MICROSCOPEC OBSERVAWONS OF IN VETRO CULTURES OF THE ANTERIOR NTUETARY FROM THE MALE RAT M. S. MECE'HGAN STATE UNEVERSéTY THEODORE E. STALE 1966 THESE LIBRARY Michigan State University (HES! MICROSCOPIC OBSERVATIONS OF IE VITRO CULTURES OF THE ANTERIOR PITUITARY FROM THE MALE RAT by Theodore E. Staley A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 1966 ACKNOWLEDGEMENTS The writer would like to extend his gratitude and appreciation to Dr. M. L. Calhoun, for her many hours of reading this manuscript, and to Dr. J. Meites, Department of Physiology, for his guidance and criticism during the course of this work. Special thanks is extended to Dr. B. L. Baker, of the Anatomy Department, University of Michigan, for his help on the staining procedures. Special mention is also due to Dr. D. Schmidt, of the Pathology Department, for the photomicrographs, and to Dr. C. Kragt, Depart- ment of Physiology, for the prolactin assays. ii TABLE OF CONTENTS INTRODUCTION . JUSTIFICATIONS FOR INVESTIGATIONS IN THIS AREA . GROSS ANATOMY OF THE RAT PITUITARY . REVIEW OF LITERATURE . . . . . . . . . . . . . . I. Classification of Cell Types . . A. Acidophils-basophils . . . 3. Greek Letter Classification C. Correlation with Hormones . . . II. Methods of Demonstrating Specific Cell Types . A. Periodic Acid Schiff Reaction B. Aldehyde Fuchsin Stain . . . . C. Extraction Procedures . . D. Trichrome Stains . . . . E. Fluorescent Antibody Technics F. deioautographic Procedures . III. Present Knowledge of the Microanatomy of the Rat Pituitary . . . . . . . . . . A. Growth Hormone Cell . . . . . B. Luteotropic or Prolactin Cell . . C. Follicle Stimulating Hormone Cell . . . D. Luteinizing Hormone Cell . . . . . E. Thyrotropic Cell or TSH Cell . F. Adrenocorticotropic Producing Cell . G. Chromophobic Cell IV. Cell Counts . . . . . . . . . . . . . . . V. The Control of Variables in the Study of Physiology and Cytology of the Pituitary . . . . . VI. Cytology of Pituitary Transplants . . . . . VII. Cytology of the Pituitary in an In Vitro Culture . iii Page mmm \l NVO‘O‘U‘IUI r-w-I‘ 0.001) 12 13 14 Page MATERIALS & METHODS . . . . . . . . . . . . . . . . . . . . . . 18 I. Animals . . . . . . . . . . . . . . . . . . . . . . . . 18 II. Culture Methods . . . . . . . . . . . . . . . . . . . . 18 III. Culture Medium . . . . . . . . . . . . . . . . . . . . . 20 IV. Culture Procedure . . . . . . . . . . . . . . . . . . . 21 V. Assay Procedure . . . . . . . . . . . . . . . . . . . . 22 VI. Histological Procedures . . . . . . . . . . . . . . . . 23 VII. Techniques for Cell Counting . . . . . . . . . . . . . . »23 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 I. Viability of Cultures . . . . . . . . . . . . . . . . . 25 II. Survival of Anterior Pituitary Cell Types . . . . . . . 26 III. Comparison of Orange G AcidOphils & Acid Fuchsin Acidophils . . . . . . . . . . . . . . . . 29 IV. Prolactin Assay . . . . . . . . . . . . . . . . . . . . 32 V. Microscopic Observations . . . . . . . . . . . . . . . . 37 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 52 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 iv LIST OF TABLES Cell Types of Normal Pituitary . . . . . . . Cell Types in Kidney Capsule Autographs . . Cell Types in Anterior Pituitary During I3 Vitro Culture . Viability of Anterior Pituitary Cells in Organ Culture 1-9 D‘y. s s s s s s s s s s s s s s s o s 0 Survival of Anterior Pituitary Cell Types in Organ CUlture 1‘9 DBYB s s s e s s s e s o s s s s Orange G Acidophils VS Acid Fuchsin Acidophils . Pigeon Crop Assay of Culture Medium 17 Page 11 15 17 28 31 36 Figure 10. 11. 12. 13. 14. 15. 16C 17. LIST OF FIGURES Viability of Pituitary Cells in Organ Culture Cell Counts of Cultured Pituitaries by PAS Orange G Technique Comparison of Acidophil Counts by Two Methods of Staining Prolactin Assay of Pituitary Culture Medium Intact Control Pituitary. Masson Trichrome & Aldehyde Fuchsin Stain lOOX . Intact Pituitary Control. PAS Orange G lOOX . Intact Control Pituitary. Masson Trichrome & Aldehyde Fuchsin Stain. 1250X Intact Control Pituitary. Masson Trichrome & Aldehyde Fuchsin Stain. 1250K. Intact Control Pituitary. Masson Trichrome & Aldehyde Fuchsin Stain. 1250X One-Day-Old Culture. PAS Orange G. lOOX Three-Day-Old Culture. PAS Orange G. lOOX Nine-Day-Old Culture. PAS Orange G. lOOX . Three-Day-Old Culture. Masson Trichrome & Aldehyde Fuchsin Stain. 1250K Four-Day-Old Culture. PAS Orange G. 1250K Nine-Day-Old Culture. Masson Trichrome & Aldehyde Fuchsin Stain. 400x . Nine-Day-Old Culture. PAS Orange. 1250X Nine-Day-Old Culture. Masson Trichrome & Aldehyde Fuchsin Stain. 1250K vi Page 27 3O 33 35 38 38 39 39 4O 40 41 41 42 42 43 43 44 INTRODUCTION The cytologic structure of the pituitary gland has undergone considerable investigation during the last few years. During this time much attention has been given to the functional changes which develop in the cells of the pituitary in relation to different physiological states. It is well known that the microanatomy of the pituitary is very difficult to interpret in the normal animal because of the complexities which exists between the pituitary, its target organs and the hypothalamus. Therefore in an effort to eli— minate some of the variables which exist in the intact animal, several procedures have been evolved to isolate the pituitary in order to better study its hormonal activity and cytology. These methods include pituitary stalk section, transplantation to the kid- ney capsule, transplantation to the anterior chamber of the eye, and ig_!i££g culture of the pituitary. This study is concerned with the microscopic anatomy of the rat pituitary under conditions of organ culture. JUSTIFICATIONS FOR INVESTIGATIONS IN THIS AREA The factors which control synthesis and release of hormones by the’ endocrine organs, particularly the pituitarygare not well under- 8t0<>d at the present time and any information in this area should be \Wery useful as a basis for future studies. Physiologic studies of endocrine organs have advanced far ahead of cytologic studies, 1 and this is particularly true in domestic animals. Since the rat is primarily a laboratory animal, most of the work has been done on this species. A basic understanding of pituitary function in this species should be helpful for studies on other species. GROSS ANATOMY OF THE RAT PITUITARY The hypophysis or pituitary gland is located beneath the third ventricle of the brain, where it is attached by the hypophyseal stalk. In most animals it is situated in the depression of the sphenoid bone known as the sella turcica. However in the rat the pituitary rests on the surface of the sphenoid bone, because of the absence of the sella turcica. Here it is covered by the dura mater. In most animals the pituitary is an ovoid gland, but in the rat it is wedge shaped, elongated laterally, and the pars distalis constitutes the major portion of the gland (Purves and Griesbach, 1951). The gland is divided into two major components: (1) the neuro- hypophysis (lobus nervosa) which is further divided into the neural stalk (infundibulum) composed of the median eminence, infundibular stem, and the infundibular process (neural lobe); and (2) the adeno- hyp0physis (lobus glandularis), which is further divided into the pars tuberalis, pars intermedia, and pars distalis. Two other terms, Which should be defined are (l) the anterior lobe, which includes Phi! pars distalis only, and (2) the posterior lobe which consists 0f the infundibular process and the pars intermedia (Maximow and Blocun, 1952; Ham and Leeson, 1961). REVIEW OF LITERATURE 1. Classification of Cell Types. A. Acidophils-basophils. Cells of the anterior lobe of the pituitary were first classified and identified by staining reactions utilizing the trichrome stains (Schoneman, 1892; Severinghaus, 1932). Those cells which stain with acid fuchsin, erythrosin, carmine, or orange G are designated as acidophilic cells, while those cells staining with aniline blue or light green were designated basophilic cells. The disadvantage of this system was that the so called acidophilic cells stain with acid dyes as well as basic dyes, and the basophilic cells stain with either acid or basic dyes, all depending on the concentration of the dye and the pH of the staining solution (Peterson and Weiss, 1955; Singer, 1952). This terminology was further extended. Those cells which stain with erythrosin were termed erythrosinophilic cells, and those staining with carmine are termed carminophilic; the samerum true for the orange G staining cells. The major dis- advantage of this system in that there appears to be a considerable di fference in the staining reactions between functionally similar ce Ills in different species (Purves, 1961). The use of the terms, "aCiJjOphIISH and "basophils" have become so deeply engrained in the litearature that it is doubtful that they will ever be eliminated comp 1 e to 1). . L\ g I B. Greek letter CldSSllltHFliflo The second method of classification was based on the use of the Greek alphabet to designate dlfterent functionally active cells. This method was first used by Bailey and Davidoff (1925) but was deveIOped to its fullest extent by Romeis (1940) in classifying human pituitary cell types. by attempting to apply this classifi- cation to different animal species,tonsiderable confusion has resulted. The problem is similar to [he a idnphll~basuphil classification in that the cells are identified by Staitlng r.artions and the reactions vary between different Species. H w.Ver, eVen with the handicaps of this method, it was a wanna thl contribution, since prior to this time no one had made a (unreutratud effort to relate cell types to their functions. Halmi (1950) in his identification Of two types of basophils in the rut used d_lfa tn designate the gonadotropic cell and REES to designate the thyruttopic cell. The only other Similar attempt was by Goldberg and Ctas.tf (1952) to identify the pituitary cells of the dog using Roman numerals. This method was not generally adopted except that the terms alpha and bets are sometimes used to designate the acidophilic and basophilic cell types. C. Correlation with_qumoneS. The third method of classification has developed over many years, 81nd is the result of hormone assays and attempts to correlate func- tiOnal changes with specific cells in t“a anterior pituitary. Among théése changes in cytology might be mentioned the absence of acido- Phi 11c cells in dwarf mice or the hyperplasia of the acidophils in humain giants or in cases of acrcm.galy. Here these cells have been CUIITéElated with the preseure or ahSente of growth, and are thus correlated with the elaboration of a growth hormone and have been designated "somatotropic hormone cells" (STH) (Turner, 1960). Simi- larly castration of rats produce hypertrophic basophilic cells and hence have been associated with the gonadotropic hormone cells (FSH and LH) (Stein, 1932-33). In addition, thyroidectomy produces hyper- trophic basophilic cells, designated thyrotropic hormone producers or TSH cells (Farquhar and Rinehart, 1954). Lactation and gestation also produos alterations in the granules of some acidophilic cells, and these were designated luteotropic or prolactin producers (LTH) (Hymer 55 al., 1961). The adrenocorticotropic hormone producing cell has been a matter of controversy. In the rat, it may be pro- duced by a chromophobe (Siperstein, 1963). From these investigations have evolved the classification of cells as STH, LTH, FSH, LH, TSH, and ACTH producing cells. 11. Methods of Demonstrating Specific Cell Types A. Periodic Acid Schiff Reaction. The periodic acid Schiff reagent (PAS) will demonstrate carbo- hydrate complexes, and in particular glycoproteins (McManus, 1946; Pearse, 1952) such as those contained in the granules and vesicles of the basophilic cells (Purves and Greisbach, 1951). This reaction is not entirely specific for basophils, for a weak reaction will occur in the acidophils, but this is generally masked by employing a counterstain which will overshadow it. The specificity of the PAS stain lies in the fact that the basophils have more glycoprotein than the acidophils, and are therefore much more prominent. B. Aldehyde Fuchsin Stain The aldehyde fuchsin stain was originally developed by Gomori (1950) as an elastic tissue stain and was thereafter utilized by Halmi (1952) to differentiate thyrotropic hormone cells from the gonadotropic hormone cells in the rat. It has since been modified by Elftman (1959c) so that it is more reliable. This procedure stains TSH producing cells specifically in the rat (Purves, 1961). C. Extraction Procedures The use of the PAS and aldehyde fuchsin stains will show the dif- ference between two types of basophils, the gonadotrophs and the thyrotroph. However it is well known from biological assay that there are two types of gonadotropic hormones produced by the anterior lobe of the pituitary (Barrnett _£_al., 1955). These two cells can not be distinguished by staining, but they have been identified by extraction procedures prior to fixation of the tissues for section- ing and staining. It has been shown that 2.5% trichloracetic acid will remove all of the staining ability of the basophils except the one responsible for the production of luteinizing hormone. This has been confirmed by hormone assay after extraction as well as t al., 1956; Barrnett, 1958). There- staining procedures (Barrnett fore by these procedures, three basophilic cell types can be dif- ferentiated and correlated with individual hormones. D. Trichrome Stains The acidophilic cells (LTH and 8TH) can readily be demonstrated and distinguished in some species by the trichrome stains (Dawson and Friedgood, 1938-39). Landing (1954) compiled a very comprehensive review on the multitudes of staining methods that have been tried in differentiating cell types of the anterior lobe of the pituitary. The demonstration of these cells is not difficult in most species, but the differentiation of cells can be more easily accomplished in some species, i.e. dog and cat (Dawson, 1946; Dawson and Friedgood, 1938-39). The rat pituitary is not easily differentiated, however, Sanders and Rennels (1959) reported that two types of acidophils can be distinguished by a modified azan stain. This stain has not proven entirely reliable by other investigators. Recently, Herlant (1960) and Pasteels (1963a) described other modifications for differentiation of acidophils in the rat pituitary. E. Fluorescent Antibody Technics By the process of elimination only one source of hormone is unaccounted for, namely the adrenocorticotropic hormone. This hor- mone is evidently produced by a different cell type in different species (Purves, 1961). In the rat some attempts have been made to use fluorescent antibodies to determine the site of its production. The results, though not entirely accepted, indicate that the basophils are the probable site of production (Marshall, 1951). Similar studies on the site of prolactin production have shown that an acidophilic cell is responsible for its elaboration, and agreed with the results of hormone assay and staining procedures (Emmart t 1., 1963). F. Radioautographic Procedures This procedure has been used to a very limited extent. Siper- stein (1963) has shown that the rate of uptake of tritiated glycine is greatly increased in a chromOphobic cell type after an animal has been adrenalectomized. It is her opinion that a Chromophobic cell type is the source of ACTH in the rat pituitary. III.Present Knowledge of the Microanatomy of the Rat Pituitary Any description of these cells is only partially correct, for they are in a constant state of transformation. The descriptions given here apply generally to the phase in which granules are present. It must be realized that the same cell may also exist in a non-granu- lar form. A. Growth Hormone Cells These cells have an affinity for all acid dyes, and the granules are the intracytoplasmic components responsible for this affinity. The cells are considered to be columnar in shape and located along the blood sinusoids of the pituitary. The nucleus is ovoid in shape and contains 1-2 nucleoli. Electron microscopy has revealed granules in the size range of 350-500 mu. The granules vary in number with the secretory phase of the cell. The Golgi apparatus has a condensed appearance and may show the ”negative Golgi” image during stages of secretion. B. Luteotropic or Prolactin Cells These cells have a similar staining affinity to the STH cell but are more ovoid in shape and are located on the interior of the cords that make up the architecture of the anterior lobe. The nuclei and nucleoli are again similar to the STH cell. One prominent dif- ference is the size of the cytoplasmic granules. in the LTH cell the granules are 600 mu in size and are considerably larger and more prominent than the granules of the STH cells. The Golgi apparatus is prominent and shows the ”negative Golgi” image during the secre- tory stages. C. Follicle Stimulating Hormone Cell This is one of the cells of the basophilic component and there- fore has an affinity for the basic dyes, and is PAS positive. These cells are round and are located on the sinusoids together with the STH cells. These cells are nearly twice the size of those of the acido- philic group. The nucleus is round, centrally located and contains one to several nucleoli. The granules are considerably smaller in size (140-200 mu). below the resolution of the light microscope and therefore give the cell a more homogenous appearance. The granule size and number again yariis with the phase of secretion of the cell. The Golgi apparatus is prominent and shows a very characteristic ”negative Golgi” image during active secretion. This cell also has large vacuoles which vary with the stage of secretion, forming a large vacuole after castration and giving rise to the term ”Signet ring cell”. D. Luteinizing Hormone Cell This cell is the second member of the basophilic group, and also the other member of the gonadotropic cells, of which there are two, FSH and LH. It has the same staining characteristics as the FSH cell, and can not be distinguished from the LH cell. This cell is round and is located in the cords of cells away from the sinusoids. The granules are of the same magnitude as the FSH cell, 100-200 mu. One characteristic feature of this cell is an elongated nucleus which is folded in the middle, giving it a kidney—bean shape. The nucleoli are not always visible. The Golgi apparatus is prominent and forms a ”negative Golgi” image during secretion. Vesicles are present and irregular in shape. E. Thyrotrophic Cells or Thyroid_§timulating Hormone Cells This is the third member of the basophilic group with the same staining characteristics as the other two. However in addition, the granules of this cell stain specifically with aldehyde fuchsin. This cell is angular in shape and has a tendency to be concentrated 10 in the center of the gland. The granules are small, 140 mu, and are not distinguishable with the light microscope. The nucleus is round and centrally located with one to several nucleoli. The Golgi appara- tus is poorly developed. F. Adrenocorticotrophic Hormone Producing Cell This cell is generally considered to be a Chromophobic cell because it does not stain. It is an irregular cell which is located on the interior of the cords of cells, but sends small projections or microvilli out to contact the sinusoids. The nucleus is generally much larger than the other cells, and is hyperchromatic. It contains one to two nucleoli. There are few granules and these are 100-150 mu in size. A Golgi apparatus has not been demonstrated. G. Chromophobic Cell This cell, if it is a separate entity, has been described morpho- logically. It may be that it is just one of the chromophilic cell types in a degranulated phase. However for purposes of classification I will include its morphological description. This cell has no stain- ing reaction, it is angular or elongated in shape, the nucleus is ovoid and hyperchromatic. No granules have been reported. There is only a slight rim of cytoplasm and the Golgi apparatus is poorly developed. This cell has no particular location but may be found anywhere throughout the anterior lobe. For purposes of clarification and simplicity, Table I includes the morphologic description of the cells of the normal anterior lobe of the rat pituitary (Farquhar and Rinehart, 1954; Herlant, 1964; Hymer, _£ 1., 1961; Purves and Greisbach, 1952; Purves, 1961; Rine- hart and Farquhar, 1953). ll m>puamoa camcosm sesamea< u .m.<«a wswfioac uwumEounuumazm Ums kHHmopro coao~m>mc cooked vcuwwcon mamauzc one mmmozm -mam meoz sauoom -mu uoz eao>o so um~=wc< ucmmn< w>uumwmz -ozomzo .moa .m.oc was mmm Houses CH >~uoom Hmum>om -H ccoom umfiswc< 3E qu mm mEmm :mH mcpou mo chHmH> cocHOM w :8 3mm acaueucH DCoCWEoum mmmzfim uoz woumwcofim bosom ooNaooH mm mEmm $4 meson DE .mom mwm -H venom venom ooN-qu a cammm 5mm mvwom uneam oomucOo USD mUHOU HUN/o DE MDQH 05C 0.3".“ wo uofluoucH comm uoz Ngfi owned umfiswmuuH Omfigoofi m>Humwmz mHo< mcpoo mo coupon :Hm uoaumuca co somewEoum -ou uoz cpo>o cuo>o :6 00¢ mm mEmm SBA mcaomscwm DE w>wuamom ecu wcofi< cwmcoccou .NsH owo>o uneasfiou oomgoom cwo< mHm scausooq uwz HHowHosz msmfiuaz fifieu mmfiscmuo wuacflmw< wm>H HHmo Hwfioo mo Hwnasz mo emucm we mamcm samum VMouoaz use .me cooEoHo .3mm mHfioo %:mz “HmEm 3mm n|.niaooosc .mm so mwsou unmmouo mom: mz mZ uoomc< mfimsoopflom 36m wagons oomcm II II Aamoav umfidowm®> HmajwmupH Umumfiscmpw .H be “sauna: .owumq m2 commouomc pommwuooo xaowcoc uoc one son AOOOaV vengeance a :mwa_fipoo m2 mz m2 ocomom. mfiwzooowom zfiocmCHEoconm . mfla:oopwom mo Aomoac obess>m mz m2 mz ucmmn< egaumascmsmse eummccueo mmflzmocaom we assay csfiomoc m2 m2 dz ucomn< newsmaoempwop pommouocw mxmc wwmp AamOHC ssmcs>a sa-©a ssmam “H-0a samEm pom nmcuz sooz and xoos umfi -eous>ouosaz 32 a2 sauce sew: sauce sew: m2 mz assoc Basso “Hoomooo powumflco AOmmfiv mfioosom mmmo wfi mxmc NH Housemmo ocm mpoocmm mom: mz umuum ozomc< noowm uoomo< xm .xcmz oumm escapades mmpo:a0Eoszo revs :ma :4 a :mm was mam zmmZQHM 2H mmmre 44mm N M1322. l6 Pasteels and Mulnard (1961), and Pasteels (1963a, 1963b) reported that the acidophilic cells can be readily identified from tissue culture explants. They have cultured pituitaries for as long as 6 weeks but report that after a few days the chromophilic cells degenerate very rapidly, except for the prolactin cell which multi- plies and retains its secretory activity. The cytology of the pituitary in culture is summarized in Table 3 (Pasteels and Mulnard, 1961; Pasteels, 1963a, 1963b; Martinovitch t al 1962; Martinovitch, __.__..'3 1950, 1954; Meites t 1., 1961; Schaberg and de Groat, 1958). THESlS l7 cmunoemm uoz u m2 Ammomv some mHHoo .mfiazoommn umouw op pom wuocmcom mo quhommz mz woumfioomnw-ooc HHmEm mflficoowflom 3mm Anmooa .mmoos mmmmmsucu songs :94 .HomHV .flm.mw mammummm mz mZ xficammp oumuooowmm pow unwoxo woumuocwwoo wanes AaooHv .Hl Ml mouse: mz mz roman oHLaouom 3mm mHHLroHom mfloumcHEovoum Amomfiv wfiflsaovflom .fll Ml couw>o:auumz mz mz comm mfiwzm0mmn 3mm omumfiocmuw zaonLH muses Aqmofiv Loofl>ocwupmz mz mz 90mmo ow5a0pum 3mm mfiwsaovwom wfioumGHEowoum >uozomuoa Aomoflv coufi>ocmopmz um oompcon< mz m2 mz cowumuocowoc w cowumfiocmuwom assessmmmm mesonsoeosao zeo< ems ma a awe new mazeaao omeu> mm.ozom:o wmeaoseom scammez< see 24 mamas m mAmm._._n=m<_> ._ .05 w>jcjb JO «ACMmDZ ._. 92 .u 00. om om 04 Om Om 0v o/o 28 TABLE 4 VIABILITY OF ANTERIOR PITUITARY CELLS IN ORGAN CULTURE 1-9 DAYS Percent Total Total Total Viable Average Day Cells Viable Non-viable Cells Percent p 1 1174 750 424 63.80 1345 967 378 71.89 978 748 230 76.48 954 751 203 78.72 72.72 i 4.88 3 1156 713 443 61.67 857 581 276 67.79 1152 820 332 71.18 66.88 NS i.3-47 4 1323 750 573 56.68 1151 873 278 75.84 1100 798 302 72.54 1029 628 401 61.03 66.25 .05 i 7.66 5 598 270 328 45.39 1470 899 571 61.15 860 399 461 46.39 760 652 108 85.78 59.67 .05 :13.78 6 1314 576 738 43.83 907 309 598 34.06 829 548 281 66.10 946 455 491 48.09 48.02 .01 i 9.08 7 1231 663 568 53.85 601 354 247 58.90 529 303 226 57.27 1819 1475 344 81.08 62.77 .05 i 9.33 8 1192 722 470 60.57 658 416 242 63.22 61.89 .01 _ 1.32 9 1374 1077 297 78.38 1042 672 370 64.49 984 536 451 54.30 1079 715 364 66.26 65.85 NS i 6.46 p - level of significance compared to day one. .41.. -J-_JC1--_& ‘Vfl FH E5513 4'»? i... -_. III. 29 to 59.14% or a 11.30% increase over the normal range. This is the only day that shows any statistically significant differences from the normal values. Days Seven, Eight, and Nine On the following three days the number of acidophils were in the normal range, with perhaps a slight increase on day 9 to 51.50%. B. Basophils These were counted using the PAS-Orange G method. No attempt was made to differentiate between the types of baso- phils as the PAS will react with all basophilic cells. The normal intact pituitary showed a 10.50% level of basophils. The relative numbers of basophils decreased at a signifi- cant rate over the 9 day period. By the 9th day the calculated percentage was down to 3.65%. This constitutes a loss of 65.23% of the total basophilic population. C. Chromophobes The normal level of chromophobes in this group of rats was 41.62%. Over the 9 day period of culture the percentage of chromophobes showed a response which was indirectly pro- portional to the response shown by the acidophils. In other words, as the acidophils decreased the chromophobes increased. Figure 2 and Table 5. Comparison of Orange G Acidophils and Acid Fuchsin Acidophils During the course of this investigation it became apparent that there was little correlation between the cell counts from the two methods of staining the cultured pituitary glands. When the values obtained at 3, 6, and 9 days of culture were plotted for each stain, [H E518 30 0.2102. uozéo mdm >m $33.55 ommauao “.0 9.2300 4.60 .N 0.... m>m 2.2300 4.110904 “.0 285.5200 .m 0.“. 33 .454 . C. .. .- fl Fla .1!.. .1311 5514.81... ....... 4‘ E. 12;. 2.23.6.3 comma: ”._fiooT o m>o OSWEDAw 1.0 mCOIHwZ 0.2.. .855: 1 sum 1000110019101 FHESIS mwc_o< 330.1%? ~0c_o< 20.1.06; mmc_o< (PT lllll mm:_o< 3920.. nuua wwwououol 01 9” O 0m.0 m_.O 0.0 mob m<>o KCEwK wmcdcu 0.3: mmeme :_._.0>.._Om£ 19.0] 36 TABLE 7 PIGEON CROP ASSAY OF CULTURE MEDIUM Response is recorded in International Units Prolactin/bird/4 pituitaries 350.033 F“: Day 1 2 3 4 0.011 0.033 0.033 0.022 0.011 0.045 0.033 0.022 . 0.011 0.045 0.090 0.066 . 0.045 0.060 9111; 9.134 0.019 0.045 0.067 0.061 4 10.012 10.006 10.034 10.039 L_J J... ”5' Day 5 6 7 8 0.022 0.044 0.033 0.033 0.022 0.066 0.033 0.066 0.044 0.088 0.099 0.234 0.066 0.134 0.168 91g19 0.038 0.083 0.083 0.150 10.015 19.028 10.052 10.101 Day 9 0.055 0.066 0.099 0.168 0.099 THESIS 37 Microscopic Observations Intact Control Pituitaries Photomicrographs of the intact control pituitary cells are included to be used as a reference in evaluating the cultured pituitary cells. Most of the photomicrographs are self-explana~ tory and so no discussion is included other than the plate descrip- tions. Cultured Pituitaries Most of the areas of necrotic tiSSue in all the sections examined are confined to the center of the organ culture; however, in many instances there were necrotic areas along the edges. These areas on the edges probably resulted from poor contact with the oxygenated medium- 38 .L____.__-_ ._____--___ _ _ Fig. 5.--Intect Control Pituitary. Masson Trichrome and Aldehyde Fuchein etein. 100x. (e) Anterior lobe. (b) Hypophyseal cleft. (c) Inter- mediate lobe. F13. 6.--Intact Control Pituitary. PAS Orange Stain. 100x. (e) Intermediate lobe. (b) PAS positive basophils. 39 Fig. 7.--Intact Control Pituitary. Masson Trichrome end Aldehyde Fuchsin stain. 1250K. (e) Large well granulated acidophil, probably LTH. (b) Large basophil presumably an LH eecretor due to the kidney shaped nucleus. Afigel Fig. 8.--Intact Control Pituitary. Masson Trichrome and Aldehyde Fuchsin stain. 1250X. (a) Large basophil with vacuoles filled with scidOphilic materiel. (b) A TSH cell contains aldehyde fuchsin positive material throughout the cell but predominately around the nucleus. 40 _—-"A———-v - he i‘W—~—_ - - Fig. 9.--Intact Control Pituitary. Masson Trichrome and Aldehyde Fuchsin stain. 1250K. (s) TSH cell, this is typical of the TSH cells found in the normal pituitary. (b) Acidophilic, columnar shape, probably STH cell. Fig. 10.--0ne-Day-Old Culture. PAS Orange G. 100x. The viability appears good except in the central area. -——-— -_ ___._ . ———~——-——.—v_——__._.. Pig. ll.--Three-Dsy-01d Culture. PAS Orange G. 100x Viable tissue is present; however, the majority of this photograph is degenerating tissue. Fig. 12.--Nine-Day-01d Culture. PAS Orange G. 100x. Viable tissue surrounds the central necrotic ares. 42 Fig. 13.--Three-Day-Old Culture. Masson Trichrome and Alde- hyde Fuchsin Stain. 1250x. In the center is a large basophil (a) containing a large vacuole (b). The acidophils (c) are dark and contain a pale area the negative Golgi image (d). Pig. 14.--Four-Day-Old Culture. PAS Orange. lZSOX. . A large hypertrophied chromophobe is located in the center, (a) the nucleus is very faint and hypertrophied. Around the nucleus is a slight rim of acidophilic material. The acidophils (b) appear active. The basophils (c) are somewhat below normal size in appear- ance e 43 L - i _ Fig. 15.-~Nine-Day-01d Culture. Masson Trichrome and Aldehyde Fuchsin Stain. 400x. Acidophils (a) are evident only along the edge. A small and degenerated TSH cell (b) is also present. A large basophil (c) displays a large vacuole. Fig. 16.--Nine-Day-Old Culture. PAS Orange G. 1250X. (a) Prominent basophil with large PAS positive granules. Remainder of cells are weakly staining acidophils. Fig. l7.--Nine-Day-Old Culture. Masson Trichrome and Aldehyde Fuchsin Stain. lZSOX. In the center is one of the very few acid fuchsin staining cells (a) present at nine days. Immediately above is a basophilic cell. (b) with a kidney-shaped nucleus, presumably a LH cell. Notice the small cytoplasmic volume of both cells as compared to the normal cells in Fig. 7. DISCUSSION The results of this investigation demonstrate that the initial loss of viable cells occurs mainly during the first 24 hours. This is probably due in part to removing the anterior pituitary, slicing it into pieces, and the adjustment to an artificial environment. The losses after this period, that is between day one and day four, are possibly due to the normal degeneration of pituitary cells with- out the accompanying replacement of cells which occurs in the normal intact pituitary 13,1119. The amount of viable tissue after three days of culture stabi- lines at the 60-65% viability level. Therefore when calculating prolactin production on the basis of pituitary weight the per cent of viable tissue should be considered. Also another consideration should be the per cent of acidophilic tissue present, since the bas0philic and perhaps the chromophobic cells do not secrete prolactin. From the results obtained here, the value of 47-48% acidophilic cells is acceptable for the majority of the one through nine days in culture. This value is also the figure determined for the intact control pituitaries. There are, however, some days when there are exceptions to this value, such as on day one, when the count is lower than the normal values. On this day, one may theorize that this is a reaction on the part of the prolactin producing cells to the absence of the hypothalamic control. Harris (1955) has shown that the hypothalamus has a direct inhibiting control over prolactin 45 secretion. This phenomenon has also been demonstrated 13 11139 by culture methods (Meites _£‘§1., 1961). It therefore seems logi- cal that upon removal of this control the pituitary would react by an immediate release of prolactin. Now if one goes on the assump- tion that the prolactin is stored in the granules of the prolactin cells, then it is not surprising to see this disappearance of acido- philic staining cells. Another day which merits some explanation is day 6. This par- ticular culture showed the lowest viability of any culture, but probably this is the result of a poor technic in culture and not a reflection of 6 days in culture. However in evaluating the cytology, the number of acidophils exceeds the number found in the normal pituitary. One may postulate that at this stage the regranu- lation of the acidophils has increased to the point that all of the prolactin producing cells in the anterior pituitary have reached a stage of production where granules are present. The assay for prolactin should verify these possibilities but due to the extreme variation in the pigeon crop assay and the limited number of observations, any variations between the days are lost. The stages of degranulation or regranulation could agree with the literature depending on the stage that the cultures were examined. Meites _£__l,, (1961) reported that at 7 days the acidophils are predominant, while Schaberg and de Groat (1958) reported few acido- phils at 12 days. At later stages the acidophils are in large numbers and heavily granulated for 21-28 days (Martinovitch, 1954). The prolactin assay in this investigation does demonstrate one important point, namely that these cultures were active and capable of producing hormones. A second point is that these were male rats 47 and therefore were not expected to produce much prolactin. The basophilic cell population decreases steadily during the culture period. This agrees with the conclusions reported in the literature. For example, the assay of the culture medium by Cutting and Lewis (1938) has shown that the basophilic hormone activity is present only during the first ten days of culture. Recently, Mittler and Meites (1964) have shown that the addition of hypothalamic extract to 3 day cultures can stimulate production of significant amounts of FSH. Ordinarily FSH is present in amounts which can be detected only for the first 3 days. After 9 days of culture, basophilic cells with large PAS positive granules were observed. (Figure 16). They were, however, somewhat smaller in size than the normal cells, but no evidence of degeneration was observed. The chromophobic cell mass can conceivably include all of the cell types, the chromOphilic cells in their degranulated stages (Severinghaus, 1937; Pearse, 1952), as well as the ACTH cells which contain granules but have no staining affinity (Siperstein, 1963), and also the chromophobic cells as such. Any massive degranulation by the acidophils would be shown by an increase in chromophobes and visa versa. ACTH production has not been demonstrated after 4 days in culture without hypothalamic stimulation (Guillemin and Rosenberg, 1955). In this investigation ACTH cells were not identified in the normal pituitary; however, after 4 days in culture, an abnormal chromophobic cell appeared (Figure 14). A cell of this type has been reported by Rennels (1962). The origin of this cell could be from three sources: an extremely degranulated acidophil, an ACTH cell, or a large chromo- phobe. As far as the acidophil is concerned, most chromophilic cells 48 do not show an increase in size when they degranulate. Secondly these cells are only present in limited numbers, which would lead one to believe that this cell came from a group which was few in number to begin with, i.e. ACTH cells. However, as previously mentioned, ACTH production requires hypothalamic stimulation, so one would expect a degeneration of ACTH cell in culture. If this cell is merely a large chromophobe, it is difficult to see why it would become more promi- nent in culture, unless it were in some stage of hyperactivity. To sum up then, the significance of this hypertrophic chromophobe is not known. When one analyzes the information obtained from the cell counts of Orange G and Trichrome stains, it is apparent that the acidophils are not of the same population. These curves may be compared to the information obtained for prolactin and growth hormone assays on cul- tured pituitaries. Nicoll (1962) reported that prolactin is produced at a relatively high level during the early stages of culture, mainly one through seven days. This corresponds to the increase of acido- phils (orangeophils) observed during the first 6 days in this investi- gation. This increase is observed only when using the Orange G stain for acidophils, therefore these cells must be prolactin producers primarily. The curve obtained using acid fuchsin as an acidophilic stain indicates a pronounced decrease in acidophils from the begin- ning of the culture but particularly from day three to day six. This corresponds rather well with the results of the STH assay form cultured pituitaries. Deuben ani Meites (1964) indicated that after the first 6 days of culture only very little STH is recovered, 19% as much as was recovered in the fresh pituitary. By calculation this investi- gation shows that after 6 days in culture, using Masson Trichrome 49' stain, there is only 9.4% of the original acidophils present. It therefore appears that these acid-fuchsin-staining cells are the pro- ducers of STH. As I have indicated the LTH cell and the STH cell can be readily differentiated in some species; however, the rat has not proven to be one of these in most cases. Some results have been reported using modifications of Heidenhain's azan stain; however, these results have been sporadic and not reliable (Dawson, 1946). Lacour (1950) using pregnant or lactating rats has shown that the LTH cells are stained with Orange C, while the STH cells stained with erythrosin or axocarmine. Rennels (1962) has reported similar results but again his procedure has not proven to be repeatable. Pasteels (1963a) has reported very good results in differentiating the prolactin and STH cells using a tetrachrome stain (acid alizarin blue) developed by Herlant (1960). In this investigation the two types of acidophils in culture could not be differentiated on the basis of cytology. The only differentiation was through the use of the two stains mentioned. There are several considerations which may be responsible for the variation in staining ability of the rat acidophil: (1) It is fairly well established that the acidophilic granules are a mixture of 4-8 proteins, (Landing, 1957) peptides, phospholipids, and hormones (Purves, 1961). (2) The staining reaction is due to the acidophilia and not to the hormone components. The hormones LTH and STH can be dissolved out without loss of staining characteristics (Purves, 1961). Acidophilia may be due to the amount of arginine and histidine present in the cell (Herlant, 1964). The histidine content does not vary much in the normal LTH and STH cells (Glenner and Bagdaymon, 1960). Recent 50 studies indicate that there is a direct relationship between the abundance of secretory granules and prolactin content (Pasteels, 1963b). However in stages of hypersecretion of one or the other cell type they may vary considerably. (3) Purves (1961) indicated that the granules vary in quality and quantity due to an alteration in the components which are affected by different rates of secretion. A well known example of this is seen in the LH producing cells of the dog or cat. When stained with Masson's Trichrome, these cells may appear red due to an excess acidophilic component, or may be green due to a change of acidophilic component, or may even be blue due to a staining by both acid fuchsin and light green. This is explained on the basis that the acidophilic and basophilic components vary during different rates of secretion. (4) Pearse (1956) in a study of esterases in the hypophysis indicated that these enzymes increase significantly during pregnancy, and the RNA decreases in amount which may influence the staining characteris- tics. All of this information points to the fact that the staining affinity of the acidophils can vary during secretion in the anterior pituitary. The outstanding point in this investigation is that by using two different histologic procedures, involving different fixatives and different acid stains, the acidophils can be shown to vary directly with the rate of hormones released. There are several variables which can be considered in determin- ing the difference in staining characteristics. Among these are the alterations in the biochemical make-up of the cell due to: secretion, culture procedures, different methods of fixation, different sites of attachment of the acid dyes, and the oxidation produced by the periodic 51 acid in the Schiff reaction. Further investigation is needed in all of these aspects before any definite conclusion can be drawn on the exact components stained in this investigation. SUMMARY This study is a semiquantitation of the microscopic changes occurring in the pars distalis of male rats during periods of short term organ culture. The parameters examined were: 1.) the amount of tissue surviving from 1-9 days of culture; 2.) the degree of survival of the major cell types; 3.) the correlation of the numbers of surviving acidophils, as identified by orange G and Masson trichrome staining, with prolactin and STH production. The initial loss of viable tissue occurs in the first 24 hours of culture. Thereafter the viable tissue stabilizes at 60-65% of the normal amount. The percentage of prolactin acidophils as detected by orange G staining increases from a normal value of 48% to 59% by the sixth day. By the ninth day the values returned to normal. The somatotropic acido- phils demonstrated by Masson's trichrome stain decrease to 5% by the sixth day where they remain. The changes in the numbers of the two types of acidophils correlate with an increase of prolactin and a decrease of STH production. The numbers of viable basophils decrease slightly over the ninth day period. In the normal rat, the two acidophilic cells can not easily be differentiated as to type, but 13.11119 during periods of hypersecre- tion of the prolactin cell and hyposecretion of the STH cell these two cells can be differentiated. LITERATURE CITED Bailey, P., and L. M. Davidoff. 1925. Concerning the Microscopic Structure of the Hypophysis Cerebri in Acromegaly (Based on a Study of Tissues Removed at Operation from 35 Patients). Am. J. Path., 15185. Baker B. L. 1962. Personal communication. D t. f A t u 1 - ha of Michigan, Ann Arbor, Michigan. 9’" ° “3 ”“3" n V“ Baker, B. L. and N. P. Everett. 1947. The Effects of Diethylstil- besterol on the Anterior Hypophysis of Thyroidectomized Rats. Endo.,,31:144. Bakke, J. L., N. Lawrence, K. P. Knudtson, S. Roy, and G. H. Needham. ' 1964. A Correlative Study of the Content of Thyroid Stimulating Hormone (TSH) and Cell Morphology of the Human Adenohypophysis. Am. J. Clin. Path., 31:576. Bardin, C. W. and A. G. Liebelt. 1960. 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A Rapid One-Step Trichrome Stain. Am. J. Clin. Path., 29g661. Guillemin, R. and B. Rosenberg. 1955. Humoral Hypothalamic Control of Anterior Pituitary: A Study with Combined Tissue Cultures. Endo., 21:599. Halmi, N. S. 1950. Two Types of Basophils in the Anterior Pituitary of the Rat and their Respective Cytophysiological Significance. Endo., 315289. 54 Halmi, N. S. 1952. Differentiation of Two Types of Basophils in the Adenohypophysis of the Rat and Mouse. Stain Tech.,|21:6l. Ham, A. W. and T. L. Lesson. 1961. Histology, J. B. Lippencott Co., Philadelphia, 4th Ed. Harris, G. W. 1955. Neural Control of the Pituitary Gland. Edward Arnold (Publishers) Ltd., London. Herlant, M. 1960. Etude critique de deux techniques nouvelles dia- tenies a mettre an evidence lea differences categories cellu- larires presentes dans la glande pituitaire.= Bull. Microscop. Appl., gm. - . - Herlant, M. 1964. The Cells of the Adenohypophysis and their Func- tional Significance. Int. Rev. Cytol,, 11:299. 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'3‘ l , It? n :4: {git r7 ’1' ' :o I -“ ‘ l) , ‘ y: 1 Cell avd Orgrc 1 IV 0‘ ‘ d 0 - J .. I‘l . . e '1: 'u 52'; ha ,- v 0 ,. m , . Va I i q k I; u 0 - V“ ’4,- 0 H1551 ‘1'. J- C. , 4“ 7‘” 1,. Maire-7:. 19‘; 1. In ’_I' ‘L: 1an of Pitui- tary Pellfcle Stimn‘af;no Farr nu Lu'ciua as h wvt:uinnic Yxfrsct. Prmt. ‘31.. l‘v‘p 29.101 . be ." S. . 7'1": ':'-.4. Nicoll, C. S. 1951’. {1.11111'33 071 Prolactin hing'Z‘citfil f"! Vii"). TE‘QBlS D fwr chree of PH 0.. l“niee“ State Hairs“3a£y, ..s: 1.-1iag, El. ’1' "3". Nikitcvftcbvwiwer, M. and J. W. Fearett. 19i9. Hist: ytcicgfc Changes in Crafts 9? Est 111 itatv on ran Kidrzy tad {pas he i.ehsglen° tarlon Unu.r the ”iquspnsion. 174*., 71:111. Psitvr‘v, 1. 1. thTa, Prune 1a filifi?ntfil? d a (e1141rs iotpcnsables (1.31 M Cf‘fh“ i ‘1’ 1‘3.” 2"”!‘51! fife“ (Yd?! 3."? +51" 28'. (fit-l 1031.168 "' ‘..‘,A ' 1. . ‘. .‘ ,., . " . I ;_ ‘ ‘ “1 ..' 11‘t"l‘. ‘.—}v ‘ t s o‘“.‘t‘a ‘l’.~.(hs .JL 1. ' .'_ f ‘.. ~J' ‘ ‘ "‘s Pasteel'n 1. L. lvh‘b. 1.1lwvrrnes mtryiuirm.:js 9 et Payerinentalce ‘U.r 1a a srsw fl‘n dw qr FM~ tits. 1:-?.. $.Cl., 3‘923?*333. --. Pr; -'.' 1,9121 14' ]_ 1,. gr» 1 I, 1",! 3.71.111"- . 196,2’ , (.,._.4.;"._;: .1359? 3,5 11 {.z’ , ._. {51'1" g ' r 4?. F."ds A‘Bde Fa. , I L., .‘- ' L“: - ‘5 . J) (in Pearse. A. G. E. 14:9. Ewe esterases of tfic ~ J‘Pl““5 “”4 Flair Functinral ff;41;1-: -e. 3. Path. a'd '- :- F‘:431. /U Peterson. R. R. and I. neiae. l‘ii. hia:ning of the fidnmiiyfdyhyfiia wit?! Aclxi and F'v4111 Piaf}. 3' We. ’zfif. . Purves. H. D. 1961. MerrnclflCY Cf 1‘1 ’"“5'J'19 C”'é ‘1 t“ it? \ - . - _ _. imzt‘lori. ' - .31? ~ , .. 2 1r. l. ~~ 1 4. ". ll . h. I ‘ f r: e p ' W . P.‘ Y‘.’ '3 H . D 3""111 hr. 1" (T 3"" . ' l '51 '7‘ 1‘ \1 L5: ‘ I F ' 7" ' " ‘1 Sfld ,1: \s ‘1‘ x. 1‘ L ‘1 l I If]: C ‘ :1 1 ( C, - . E" Q § .' I ' :2 a I ‘ I...:. A 0., a 8 - 9 ~ ~ 0 - 3 :{ 1L "1'1 ‘g ~:" 3 ‘il‘r, ‘I\r (1' 7” l ‘1’! '1. g 11‘" " ‘ LP 1 anvrs “. D. and d. ". L'.i.hé i il‘l. I ~ "**6 :f A ‘F‘Phiis in 56 Quilligan, E. J. and I. Rothchild. 1960. The Corpus Luteum-Pituitary Relationships: The Luteotrophic Activity of Homotransplanted Pituitaries in Intact Rats. Endo., 21:48. Reece, R. P. and C. W. Turner. 1937. The Lactogenic and Thyrotropec Hormone Content of the Anterior Lobe of the Pituitary Gland. Missouri Univ. Agr. Exp. Sta. Research Bull. No. 266. Rennels, E. G. 1962. An Electron Microscope Study of Pituitary Auto- graph Cells in the Rat. Endo., 11:713. Rinehart, J. F. and M. G. Farquhar. 1953. Electron Microscopic Studies of the Anterior Pituitary Gland. J. Histochem. Cyto- chem., 1193. Romeis, B. 1940. Die Hypophyse, In Handbuch der mikroskopischen Anatomie desldenschen, von Mollendorf, Ed. Vol. 6, part 3. Berlin: Julius Springer. Sanders, A. E. and E. G. Rennels. 1959. Evidence on the Cellular Source of LTH Derived from a Study of Rat Pituitary Autographs. Z. Zellforsch”.32:263. Schaberg, A. and C. A. de Groat. 1958. The Influence of the Adeno- hypophysis on the Morphology and Function of the Adrenal Cortex lfl Vitro. Ethl. Cell Research, 12:475. Schonemann, A. 1892. Hypophysis und Thyroida. Arch. path. anat., 129: 310. Severinghaus, A. E. 1932. A Cytological Technique for the Study of the Anterior Lobe of the Hypophysis. Anat. Rec., 22:1. Severinghaus, A. E. 1937. The Cytology of the Pituitary Gland, in: The Pituitary Gland, Proc. Assn. Research Nerv. & Ment. Dis., 17:69. Singer, M. 1952. Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes. Int. Rev. Cytology, 1:211. Siperstein, E. R. 1963. Identification of the Adrenocorticotrophin Producing Cells in Rat Hypophysis by Autoradiography. J. Cell Biol., 11:521. Smith, A. 1962. Tissue Shrinkage Caused by Attachment of Paraffin Sections to Stick: Its Effect on Staining. Stain Tech., 21:339. Smith, H. A. S. and T. C. Jones. 1961. Veterinary Pathology. Lea & Febiger, Philadelphia, Penna. Snedecor, G. W. 1946. Statistical Methods. The Iowa State College Press, Ames, Iowa. Stein, S. I. 1932-33. Experimental Studies on the Hypophysis Cerebri. II. The Effect of Castration in the Male Albino Rat. Anat. Rec. 56:15. 57 Stockard, C. R. 1941. The Genetic and Endocrine Basis for Differences in Form and Behavior. Am. Anat. Mem. No. 19. The Wistar Inst. of Anat. of Biol., Philadelphia. Turner, C. R. 1960. General Endocrinology. 3rd Ed. Philadelphia, W. B. Saunders Co. Wolfe, J. M. 1935. The Normal Level of the Various Cell Types in the Anterior Pituitary of Mature and Immature Rats: Further Observa- tions on Cyclic Histological Variation. Anat. Rec., 211321. Wolfe, J. M. and R. Cleveland. 1933. Cyclic Histological Variations in the Anterior Hypophysis of the Albino Rat. Anat. Rec., 22:233. APPENDIX Histological Procedures Four glands were removed on each successive day and g of each gland was placed in Bouin's fluid and the other % in formol-sublimate. A. Masson's trichrome and Aldehyde Fuchsin (Baker, 1962). This procedure best demonstrates the acidophilic cells and the thyro- tropic cells. 1. The gland is fixed for 24 hours in Bouin's fluid, prepared as follows: Bouin's fluid: 750 m1. of saturated picric acid in water. 250 ml. of 37-40% formaldehyde. Add 1 ml. of glacial acetic acid to 20 m1. of the above solution, prior to use. Use 40 times as much fixative as tissue volume. Dehygration a. 50% ethanol for 1 hour. b. 70% ethanol for 1 hour or may be stored in this solution. c. 80% ethanol, 2 hours or may be stored in this solution. d. 95% ethanol, 2 changes of 2 hours each. e. 100% ethanol, 2 changes of 2 hours each, render absolute with anhydrous MgSO4. Clearing a. 100% ethanol plus carbon disulfide (C82) in (1:1) overnight. b. CS2, 2 changes during 24 hour period. 58 59 (1) First change of C82 must not be cloudy. (2) The tissue at first will float and ideally eventually sink. Slight warming helps. Infiltration with Paraffin a. CS2 plus paraffin (1:1), use 56-58O Tissuemat. (1) Be Sure Paraffin is cold when the C32 is added. (2) csz IS EXPLOSIVE, ABOVE 460C. (3) Keep in a warm place, such as on top of a 60°C oven. You must watch the stoppers on the vials containing the mixture so that they don't blow off and the C82 evaporate. Allow 1-3 days for infiltration. b. Place into the Paraffin at 56-58°C and draw a vacuum, for 15-20 minutes, or until the bubbling stops. Requires 25 pounds of pressure. Without a vacuum oven, four changes of Paraffin at 15 minutes per change. (1) Sectioning is best after a vacuum, otherwise the tissue tends to harden too much due to the prolonged heat. Qgsting the infiltrated tissue in the block a. Molds (1) Glass concave molds work very effectively. They are heated and are coated with a film of glycerine. (2) Hot paraffin (56-580) is poured directly into the mold and the tissue transferred before the paraffin cools enough to form a film over its surface. (3) Metal molds may be used, it is not necessary to have glass molds. 60 b. Orientation of whole pituitaries in the mold (1) (2) The block is cast with the pituitary orientated so that the pars nervosa is directly flat on the bottom. Be sure that heated forceps are used to transfer the pituitary to the mold, as bubbles may form around the gland and this makes sectioning very difficult. The pituitaries taken from culture can not be orien- tated any particular way, because of the lack of identifying features. 6. Sectioning a. Methods for obtaining representative samples from a whole pituitary gland. (1) The pituitary is cut so as to take three representa- tive levels through the gland. One level % of the distance through the gland, a second 5 the distance through the gland, and a third level 3/4 the dis- tance through the gland. (a) For immature rats (22 days) begin by taking 12 sections at 6 microns to the % level, then 30 sections at 4 microns. Then another 12 sections at 6 microns to the a level, then 30 more sec- tions at 6 microns, and then 30 sections at 4 microns. (b) For adult rats, begin by taking 30-35 sections at 6 microns to the % level, then 30 sections at 4 microns. Then another 30-35 sections at 6 microns to the 3/4 level, and then 30 sections at 4 microns. b. Required number of sections and thickness of sections from 7. 8. 61 each level to obtain a representative sample. (1) Thirty sections cut at 3-4 microns, in ribbons of 10 sections are taken at each level. Procedure for mounting sections: (1) One ribbon of 10 sections from each of the three levels is mounted on a separate slide, so that three slides are obtained from a single gland, each contain- ing a representative sample from each level. (2) The 6 micron sections can be saved and mounted to be used as controls in the staining procedure. Comments on sectioninggand mounting of sections a. In order to cut paraffin (Tissuemat) at 3-4 microns, the block must be kept cold at all times as must the knife. (1) In order to do this, ice water is dripped on a piece of cheese cloth which is wound around the block. (2) Also an ice cube is affixed to the surface of the knife blade to maintain a cold blade. (3) The blade is wiped off periodically with chloroform to maintain a dry cutting surface. The sections are affixed to the surface of a slide with albumen and allowed to dry one to several days at 37°C. The sections are not heated in any manner so as to melt the paraffin. Heating of the sections interfere with the selective staining of the trichrome stains. (Smith 1962). Staining_with Aldehyde Fuchsin and Masson's Trichrome b. C. Xylol, 3 changes, 5 minutes each. Absolute alcohol, 2 changes, 5 minutes each. 95% ethanol, one change, 5 minutes. 62 80% ethanol, one change, 5 minutes. 70% ethanol, one change, 5 minutes. Stain in Aldehyde fuchsin 20 minutes, or until the beta cells are stained. (l) Slides must be rinsed briefly in 70% alcohol and examined with the microscope. (2) A control slide should be processed at the same time. Rinse in two changes of 70% ethanol, until the excess stain is removed from the background. (1) There should be no background staining if the stain is ripened properly. If the background does stain, there is little that can be done to remove it. The slide may be bleached, but this will change the selective staining ability of the aldehyde fuchsin stain. It is best to discard the slide and start again. The background should be absolutely clear. Rinse in distilled water, 5 minutes. Place in Masson A solution for 30 minutes, check with the microscope to determine the degree of staining of the alpha cells. (1) The slide may be rinsed in water briefly before examination. Rinse in distilled water, as brief as possible. Place in Masson B 30 minutes. This can be left as long as 13 hours if necessary. Place in Masson C for 45 minutes until the basophils are stained. Rinse in distilled water to remove excess Masson C. 10. t. 63 Place in Masson B for 2 minutes. Place in 1% acetic acid, 2 minutes. Rinse in distilled water. Rinse in 95% ethanol, this must be brief, just long enough to remove the excess light green stain. About 10 seconds. Place in absolute alcohol, 2-3 minutes, 2 changes. (1) Too long will extract the light green. Change rapidly to xylol for the absolute alcohol will pick up moisture from the air and the section will not clear in xylol. On microscopic examination the section will appear cloudy due to the moisture in the xylol. (1) Change the slides through 5 changes of xylol of 5 minutes each. Mount with Permount or Canada balsum. Comments on staining Acidophils will stain orange to red, but can't be distin- guished as to type. The thyrotrops or Beta cells of Halmi stain deep purple and are generally heavily granulated. The gonadotropis or Delta cells of Halmi stain green with a flocculant cytoplasm. Chromophobes may have a faint green cytoplasm, but the color is homogenous and distinct from the gonadotrop. No nuclear stain is used as the acid fuchsin will generally stain the nuclei and nucleoli red; however, sometimes the light green will predominate in the nuclei. Methods of Preparation of the stains Aldehyde fuchsin (Elftman, 1959c). 64 (1) 500 mg. of basic fuchsin is dissolved in 100 m1. of 70% ethanol. (2) Then add: 0.75 ml. of paraldehyde, and 1.25 ml. of concentrated hydrochloric acid. (3) Allow the stain to ripen 26 hours in the oven at 37°C or 3 days at room temperature. (a) The shade of the mixture will gradually turn to a deep purple, almost indistinguishable from gentian violet. As soon as this change has taken place the dye is ready for use. (b) The stain retains its selective staining ability for about 3 days. Control slides should be processed before each group of experimental slides. b. Masson A (1) Acid fuchsin, 0.3 gm. (2) Ponceau de xylidine, 0.7 gm. (3) Distilled water, 100 ml. (4) Glacial acetic acid, 1 ml. to be added just prior to use. c. Masson B (1) Phosphomolybdic acid, 1.0 gm. (2) Distilled water, 100 m1. d. Masson C (1) Aniline Blue to saturation (about 2-3 grams), or Light Green to saturation, (about 2-3 grams). (2) Distilled water, 100 ml. (3) Glacial acetic acid, 2.0 ml. per 100 m1. of stain to be added just prior to use. 65 Periodic Acid Schiff and Orange G Method. (Purves and Greisbach, 1951). This procedure best demonstrates the basophilic cell types, but is perhaps the most useful in making cell counts. 1. Fixation a. The glands are fixed 24 hours in formol-sublimate which is prepared as follows: (1) Mix 9 parts of saturated mercuric chloride with 1 part of 40% formaldehyde. b. After 24 hours the glands are washed in 95% ethanol. 2. Dehydration a. Dehydration is continued from 95% ethanol and is the same as previously described under aldehyde fuchsin and Masson's trichrome stain. 3. The procedure for embedding and sectioning is the same as previously described. 4. Staining procedures (Elftman, 1959a) a. Xylol, 3 changes, 5 minutes each. b. Absolute ethanol, 2 changes, 5 minutes each. c. 95% ethanol, 1 change, 5 minutes. d. 80% ethanol, 1 change, 5 minutes. e. 70% ethanol, 1 change, 5 minutes. f. Lugol's iodine, 5 minutes. g. Rinse in distilled water. h. 5% sodium thiosulfate, 3 minutes. 1. Wash in running tap water, 15-20 minutes. j. Distilled water, one change, 2 minutes. k. 1% periodic acid, 15 minutes. 1. Rinse in 3 changes of distilled water, 2 minutes each. V. 66 Place in sensitized Schiff reagent (Elftman, 1959b), 20 minutes. Rinse in tap water until there is a maximal development of color, 2-5 minutes. Rinse in clear water. Stain in Harris hematoxylin, 2 minutes. Rinse in tap water and follow by rinse in 1% HCl. Wash in tap water, 10 minutes. Stain in 3% Orange G at pH 2.0 for 10 seconds. Rinse in tap water briefly, the intensity of the Orange G can be regulated during this and the next step. Rinse in 95% ethanol briefly. Absolute alcohol, 2 changes, 2 minutes each. Xylol, 3 changes, 5 minutes each. Comments on staining Lugol's iodine is used to remove the mercury from the tissue sections. The mercury will show up on microscopic examin- ation as dark crystals if not removed. The sodium sulfate reduces the iodine to a colorless form which is more readily soluble and is removed by washing in tap water. Schiff's reagent selectively stains all glycoprotein con- taining compounds in the pituitary. This includes 2 types of gonadotrophs; LH cells, and FSH cells as well as the TSH cells. Also the Schiff reagent will stain degenerative or necrotic cells. The Orange G is a counterstain and as such stains every- thing that is not stained by the Schiff reagent. It does not, however, stain the chromdphobes. e. 67 The Harris hematoxylin stains the nuclei of all cell types. 6. Preparation of stains b0 Schiff Reagent. (Elftman, 1959b). (1) (2) (3) (4) (5) (6) (1) A stock solution is prepared by dissolving one gram of basic fuchsin in 100 ml. of distilled water. The solution is warmed to produce complete dissolution of the dye. The solution is then cooled and filtered. The next step is to produce sulfur dioxide by drop- ping sulfuric acid on sodium bisulfite in a closed container and then passing it through the basic fuchsin solution until decoloration occurs. The amount of dissolved sulfur dioxide determines the sensitivity of the Schiff reagent. The 802 can be measured by titration against Lugol's iodine. The most effective Schiff reagent for staining rat pitui- taries is produced when the titration requires 0.8- 2.0 ml. of Schiff reagent to decolorize 1.0 ml. of Lugol's iodine. The amount of 502 can be decreased by bubbling air through the decolorized Schiff reagent. The Schiff reagent must be stored in the refrigerator tightly stoppered to prevent deterioration. The solution remains effective over several months, but should be titrated periodically before use. Orange G Three grams of Orange G is dissolved in 100 ml. of distilled water. Then the solution is adjusted to a pH of 2 with concentrated hydrochloric acid. C. Harris Hematoxylin (l) Dissolve 40 grams of ammonium alum in 400 m1. of distilled water by heating. At the same time dis- solve 2 grams of hematoxylin in 20 m1. of absolute alcohol. Combine the two and bring to a boil. Add 1 gram of mercuric oxide slowly. After mixing, cool the solution in cold running water. Filter before use .