination, they found that the releasing factor elevated serum LH of rats in every stage of the estrous cycle. Although there were no significant changes in pituitary responsiveness during the estrous cycle, their results were suggestive of possible enhancement on the morning of proestrus. More precise assessment of pituitary responsiveness for LH release awaited the report of Matsuo at al. (1971) 28 describing successful synthesis of the decapeptide (pyro)Glu-His-Trp- Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (LRH), which was shown to have potency comparable to that of the natural porcine LH-releasing factor (Arimuri et aZ., 1972). Synthetic LRH was demonstrated to have a circulating half life of about seven minutes, and was shown to be selectively taken up by the pineal and the pituitary as well as by kidney and liver (Redding and Schally, 1972). Intravenous injection of LRH was reported to be effective in inducing ovulation only on the day of proestrus in both normally cycling and in pentobarbital blocked pro- estrous rats (Rippel et aZ., 1973). Similarly, injection of synthetic LRH into cycling female rats preferentially enhanced pituitary sensi- tivity to the releasing hormone on the afternoon of proestrus (Cooper et aZ., 1972; Martin et aZ., 1974). The infusion of small doses of synthetic LRH directly into the anterior pituitary by way of a hypo- physial portal vessel was also shown to effectively elevate serum LH levels (0ndo at aZ., 1973). In a pair of papers, Debeljuk et al. (1972a and 1972b) demonstrated that such LRH administration was more effective in inducing pituitary LH secretion in immature rats of both sexes than in corresponding adult animals. These data suggest that pituitary responsiveness differs in animals of different physiological conditions. Direct effects of ovarian steroids on pituitary sensitivity to exogenous LRH have recently been measured. In 1971 Arimura and Schally reported that estradiol benzoate given on day one of diestrus, 24 hours before LRH treatment, caused increased pituitary sensitivity to LRH 29 without altering basal LH levels. Similarly, when estradiol benzoate was given on the morning of estrus, pituitary response to LRH therapy was increased 48 hours later (Debeljuk at aZ., 1972c). Using a differ- ent experimental approach, Clemens et a1. (1972) provided indirect evidence that estradiol may increase pituitary responsiveness to LRH with the demonstration that estradiol benzoate augmented pituitary LH release in response to electrochemical stimulation in steroid blocked ovariectomized rats. On the other hand, Debeljuk at al. (1972c) demon- strated that treatment with progesterone or estradiol benzoate and progesterone on the day of estrus resulted in suppression of pituitary responsiveness to LRH treatment 48 hours later. Likewise it has been shown that when progesterone is given to cycling rats on the day of proestrus, basal LH concentrations are not different from those of rats receiving control oil treatment, while pituitary reactivity to LRH is suppressed when measured 48 hours after steroid therapy (Arimura and Schally, 1970). Using in vitra techniques, Schally et a1. (1973) also indicated that steroids can alter adenohypophysial responsiveness to LRH. Their work suggests that low levels of estradiol enhanced basal LH release without affecting the response to LRH. 0n the other hand, higher estradiol doses acted to suppress both resting LH release and the response to LRH co-incubation. They also reported that progesterone decreased the response to LRH without changing basal LH release rates. In evaluating the effects of steroids at the hypophysial level these studies generally indicate that in in viva conditions estrogen enhances pituitary responsiveness to LRH stimulation while progesterone has a 30 suppressive effect. However when examined in vitro, high levels of both steroids act to lessen the ability of LRH to induce pituitary LH secretion. In female rats the ability of ovarian steroids to enhance pituitary responsiveness to LRH is altered after ovariectomy, where in addition to estradiol benzoate's effect to suppress the already elevated basal serum LH levels, three days of treatment with the hormone results in decreasing pituitary capability to respond to LRH when compared to intact control animals (Negro-Vilar at aZ., 1973). Libertun at at. (1974) presented both in viva and in vitra evidence suggesting that ovariectomy decreased pituitary responsiveness to LRH, and that this suppression could be overcome following 3 days of pretreatment with subcutaneously administered estradiol benzoate. Rats also show a sex related difference in steroid feedback characteristics at the pituitary level. Debeljuk at al. (l972d) reported that 48 hour pretreatment with estradiol benzoate or testosterone depressed pituitary response to LRH in intact male rats. However after castration estradiol benzoate acted to facilitate the ability of LRH to elevate serum LH levels (Debeljuk et aZ., 1973)° METHODS Experimental Animals Long-Evans rats were maintained in a temperature controlled colony having a twelve hour light/dark schedule, with lights on from 6 a.m. to 6 p.m. They received Wayne Lab-Blox diet for rats and mice, and water ad Zibitum. All experimental animals were of similar genetic constitution, having been bred and maintained within the Endocrine Research Unit's rat colony. Daily smears of vaginal cytology were taken to determine patterns of each rat's reproductive status within the estrous cycle. Endocrine studies were performed using 4 to 6 month old proestrous, estrous and second day diestrous, as well as 23 to 30 month old constant estrous and constant diestrous female rats. 01d rats were considered to be in constant estrus or constant diestrus if they showed at least 10 consecutive days of cornified or leucocytic vaginal smears, respectively. Following experimental manipulation of any rat, a recovery period of at least 3 weeks was allowed to assure adequate hematocrit and blood volume recovery before the same animal was considered suitable for further experimentation. 31 32 Hormone and Drug Treatments Rats in each of the previously defined reproductive states were treated with various doses of L-dOpa (Hoffman—La Roche Inc.; Nutley, N.J.) or synthetic LRH (Eli Lily Inc.; Indianapolis, Ind.). In other experiments administration of L-dopa or LRH was coupled with subcuta- neous pretreatment with estradiol benzoate (Upjohn Co.; Kalamazoo, Mich.) or progesterone (Mann Research Laboratories; N.Y., N.Y.). For experi- ments involving solely L-dopa therapy, intraperitoneal (i.p.) injection of 0.5 ml of saline or an equivalent volume saline suspension containing 3 or 30 mg L-dopa was made between 1:30 and 2:00 p.m. Intravenous (i.v.) treatment with LRH alone using 0.5 ml saline solutions of 0, 5, 50, and 500 ng LRH was performed during light ether anesthesia between 10:30 an 11:00 a.m. Similar experimental regimes were then performed coupling subcutaneous pretreatment of 5 mg progesterone dissolved in 0.2 ml corn oil 2 hours before i.p. administration of L-dopa at 30 mg per 0.5 m1, i.v. LRH at 50 ng per 0.5 ml, or suitable saline control. In addition, 24 hour pretreatment with subcutaneous estradiol benzoate at a dose of 20 pg in 0.2 m1 corn oil was followed by L-dopa or LRH administration to young diestrous and old constant diestrous groups in the manner described for the progesterone treatment regime. The duration of estradiol benzoate pretreatment was 2 hours for rats in all other reproductive states of concern. All experiments using steroid priming were conducted in a manner to allow initiation of L-dopa and LRH therapy between 10:30 and 11:00 a.m. 33 Blood Collection Immediately after initial cage disturbance, pretreatment blood samples of about 1.5 ml were obtained during light ether anesthesia by suborbital sinus puncture using heparinized capillary tubes. This bleeding technique was previously shown to cause minimal stress effects on serum hormone concentrations (Riegle, unpublished). In all experi- mental designs, acute treatment with various doses of L-dopa or LRH was made about 10 minutes after the pretreatment bleeding. All animals receiving L-dOpa therapy were serially bled at 15, 60 and 120 minutes after drug administration. Similarly, rats were serially bled at 15, 30 and 60 minutes after LRH treatment. The resulting whole blood samples were then allowed to clot at room temperature for between 30 and 120 minutes. The samples were refrigerated overnight and then centrifuged to expedite serum collection. Serum samples were then frozen at -15°C for subsequent analysis of radioimmuno-assayable LH and prolactin. Radioimmuno-Assay for LH and Prolactin The respective radioimmuno-assay procedures for LH and prolactin determination are those described and validated by Monroe at al. in 1968 and Niswender et al. in 1969, and routinely used in the laboratory of Dr. J. Meites at Michigan State University. Purified rat prolactin (H-lO-lO-B Prolactin) and LH (LER 1056 LH) were radio-iodinated with 125I at Dr. Meites' facilities, and subsequently eluted through a l x 15 cm Bio-Gel P60 column. The labeled hormone elutant was then 34 diluted with 0.1% gelatin phosphate buffered saline (PBS) solution to a concentration of about 30,000 counts per minute (CPM) per 100 pl as counted in an automatic gamma well counter (Nuclear-Chicago, model 10851.; Des Plaines, 111.). Anti-rat LH antiserum had been prepared by immuniza- tion of rabbits with the purified hormone, and was then diluted to a working concentration of 1:28,000. The anti-rat prolactin antiserum, which had been produced by immunization of rabbits with purified pro- lactin, was used at a working dilution of 1:5,000. Precipitation of the antigen-antibody complexes of either LH or prolactin was performed using a sheep antiserum resulting from specific immunization against rabbit gamma globulin. The ovine anti-rabbit gamma globulin serum was routinely used at a 1:60 dilution. The radioimmuno-assay procedure is virtually identical for determination of LH and prolactin except for the use of different puri- fied and labeled hormone species, and the administration of specific rabbit antisera for combination with each hormone. Duplicate aliquots of unknown serum samples were generally run at two dilution volumes for hormone determination in both prolactin and LH assays. In all cases, the selected volume of serum from samples to be tested was placed in 12 x 75 mm diSPo culture tubes (Scientific Products; McGaw Park, 111.). All samples were diluted to a volume of 0.5 ml with 0.1% gelatin PBS. A volume of 0.2 ml of the working rabbit antiserum specific for rat LH or prolactin was then added to all culture tubes. Tubes were briefly vortexed and placed in a refrigerator at 4°C for 24 hours to allow equilibration of the complexing reaction between available hormone 35 antigen and the exogenously administered antibody. At the end of the incubation time 100 pl of radio-iodinated hormone with a total activity of about 30,000 CPM was pipeted into each tube, briefly vortexed, and re-incubated at 4°C for an additional 24 hour period. During this time the radio-labeled hormone competes with the unknown amount of native hormone within the original serum sample for available binding antibody in an equilibrium manner. Following the incubation 200 pl of the pre- cipitating ovine anti-rat gamma globulin antibody was added to each tube, followed by short duration vortexing. A 72 hour incubation at 4°C was then carried out to allow near maximal antigen-antibody com- plexing and precipitation. At the end of this incubation period all tubes received an additional 3 m1 of cold PBS, and were then centrifuged at 2200 revolutions per minute for 30 minutes in a refrigerated centri- fuge (National Equipment Co., model K). After discarding the superna- tant and drying the tube walls with tissue paper, the tubes were placed into plastic holding jackets and counted in the automatic gamma well counter. In addition to tubes with unknown amounts of serum hormone to be assayed, sets of culture tubes containing known amounts of purified hormone were included in the assay procedures and counted to serve as reference standards. Standards for the prolactin assays consisted of triplicate samples containing 16 different quantities of purified NIH rat prolactin RP-l ranging from 0.4 to 40 ng. Similarly, LH reference standards consisted of culture tubes containing triplicate samples of 16 different doses of purified NIH LH RP-l ranging from 0.8 to 40 ng. 36 Determinations of general binding characteristics of the assays were accomplished through inclusion of total count tubes, normal rabbit serum (NRS) tubes, and total antibody binding tubes into the assay procedure. Total count tubes received only the radio-iodinated hormone, and were a reflection of total efficiency of count recovery. The NRS tubes contained 200 p1 of diluted rabbit serum in 0.1% gelatin PBS rather than the hormone specific antibody, thus corresponding to non- specific binding activity. Total antibody binding tubes were equivalent to "zero hormone" standards. The counting time for all tubes in a given assay was calculated to equal 10,000 counts in the effective "zero hormone" standards (counts in total antibody binding tubes less those occurring in the NRS tubes). The non-specific activity represented by NRS tube counts was subtracted from all sample tubes counted by proper adjustment of the gamma counter's background setting. Standard curves were drawn on 3 cycle semi-logarithmic paper correlating CPM with the log of reference standard hormone doses. The standard curves thus generated exhibited 50% cold hormone binding at 3.3::O.l ng for prolactin determinations and 15.6:t0.8 ng in LH assays. Tabular representations were then derived from the standard curve to facilitate translation of CPM data into corresponding hormone content for all unknown serum samples tested. Quantitative serum hormone data were then transformed to ng/ml concentration units. 37 Statistical Analysis Daily vaginal smear data were recorded for all surviving rats in each of the experimental protocols used. Effects of the various treatments on the resultant vaginal cytology patterns were assessed after determination of the percentage of rats in each group whose patterns remained unchanged. Tests for differences of post-treatment cycling characteristics among animal groups were made using the non- parametric Kruskal-Wallis test for differences of location in ranked data (Sokal and Rolf, 1969), critical to a 5% significance level. Analysis of serum hormone data for dose response effects of acute treatment of L-dopa or LRH, and for steroid pretreatment effects was performed using analysis of variance. Use was made of a multivari- ate analysis of variance program which incorporated a transformation matrix to accommodate a blocking effect of hormone measurements through time on individual rats. The statistical program had been modified for use on Michigan State University's CDC 6500 computer by the Office of Research Consultation, School for Advanced Studies, College of Education, Michigan State University. A critical alpha probability value of 0.05 was selected for these analyses. In those studies where significant response variability for specific experimental factors was demonstrated, Duncan's Multiple Range Test was used to test for differences among means . RESULTS Vaginal Cytology Data The data in the first table summarize the various treatment effects on patterns of vaginal cytology. When no steroid pretreatment was given, neither acute L-dopa nor LRH therapy affected estrous cyclic- ity in 871t2% of the young rats independent of their reproductive status at the time of experimentation. However 500 ng of LRH given either on the morning of proestrus or day two diestrus usually delayed onset of the succeeding vaginal estrus by 2 or 3 days. Those young rats expe- riencing altered vaginal cycling patterns following any of the experi- mental regimes typically became pseudopregnant. Likewise, cyclicity was generally not affected either by reproductive status or acute drug therapy in young rats which had been primed with 5 mg progesterone two hours before an experiment. The only difference was that progesterone pretreatment to young rats on the second day of diestrus usually either delayed the next estrus by 1 to 4 days or caused the next day's pro- estrous smears to be followed by cytologic profiles typical of diestrus rather than the expected cornified patterns characteristic of estrus. 0n the other hand, pretreatment of young rats with 20 pg estradiol benzoate invariably caused appearance of 2 to 3 days of cornification, followed by onset of pseudopregnancy which lasted from 9 to 15 days regardless of acute L-dopa or LRH administration. 38 39 Acute treatment with either L-dopa or LRH did not alter vaginal cytology patterns of old female rats when compared to the corresponding control groups (Table 1). While estradiol benzoate priming consistently precipitated development of pseudopregnancy in all young rats, the treatment was incapable of causing similar responses in aged rats. Pretreatment with estradiol benzoate was also unable to significantly increase the frequency of cyclicity induction in old rats. Although estradiol benzoate therapy appeared to partially restore estrous cycling in the aged constant diestrous rats, this change was not significant. Progesterone pretreatment likewise had no effect on vaginal cytology patterns in senescent rats when compared to animals which received no pretreatment. However simple experimental manipulation significantly restored vaginal cyclicity of old rats regardless of steroid pretreatment or acute drug therapy. Further, nonspecific stressors were more effec- tive to cause resumption of estrous cycles in constant estrous than in constant diestrous rats, where 76:t6% of old constant estrous rats showed some recovery of cyclicity compared to 43:t7% of old rats orig- inally in the constant diestrous state. However, although old cycling rats typically failed to show characteristic proestrous smears, normal cycle lengths of between 4 and 6 days were observed in 59 of 97 constant estrous rats and 31 of 40 rats that were normally in constant diestrus. 4C1 .mcppuau peewme> emeeumus m:_3esm was; epe we eevuceeegeo .uemsueegu peacuepseexo mevzeppee xupuppuxu peerme> unsung; mevzegm mung epe me cmesaze .meguuuee Paevme> eomeeguee unwavevgxe mung ee sewageeeseu .A=e= ee Homeemv acmEuemcu pavemewcoexm Levee megouuee peewme> cw «usage on emzegm cove: mums me goeseze .ueesueegu emuecmvmee em>meuec gore: macaw m>+uueeegemg =e>wm as» :_ man; we geese: "cache AN~.V m\~ Moa.v o,\a M-.w m\~ Aoo.w ep\o Mom.w op\m Amm.v m\m Ame.w m\k _ Ian a: om AaN.V k\~ om.v op\m _h. “\m Ace. e.\o om. op\m Aeo.Pv e\e Awe. m\k mace-a as on Ao¢.v o_\¢ Aom.v op\m Ace.v op\e Aoo.v e_\e Aon.v op\~ Ace.v op\~ Aea.v o_\m ammmwmmwmmwuu .oeeceumm one Aek.v m\k Aem.v m\~ A-.e a\~ ANF.V e\_ Ace.v o_\o Ace.v op\o Ace.v a\o :aa m: cm Moe.v e_\¢ Aem.v m\m Aom.w op\m Ae¢.v m\¢ Ace.v o_\o Mee.w m\o Moe.v o_\o uaoe-a as on mk.v a\k Ame.v m\m ANN. m\~ Am~.V e\~ Ace.v a\o oe. e\o oo.v pp\o a mhwgwewwe " c e L a euee~eom —emeecumu Ao~.V op\~ Aom.v op\m Ace.v o.\m Aoo.v “\e Ace._v m\m Aoo._v e\m Ace.v ~\e zap a: com Mem.w p.\e Mm¢.w “\m Ace.v pp\~ Ap_. o\_ Ao~.v op\a Ace.v ep\e MFa.V _P\ep zaa a: em om. e\¢ em. a\m Aom.v e\¢ Ace. e\o Ace.v op\a Aom.v ep\e Ne. .m\e I“; a: m Me¢.w a\¢ Mom.w op\e Rem.w a\m Mop. eP\P Moe.Fv ep\ep Mme.w m_\F. Mom. e_\a mace-a es en mm. m\m om. op\m Ase. m\e co. e.\o ek.v ep\¢F Ne. e_\e_ mm. ep\m_ mace-a as m AP~.V e~\m aAmm.v k~\emm Am~.v e~\mp Aeo.v -\F Ama.v GM\mm hem.v QM\em afiem.v mMM\am~ .aceEHawmuwuneoz mecumewe mecumm mzcummwe assume mecummwo megumm mzcumeega anesugu penanceu “enumeeu acepmceu acoumeeu wage ecu «cease: xuvuvpuxu we ummco mccmuuee mewmeesuea museum o>wuu=eeceos meeree> e? xwepeuxu peewme> we megeuuee xuepeuxu Peevme> we megouuoe ee ececmummmece as m to mueeNeoe Feveecumm m: cm saw: pewsuemguege we use .xeesegu xx; ce eeeeie uueuu we muuummw .p e—ee» 41 Effects of L-Dopa on Serum Prolactin The data in Table 2 and Figures 3 and 4 indicate that basal serum prolactin concentrations were different among the reproductive states studied. Prolactin levels were highest during proestrus and estrus, intermediate in both groups of aged rats, and lowest during the second day of diestrus. Injection of the saline vehicle did not affect serum prolactin in any of the groups other than that of young rats on the day of estrus (Table 2). Saline treated control estrous rats showed prolactin reduction 15 minutes after injection which was maintained for at least 2 hours. However statistical analysis indicated that there was no significant change in serum prolactin levels through time when evaluated across all reproductive states. As is shown in Table 2, intraperitoneal administration of both 3 and 30 mg L-d0pa generally caused marked suppression of circulating prolactin levels by 15 minutes in rats of all reproductive states (Table 2; Figures 3 and 4). Although the high dose of L-dopa invariably caused maximal lowering of serum prolactin for at least 2 hours, the 3 mg drug dose was less effective, as indicated by significant inter- action between drug dose effect and time after administration (Figure 1). Analysis of the data indicates that, across all reproductive conditions, the low L-dopa dose was only effective in reducing prolactin at 15 min- utes after injection. Further, not all reproductive states were equally susceptible to L-dopa induced inhibition of prolactin secretion as indexed by serum hormone levels. Figure 2, illustrating the interaction between drug dose and reproductive status, shows that 3 mg L-dopa was 42 more effective in decreasing prolactin in young proestrous and estrous rats than in those on the second day of diestrus or in either group of aged rats when assessed across all of the post-injection blood sampling times. The apparent diminutive effect of the low L-dopa dose on the diestrous group was likely due to the presence of already low basal prolactin levels in this status. Figure 3 shows the existence of a three way interaction among drug dose, reproductive status and time after L-dopa injection in young rats. The data depicted in this figure suggest that the 30 mg L-dopa dose was optimally effective in reducing serum prolactin for the entire 2 hour period after injection in all groups tested. Similarly, the low L-dopa dose caused depression of serum prolactin to comparable levels in all young rats throughout the ensuing 2 hours. Conversely, the ability of L-dopa to lower serum pro- lactin was more transient in both groups of aged rats, as is shown in Figure 4. Serum prolactin levels were restored to approximately those of saline injected control values by 60 and 120 minutes after 3 mg L-dopa administration. By 120 minutes after injection, mean serum hormone concentrations tended to rebound above corresponding saline controls, though this didfference was not significant. Data in Tables 3 and 4 depict the effects of subcutaneous pre- treatment with 20 pg estradiol benzoate and 5 mg progesterone, respec- tively. As is shown in these tables and in Figure 5, pretreatment with either of these ovarian steroids was equally able to depress resting prolactin levels in rats on the days of proestrus and estrus, but did not change those hormone concentrations characteristic of the other 43 reproductive conditions which were studied. Neither of the steroid pretreatment regimes generally altered the ability of L-dopa to inhibit prolactin secretion in any of the reproductive conditions tested. However the 30 mg L-dopa dose used in this study was found to be less effective across time in young estrous rats than in any of the other reproductive states studied. This decreased responsiveness was mainly due to response refractoriness of estrous animals which had been primed with estradiol benzoate (Table 3). The presence of a significant interaction between drug treatment and time after injection (Figure 6) also indicates that the suppressive effect of 30 mg L-dopa had gener- ally begun to wane by 120 minutes after treatment in the steroid primed rats, a response which was not apparent in rats that received no steroid priming (Table 2; Figure 1). 44 .ewepm e>wuueeeceec em>wm e cw mucuspeega Fpe cow empeee wee mmepe> Peepeeo uemsueegpecae .Z.m.m H :me mm CZOnm wLm m:OPHMLHcm0:ou UCOELOI E? #8 ma a.m.: at... 5mm 2 88; as on 583.08 313.2 a.mimam 2 88; as m a.m.: a.m.: :3 we: c.3352 933.9: : 63:8 ”ngumwwo accumcoo UPC 3 a EN 3. a 2: m.m a EN 2 88; as on we? :3 We? :3 a. z... 95 a 8%; as m 933.2: 98358 4.83.5 $75.8, mm 5.28 ”ngpmm pcmpmcou UPC 3 3.: me 4. 2 E 3.2 S 88-.. as o... 9:“ ma... :2 we... 92“.. 92 S 88; as m 923.3 2352 Rename: 533.8 2 3.528 “magummwo em no.2 to 3.3 in new 2 88; S on 3: <3 :3 7mm 9:... 9mm 2 88; as m 533.8, 28.58 9mm.” <2: Néamemm E 2:28 ”magpmm we: 4.8 :3 0.8 Ni... 4.3 S 88; 9.. on 323.2: 923.5 «.23.: S 88-.. as m 0.83.4.3 2228 58438 ”330.28 S .828 ”magpmmosa CE 8 3 S. 2 33.. «383281 Ammpzcwev wave Ape\mcv cmyeepece Ezgmm meet epeEew “seemecem eee maze» to mpm>ep ewpeeFeLe Ezcmm ee “casueegu eeeene me mpoewmm .N epeeh 45 .pceEuemgpece epeeNeee peweecemm Lee; Leewuapeezwu .pcesueegaece eueeNeee Feweegume gee; ezwe .epeum cewuoaeeceee em>wm e cw mpcespeegp Ppe Lew eeweee age meepe> weepeee pemsueeguesae .Z.w.m H :mmE mm CZOzm wLm mcomechmucou mCOELOI EN“ 4.: 984. 9: 3 a 4.0m 2 38.; a... on 933.2: memaogm: 452:2 0.33.2: a 33:8 uumsLummwo unmumcou UFO 0.2.4 a.mm m: a.m.NN to a 92 m 88-.. as on cam“ a.mmm 922:: 0.33.2: 92.222 2 63:8 enmecumm uceumeeo ewo em 2.: Z 3.: m: 3.2 2 38.; me on 523.42 .22... e2 ”H.833: 93:52 2 :228 unmasumewo e238: 2398 @3382 a 88; as on :33: 310.48 c.8432 :1 :2 a . 295:8 enmecumm 92.. 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"meeumewo ucepmeeu epo 3.23.3 3?. 2.8 «3.23.; 2 88; 2 cm :2332 :3 3% :23: 92... m2: 2 2228 ”weepmm peeumeeu epo 33 mam :.23.$ 9:32 : 88L 2 cm 3.2.3.8 3.4.3:: 923.2: 3.23.8 2 2228 ”mesummwo 223.2 :3 3.2 3 a 3.2 2 823 2 cm 983.83 3m... :2 923.2: 223.02 2 :228 "ngumm 933.2 o.m 3.8 3 3.: 2 38; 2 cm c.2352 33:2 c.3322 “333.: 2 2228 ”mecummeea o2 8 2 2- a 33m 2568883 Amepecwsv esww AFE\mev cwyuepece Eagem ecegmpmemece mg m cpwz acesueegumce Lee; exp mew>weoec was: eweEew pneumecem ece maze» we mwm>mw ewuoewege Eecem co xmecmsp eeeeum we mueewwu .e eweew Figure l. 47 Serum prolactin levels in female rats after administration of various doses of L-dopa, without regard to reproductive status . The height of each bar represents mean serum prolactin con— centration expressed as ng/ml, with brackets corresponding to the standard error of the mean. Each cluster of graphs indicates measured prolactin concentration at l5, 60 or 120 minutes after drug injection. Open bars depict control responses while striped and shaded bars correspond to 3 and 30 mg L-dopa treatment, respectively. PROtACTIN 48 . .‘ - i ——l E. E 'r .5: E —————1 § [Lu/Bu g ° mg L-DOpa Figure l Figure 2. 49 Serum prolactin concentrations in young and aged female rat:xs; after administration of various doses of L-dopa. The height of each bar represents mean serum prolactin con- centration expressed as ng/ml, with brackets corresponding to the standard error of the mean. Open bars signify responses to saline control while those which are striped or shaded correspond to prolactin levels measured after 3 or 30 mg L-dopa, respectively. The effects of the various L-dopa doses on serum prolactin levels are shown for young rats on the days of proestrus, estrus and the second day of diestrus, as well as in aged constant estrous and constant diestrous rats. Data illustrated in the figure represent pooled prolactin values from 15, 60 and l20 minutes after L-dOpa therapy for the appropriate reproductive conditions. 0'd c. o. O'd C. E. Diostru, 50 > F1gure 2 Figure 3. 51 Effects of L-d0pa therapy on serum prolactin concentrations in young cycling female rats. These data illustrate the effects of L-dopa treatment on serum prolactin levels in young proestrous, estrous and second day diestrous rats. Mean serum prolactin levels expressed in ng/ml and their standard errors appear on the ordinate as a function of time relative to injection of various L-dopa doses. L-dopa administration is depicted by a caret on the abscissa, with blood samples taken l5 minutes before drug treatment, and at l5, 60 and 120 minutes afterwards. Solid circles represent control values while open circles and open squares signify 3 and 30 mg L-dopa treatment, respectively. 52 m ecemwm “Ob—acme eN— cc 3 m7 e- 8 3 m7 o :08: a... BMW“: enlnfigo AIM .m m ImAa/ue‘mm\ H s: a jee.H\ m sees” =3 H .L/J 3.805 as 3me 2: eezeooi nu] ugpolmd Bu g 9 O m Figure 4. 53 Effects of L-dopa therapy on serum prolactin concentrations in aged female rats. These data illustrate the effects of L-dopa treatment on serum prolactin levels in senescent constant estrous and constant diestrous rats. Mean serum prolactin levels expressed in ng/ml and their standard errors appear on the ordinate as a function of time relative to injection of various L-dopa doses. L-dopa administration is depicted by a caret on the abscissa, with blood samples taken 15 minutes before drug treatment, and at l5, 60 and 120 minutes afterwards. Solid circles represent control values while open circles and open squares signify 3 and 30 mg L-dopa treatment, reSpectively. 54 =- co m— H\m3m E .- H ll\ :2 .u. 2:505 2:32.50 o._o e weaned 2-.. e: s a 2.: n a: as: a la H .IH ll \ H a: .w H H em~ emN \ $39.5 u an M nu el D m. [I m. 253 22200 =3 9.5 2.... Figure 5. 55 Effect of steroid pretreatment on basal serum prolactin concentration in young and aged female rats. The height of each bar represents mean serum prolactin concentration expressed as ng/ml, with brackets corresponding to the standard error of the mean. Each bar indicates pro- lactin levels in serum samples taken at about 15 minutes before acute L-dopa treatment. Open bars depict responses in rats receiving no steroid pretreatment while shaded and striped bars correspond to subcutaneous pretreatment with 20 ug estradiol benzoate and 5 mg progesterone, respectively. Two hour progesterone pretreatment was employed in all repro— ductive states. Similar two hour estrogen priming was per- formed in all groups except young rats on the second day of diestrus and aged constant diestrous animals, which were given 24 hours of estrogen pretreatment. 56 m weave; ouohm om.“ w .9; .3 w . . u .eo... .3 u to... . V L 30 .w .u 20 3:35 2:3 3:32.. .nu Av 2.51.2... .223 Figure 6. 57 Effect of steroid pretreatment on the ability of L-d0pa therapy to suppress serum prolactin concentration in fema'l e rats, without regard to reproductive status. The height of each bar represents mean serum prolactin con — centration expressed as ng/ml, with brackets corresponding; to the standard error of the mean. Each cluster of graphs: indicates measured prolactin concentration at 15, 60 and 120 minutes after drug injection for untreated rats as wel 1 as those receiving pretreatment with 20 ug estradiol benzoate or 5 mg progesterone. Open bars signify prolactin levels “" saline treated controls at various times after injection. Serum prolactin levels following 30 mg L-dopa treatment ar’£3 designated by squared, shaded and striped bars in rats receiving no steroid, estradiol benzoate and progesterone priming, respectively. Two hour progesterone pretreatment was employed in all reproductive states. Similar two hour estrogen priming was performed in all groups except young rats on the second day of diestrus and aged constant diestrous animals, which were given 24 hours of estrogen pretreatment - A. 58 o egemwm .EE efi SE 3 2.3-. 9.. e n E” E SE 2 =2 l‘" / ugpqoad Bu a 6 N 59 Effects of L-Dopa on Serum LH The series of experiments designed to assess the influence of systemic L-dopa treatment on serum LH levels involved drug admin- istration at about l:30 p.m. As is seen in Table 5 and Figure 7, basal LH levels at about lO minutes before L-dopa therapy were markedly elevated and quite variable in proestrous rats. This finding indicates that the preovulatory gonadotrOpin surge had already begun to occur in several of the rats studied. Of the low levels representative of all other reproductive states, LH concentrations of old constant estrous rats were slightly greater than those of either estrous or diestrous, which in turn exceeded those found in aged constant diestrous animals. The effect of drug treatment through time depended on dosage used. Figure 8 shows the interaction between L-dopa dose and time after injection as averaged for all reproductive conditions. Although the data depicted in this figure were highly variable, they suggest that the 30 mg L—dopa dose elevated serum LH at l5 and 60 minutes, but did not change LH from corresponding control levels at lZO minutes post- injection. On the other hand, the 3 mg dose appeared to cause moderate LH elevation which was maintained for the entire 120 minute sampling period. Analysis of variance showed that the effectiveness of L-dopa to elevate serum LH depended on the reproductive status of animals studied. Data in Table 5 and Figure 9 indicate that 3 mg L-dopa optimally stimulated pituitary LH release in young diestrous rats, significantly increasing serum LH for at least 120 minutes. However, this drug dose did not cause significant LH changes in any of the other 60 reproductive conditions studied. The effectiveness of 30 mg L-dopa also varied as functions of time after injection and of reproductive status. Figure 9 shows that the high dose of L-dopa nearly doubled the already greatly elevated serum LH concentrations in proestrous rats at 15 and 60 minutes post injection. Thirty mg of L-dopa also markedly augmented serum LH after l5 minutes in the young estrous group. On the other hand this drug dose caused only moderate LH elevation in young diestrous rats, though the effect was evident for the entire 120 minute sampling period. In contrast, data in Figure 10 indicate that old rats responded differently to 30 mg L-dopa than did younger ones. While L-dopa caused LH enhancement in constant estrous rats which was com- parable to that of young rats on the day of estrus, the response of old animals was not significant until 60 and 120 minutes after treat- ment. Old constant diestrous rats were completely unresponsive to 30 mg L-dopa. Figure ll depicts basal serum LH levels as a function of pretreatment with estradiol benzoate or progesterone. Estradiol benzoate therapy suppressed resting serum LH levels below those char- acteristic of untreated controls when assessed across all reproductive conditions. Evaluation of responsiveness within each reproductive status showed that this depressive effect was significant in proestrous, diestrous and aged constant diestrous rats. Although progesterone priming tended to decrease basal serum LH in all groups, this differ- ence was not significant. Pretreatment with estradiol benzoate blocked L-dopa's ability to elevate serum LH by l5 minutes after injection in 61 all reproductive conditions other than the young proestrous group (Table 6; Figure l2). Direct comparison of responses between untreated control rats on the day of proestrus and those receiving estradiol benzo— ate pretreatment was not made because the experiments involving steroid priming were initiated at 10 a.m. in order to avoid the demonstrated early afternoon preovulatory LH rise in proestrous rats. However, response similarity between control and estradiol benzoate pretreated proestrous rats is suggested since in both groups L-dopa caused signif- icant elevation above corresponding saline injected controls at 15 and 60 minutes after administration. On the other hand, prior therapy with exogenous progesterone did not significantly affect the ability of 30 mg L-dopa to alter serum LH at l5 minutes after injection in any of the reproductive conditions studied (Table 7; Figure 12). However, compar- ison of data in Table 7 with that in Table 5 indicates that progesterone priming increased the duration of L-dopa's effect in young rats since pretreatment resulted in L-dopa induced LH elevation which lasted for the entire 2 hour period in these animals. Conversely, Table 7 shows that progesterone inhibited the effect of L-dopa to raise serum LH levels through time in unprimed constant estrous rats, whose responses are summarized in Table 5. 62 .epepm e>wueeeeceec ce>wm e cw mucuseeegp Ppe Lew eeweee ewe meepe> Fegueee peeEueeweesae .z.w.m H ewes we :zezm ewe mcewuegpeeecee eeeEce: ed 3.: 3 3.2 we 35 2 88; as on me 3.“ o; a: we 36 3‘ 35 2 5:8? ”magpmmwo accumcou US a.mp “He.em m.e2 “ne.ee _e.__ a _.Ne 02 aeoe-4 as on E 3.8 we 3.8 m.“ 333 2 382.23 a.m. 3.5 E 3.33 S 3.33 .3 353 mm .828 nmzwpmm ucmumcou US e.m “He.m. ¢.e H.¢.2~ m.e Mme.~N e. mace-4 me em 0.x aH~.mN e.m “He.mm _.e_ “ne.m~ N2 aeoe-e as 3 To 3.2 we 3.: m; 3.2 3 3E : 2828 umzwummwo m._ M”~.N2 m.e HUN.~P e.h_ “Ho.em o2 aeoe-4 as on e;e_ H _.em m.e_ “Hm.em m._~ “um.ee 22 mace-4 as 3 92 3.8 _.m 3.: ea 3E 3 3.: a 2228 "mewpmm e.em “no.3mm n._mmsnm.m- e.eemsnu.wmn o2 mace-4 as on e.em2num.2mm 3.242aum.eem e.mm_une.emm __ mace-4 as m a.mm “Hm.wm e.wo_flum.eam m.ompsnm.mmm me.oo_sum.eem .N _oee=oe "mawpmwoga emp em mp mp- : epepm e>wue=eegeem Ameuecwev mew» A_e\mev :4 Ezcmm meme mFeEew eceemeeem eee maze» we mpe>ep :4 Eegem ce meagegu eeeeue we mpeewwu .m eweew 63 .paeeueeapeae epeeNaee Peweeaume azea azew-»pae3we .paeEueeapeae eweeNaee Peweeaume azea ezw e .eueum e>wpezeeaeea ae>wm e aw meaesueeau ppm aew empeee mam mezpe> Feapaee paesueeaueaee .z.m.m H aeee me azeam mam maewpeauaeeaee eaeEae: tom r—W \OLD LOO OLD KOO r-F- NLD NED 00.— FF +l +l +| +| CDGD «ac: 3K>d3 Fm +I +| Vd’ Q'N \ON ‘01? 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"mzaumm pacemaee e_o 0.23.; 923.33 93 3.2 : 38.4 2. em 9m “92 ed “mg TN “N.“ e.m “9: S .93an ”mzaumewe c.32auo.ew o.e_anm.me m.emflum.me op meeenz me om edgimm w.~ ANA: m.m H52 m.m HT: 3 3.5an ”mzapmm m.epnflm.ew «.mpnflw.ww e.m Hflmze op eeeeuz me on we Hmzm m.m “Tme m6 “ode em..\. “Tee 3 35:8 ”mzapmeeaa emp om mp mp- a euepm e>wpezeeaeem Amwp=Cwsv aaww A_a\mcv Iz azamm eaeaeememeae me m apwz paeEeeeapeae azea wee; eweEew uaeemeamm eae maze» we mpe>ep :4 Ezaem ae »eeaeau eeeeu4 we mpeewwm e3» maw>weuea .n eweew Figure 7. 65 Resting serum LH levels in young and aged female rats. The height of each bar represents mean serum LH concentration expressed as ng/ml, with brackets corresponding to the standard error of the mean. The number within each bar denotes that group's sample size. 66 u:..n0_n Jacou vZU aaawum £ucou tzu w egzmww n:..uo_o naaunm naa.aooam 1 6 N IUU./ l4] 6m! °\' as men Figure 8. 67 Serum LH levels in female rats after administration of various doses of L-dopa, without regard to reproductive status. The height of each bar represents mean serum LH concentration expressed as ng/ml, with brackets corresponding to the standard error of the mean. Each group of three bars indi- cates measured LH concentration at 15, 60 and l20 minutes after drug injection. Open bars depict control responses while striped and shaded bars correspond to 3 and 30 mg L-dopa treatment, respectively. 68 m 95m: oaom-.. GE 3 e SE :2 EE 3 EE 3 Figure 9. 69 Effects of L-dopa therapy on serum LH concentrations in young cycling female rats. These data illustrate the effects of L-dopa treatment on serum LH levels in young proestrous, estrous and second day diestrous rats. Mean serum LH levels expressed in ng/ml and their standard errors appear on the ordinate as a function of time relative to injection of various L-dopa doses. L-dopa administration is depicted by a caret on the abscissa, with blood samples taken 15 minutes before drug treatment, and at 15, 60 and 120 minutes afterwards. Solid circles represent control values while open circles and open squares signify 3 and 30 mg L-dopa treatment, respectively. 70 m mazmwe 335:. :2 3 2 2. m =2 3 m— 1 M/ LIL aem / m/H 2H /Hszae a IVauH as u .» sea...” \ H 3 a zzusmzfl 3 P g as. - . a 8: «milk .W 239: 3% 1: H m. .. Lu .2 2:305 3 3:3 3:30.... am 32 Figure 10. 71 Effects of L-dopa therapy on serum LH concentrations in aged female rats. These data illustrate the effects of L-dopa treatment on serum LH levels in senescent constant estrous and constant diestrous rats. Mean serum LH levels expressed in ng/ml and their standard errors appear on the ordinate as a func- tion of time relative to injection of various L-d0pa doses. L-d0pa administration is depicted by a caret on the abscissa, with blood samples taken at 15 minutes before drug treatment, and at 15, 60 and l20 minutes afterwards. Solid circles represent control values while open circles and open squares signify 3 and 30 mg L—dopa treatment, respectively. op 8:3“. ”3:58 :2 S . 3 m7 :2 8 3 m? a o 2 w C‘U/Olh lm Misses: 2 :3 H, H as? l H V.IH 2 H m H\ s u B 1 /\ H 2 as: / A V W 3:33 Enacou 1.0 3 mafia 2.2225 1.0 S ‘/ Figure ll. 73 Effect of steroid pretreatment on basal serum LH concentration in young and aged female rats. The height of each bar represents mean serum LH concen- tration expressed as ng/ml, with brackets corresponding to the standard error of the mean. Each bar indicates LH levels in serum samples taken at about 15 minutes before acute L-dopa treatment. Open bars depict responses in rats receiving no steroid pretreatment while shaded and striped bars correspond to subcutaneous pretreatment with 20 ug estradiol benzoate and 5 mg progesterone, respectively. Two hour progesterone pretreatment was employed in all reproductive states. Similar two hour estrogen priming was performed in all groups except young rats on the second day of diestrus and aged constant diestrous animals, which were given 24 hours of estrogen pretreatment. Basal L" Consi. Const. Esirus 200! Diestrus Esirus DiesirUS Proesirus Ilu /BU 100 ' 74 E. 5' O L n: In l J c Figure 11 Figure l2. 75 Effect of steroid pretreatment on the ability of L-dopa therapy to increase serum LH concentration in young and aged female rats. The ordinate portrays the difference in serum LH concen- tration in ng/ml at 15 minutes following intraperitoneal administration of 30 mg L-dopa or saline. Positive values indicate that serum LH levels in rats receiving L-dopa exceeded those in corresponding controls, while negative values (those falling within the striped area) demonstrate the reverse relationship. Those bars in the left portion of the figure illustrate responses in untreated control rats of all reproductive states tested. The center cluster of bars shows responses following two hour pretreatment with 5 mg progesterone. In the set of bars on the right, 24 hours of pretreatment with 20 pg estradiol benzoate was used in aged constant diestrous rats and young rats on the second day of diestrus, while rats in the remaining repro- ductive conditions received 2 hours of estrogen priming. mesa? 3:. . m .35... dd dd 5.5 .3” .o wV/J 77 Effects of LRH on Serum LH The responses of the various groups to intravenously admin- istered synthetic LRH are summarized in Table 8. Although the 5 ng dose was ineffective, 50 and 500 ng LRH rapidly increased serum LH in a dose related manner. Figure l3 relates the effect of LRH at the blood sampling times of 15, 30 and 60 minutes as averaged across all repro- ductive groups. Data in this figure also demonstrate that both 50 and 500 ng LRH caused maximal effect in the blood sample taken l5 minutes following injection. Further, the existence of an interaction between LRH dose and reproductive status of the experimental subjects is de- picted in Figures l4 and l5, where serum LH data is averaged across the post-injection bleeding intervals for animals of each physiological sta- tus. As is most clearly shown by the response to 500 ng LRH, the syn- thetic polypeptide was most effective in young proestrous and estrous rats, intermediately so in young diestrous and old constant estrous animals, and minimally effective in the old constant diestrous group. Data in Table 8 and Figures l4 and l5 also suggest the existence of an additional significant interaction between drug dose and reproductive condition through time after injection. Although the magnitude of response at 15 minutes was greater in proestrous and estrous rats than those of the other groups, the time course of the effect was more transient as evidenced in decline of serum by 50% toward baseline levels by 60 minutes after injection. In contrast, the slight elevation characteristic of young diestrous and senescent rats tended to be pro- portionally more sustained through time. However, even at 60 minutes 78 after LRH injection, absolute serum LH levels were still higher in young proestrous and estrous rats than in those of the remaining reproductive conditions. Pretreatment regimes incorporating either progesterone of estradiol benzoate injection (Tables 9 and 10) caused significant changes in pituitary responsiveness to exogenous LRH stimulation as indexed by serum LH levels. Data in Figure 16 describe the interaction of steroid pretreatment on the degree of LRH induced increase of serum LH as averaged through time after injection for all reproductive condi- tions studied. Statistical analysis shows that although priming with either of the ovarian steroids did not greatly affect basal serum LH, it did significantly enhance the ability of LRH to elevate serum LH. Differences were found in the ability of progesterone to increase pituitary sensitivity to LRH stimulation when administered to rats in different reproductive states. Figure 17 summarizes changes in serum LH occurring at l5 minutes after injection of 50 ng LRH (that time characteristic of maximal serum LH increase) in all experimental groups following steroid pretreatment. Estradiol benzoate therapy in- creased pituitary responsiveness during estrus and to some degree in diestrous rats, but did not increase the already high responsiveness occurring on the day of proestrus. The steroid also restored pituitary response capability of old constant estrous rats to that characteristic of young proestrous animals. 0n the other hand, old constant diestrous rats, like young rats during diestrus, did not exhibit greatly enhanced pituitary responsiveness while under the estrogen influence. In young 79 rats progesterone priming consistently increased pituitary responsiveness to LRH, with the effect being most predominant on the days of proestrus and estrus. However in aged rats progesterone therapy did not significantly alter the ability of LRH to increase serum LH when compared to the response of control animals which received no steroid priming. Effects of LRH therapy on serum LH levels of young and senescent female rats Table 8. Serum LH (mg/m1) Time (minutes) 15 60 30 -15 Reproductive state Proestrus oouno OLOt—N r—r—r—N +1 +1 +1 +1 0000 @0500 SODNLO N LOLOQ'Q “DI-Om“) F- O +1 +1 +1 +1 LDNOO NQMF- Q'Q'NN V moooo ngN F +1 +1 +1 +1 Cd'wo Fr—d't— LOQ'OF PM) 38.4 i 6.83 Contro 5 ng LRH 50 ng LRH 500 ng LRH Estrus 12.1 +1 50.1 11.5 +1 35.8 8.8 +1 33.1 1.6 +1 22.6 10 ontrol 5 ng LRH 50 ng LRH 80 Che—w #NLD m +1 +1 +1 @000 Lowd- PNM m VOL!) NOON PM) +1 +1 +1 l—OO ooém NED“) <' Nooo mNLo u—u—m F +1+1+1 Nooo use va-ooo CO 10 500 ng LRH Diestrus NNtDm (VI—NOW N +1 +1 +1 +1 LOMNO moooun m P MNNLO Oil—em ‘0 +1 +1 +1 +1 051131.00 GNP-£0 r—m N omootx mome- <2- +1+1+1+l oqmoo 0066K: Now N 18.1 i 5.2 10 10 10 I In: Im—J I—m_.l 0.1 U5 L 0": +953: C: O O CO ummm 01d Constant Estrus ”NF-m @5000.— I- F-d' +1 +1 +1 +1 d'mmo [\mmr— LOP-MN P fiON‘O émmm u—-l- +1 +1 +1 +1 @0300 omoocr Q’FWN F Ofidztom d’l-DNI— F'l-‘N +1 +1 +1 +1 [\Q'Q'O mpzoim NVLON F 37.9 i 2.3 030100 3: Im Int-J PEI—l 0—1 0" L O): HO: C: o O CO Qmmm 01d Constant Diestrus DECO” OONCQ ,— +1 +1 +1 +1 NNNO wako Nr—I—Q OOr—LD 0K0: +1 +1 +1 +1 NOCDOS Nd’l-OOO. l—F-Nw \OQ'LDM Kn—ooso +1 +1 +1 +1 moooua [\l\.I—O‘; l- cm 22.0 i 3.7 5 ng LRH 50 ng LRH 500 ng LRH Control Hormone concentrations are shown as mean i S.E.M. state. 0 1V8 duct in a given repro aPretreatment control values are pooled for all treatments 81 .ucmsummgamga mumo~cma Forumsumm Lao; Lzowuzucmzhu .ucmEpmmgpmga wpmo~cmn Fowumgumm Lao; ask a .mpmum m>wpuauocamg cm>wm a cw mangpmmgp ppm so; empooa mew mmapm> Fogpcoo pcmsumwgumgam .z.m.m A cums mm czogm men mcovpmgacmucou mcoeso: 8;: EN :3 8.: ma: 8.3 S :3 9. S 3 3.2 -- m: 2; o; no.3 S 2:28 unngummmo acmumcou 2o N.¢anm.mm _.NN..N.NFF m.m~.”m.opp o_ I“; a: om 9m 3.: -- $23.8 983.: 2 P828 nungumm pcmpmzou US 3: 8.3 733.: 32:35.8 2 =3 9. 8 3 “9m 1 2 $8 3 23 S 2:28 unmagummvo .8 3.3 2.3.8 33:: 2 5: a: om 3 3.8 -- ma 3.: 3:33 m :55 numagumm :w a. :m 03 no.8 2398, S :5 9. om ¢.m sum.NN -- o.m “ne.ep mo.m_.”_.mm __ Poebcou ungummogg 5 oo om mp m_- c mumpm aspuuaeoeamm Ammuzcwev wave A_E\mcv I; Eacmm acmsummgumga mumoNcma Fowumgumm m: cm mcw>rmumg mum; mFmEm» ucwommcmm use acne» we mFo>mF I; Ezgmm co xamgmgu zm4 eo muumwem .m «pack 82 .mpmum m>wuu=uoggmg cm>wm 8 av mucmsummgp PPM com umpooa mgm mmzpm> Fospcoo pawsummgumsam .£.u.m H :mmE mm :305m mgm mCvaMchmucou wcoELOI 3 3.3 5833 3:“ :3 m 5: 9. cm as 3.2 2. 3.23.: 3.23.5 2 25:8 ungummwo accumcou UPC :3 3m 3.33.33 3.33 3.3m 2 =5 9. cm me 3.2 -- M: 3.2 3 n is 2 25:8 ”msg#mm vcmumcou UPC 3 3.5 3 3.3 373.3. 2 5: 9. om m.~ 3.3 -- 3 3.3 8.23.: S .828 "ngpmmwo 933.8 3.83.3 3.83.33 2 :5 3: om 3.23.: 1 3 3.2 3:“ EN 2 35:8 "masumm 3.23.2: 3.23.2: 3.3352 2 :3 9. cm 3 H E: -- m8 3.3 To 3.8 2 F828 "magpmmogq 8 cm 3 2- c 38% 8382328”. Ammpzcwsv were Aps\mcv :4 Eagmm gemspmmgumcg mcogmpmmmoga as m gpwz ucwapmmgumga Lao; o3“ m:_>woumg mpmc m—memw pcmummcmm new acne» $0 mpm>mp :4 Ezgmm co xamgmcu Ix; we muumewm .o_ anMF 83 Figure 13. Serum LH levels in female rats after administration of various doses of LRH, without regard to reproductive status. The height of each bar represents mean serum LH concen- tration expressed as ng/ml, with brackets corresponding to standard error of the mean. Each cluster of bars indicates measured LH concentration at 15, 30 or 60 minutes after LRH injection. Open bars depict control responses. Horizontally striped, diagonally striped, and shaded columns correspond to 5, 50 and 500 ng LRH treatment, respectively. 84 .EE 3 8m m3 wgsmwm Is.— m: on m .5... a .5... m: :2 "Wm Bu Figure 14. 85 Effects of LRH therapy on serum LH concentrations in young cycling female rats. These data illustrate the effects of LRH treatment on serum LH levels in young proestrous, estrous and second day diestrous rats. Mean serum LH levels expressed as ng/ml and their standard errors appear on the ordinate as a function of time relative to injection of various LRH doses. LRH administration is depicted by a caret on the abscissa, with blood samples taken about 10 minutes before hormone treatment, and at 15, 30 and 60 minutes afterwards. Solid circles represent control values while open circles and open squares signify 50 and 500 ng LRH treatment, respectively. 86 3 853“. a o . 2 SE a 8 on m— 2 ”III. S—WHU “Ill“ 0". / as 9. a n H k m/ 8:23... H is. 3 a. a: co... he mDL~»O_O magnum I.— A: 48. I1 E o .2 u o. Hillel» lllll'n/ a: a: 3m 3. D: OOn mac.»oocm 3 an 2 S- i. I 2; I“! /Bu Es Figure 15. 87 Effects of LRH therapy on serum LH concentrations in aged female rats. These data illustrate the effects of LRH treatment on serum LH levels in aged constant estrous and constant diestrous rats. Mean serum LH levels expressed in ng/ml and their standard errors appear on the ordinate as a function of time relative to injection of various LRH doses. LRH administration is depicted by a caret on the abscissa, with blood samples taken about 10 minutes before hormone treatment, and at 15, 30 and 60 minutes afterwards. Solid circles represent control values while open circles and open squares signify 50 and 500 ng LRH treatment, respectively. 88 m3 mczmwu $35.: 33 an m“ e_- 39 em mu , °_- c.1111” - “11:... . F . 1 3 . I I I a: on 0 1mAon n - . 133.. H - . H m 9. co» m L n 60¢ . 3235 33300 20 3: -_._ 3.23 “c3200 20 :3 lw/fiu 89 Figure 16. Effect of steroid pretreatment on the ability of LRH therapy to elevate serum LH concentrations in female rats, without regard to reproductive status. The height of each bar represents mean serum LH concen- trations expressed as ng/ml, with brackets corresponding to the standard error of the mean. Each cluster of bars indicates the average LH concentration measured between 15 and 60 minutes after LRH injection for untreated rats as well as those receiving pretreatment with 20 pg estradiol benzoate or 5 mg progesterone pretreatment. Two hour progesterone priming was employed in all reproductive groups. Similar two hour estrogen pretreatment was per- formed in all groups except young rats on the second day of diestrus and aged constant diestrous animals, which were given 24 hours of estrogen pretreatment. Open bars signify LH levels in saline treated controls while shaded bars correspond to LH titers measured in rats having received 50 ng LRH. o— osampu :5 a: a... 90 .m ._O_o