The Genesis of Cell Types in the Adenohypophysis of the Human Fetus As Observed with Immunocytochemistry ’

The Genesis of Cell Types in the Adenohypophysis of the Human Fetus as Observed with Immunocytochemistry ’

BURTON L. BAKER AND ROBERT B. JAFFE2J Department of Anatomy and Department of Obstetrics and Gynecology, The University of Michigan Medical School, Ann Arbor, Michigan 481 04

ABSTRACT Hypophyses of 21 human fetuses, ranging in gestational age from 6 to 23 weeks, were studied by immunocytochemical and histological staining to ascertain (1) the time of origin of specific cell types and (2) the development of parenchymal cell zonation in the pars distalis. No hormones were identified at six weeks. Probable corticotrophin-containing cells appeared at seven weeks. Somatotrophs were observed first at 10.5 weeks; correlation with other reports indicates that they appear at eight to nine weeks. Melanotrophs were detected at 14 weeks; the cells containing melanotrophin were far fewer than corticotrophs. The youngest fetus to possess gonadotrophs was 10.5 weeks old. In all specimens gonadotrophs (LH-cells) stained well with immunocytochemical procedures but poorly with histological methods. Thyrotrophs first occurred at 13 weeks. Zonal distribution of cell types in the pars distalis was evident almost from the time of their appearance. Somatotrophs were most numerous laterally and immediately anterior to the residual cleft. At 10.5 weeks corticotrophs were con- fined chiefly to the borders of vascularized connective tissue (trabeculae) and to the lateral peripheral region of the pars distalis. Thyrotrophs appeared chiefly in the anteromedian zone, particularly in its superior portion, but were found laterally also. In the older specimens, gonadotrophs generally occurred throughout the pars distalis but were less numerous near the trabeculae and in the anterolateral region. There was good correlation between the time of appearance of various cell types and published data on secretory capacity of the gland.

With increasing attention being given human fetal adenohypophysis as revealed to the endocrinology of the human fetal- by immunocytochemistry correlated with placental unit, a more precise understand- histological ~taining.~The exceptional sen- ing of the development of secretory compe- sitivity and specificity of immunocyto- tence by the fetal hypophysis is desirable. chemistry should enable one to ascertain Particularly critical is determination of with considerable accuracy the time when the earliest time at which each hormone hormones first appear in the gland. Indeed, is secreted by the adenohypophysis. In- little is known about the development of vestigators have attacked this problem by corticotrophs, mammotrophs, and melano- using several technical methods, includ- trophs; evidence pertaining to the status of ing assay for the presence of hormones in umbilical blood, in the pituitary gland Accepted January 17, ’75. 1 Supported in part by research grants from the Na- itself, or in medium in which the gland tional Institutes of Health, HD-03159-06 to Dr. B. L. has been cultured. Others have utilized Baker and HD-08478 to Dr. R. B. Jaffe. 2 Dr. Jaffe’s present address is: Reproductive Endo- staining procedures and immunofluores- crinology Center, Department of Obstetrics and Gyne- cence for demonstrating the time in ges- cology, School of Medicine, University of California, San Francisco, California 94143. tation when specific cell types appear 3 We express our appreciation to Mrs. Ya-Yen Yu and MIS. Frances Wicks for their expert technical as- (table 1). sistance. The study reported here is concerned 4Histological staining is used in this paper to en- compass all staining procedures other than immuno- with the genesis of specific cell types in the cytochemical techniques. AM. J. ANAT., 143: 137-162. 137 138 BURTON L. BAKER AND ROBERT B. JAFFE

TABLE 1 Reports on earliest detection of hormones in the human fetal hypophysis or in the medium of pituitary cultures (week of gestation)

Hormone Growth Pro- Cortico- Thyro- Author Method hormone lactin trophin MSH LH FSH trophin Dubois et al., '73 IF 7 Ellis et al., '66 IF 12 Fukuchi et al., '70 RIA 12 BA 14 Gailani et al., '70 TC-RIA 8 BA Gitlin, Biasucci, '69 TC-IE 9 14 14 Groom et al., '71 TC-RIA 13 13 Harteman et al., '73 TC-RIA 71 72 14+ Ivanova, Levina, '66 BA Kaplan et al., '72 RIA 9 Keene, Hewer, '24 BA 8 Levina, '68 BA 15+ 18+ 10 13+ ?13+ 13+ s 20+ Levina, Ivanova, '64 BA 19 18 Pavlova et al., '68 PH-BA 9 9 BA, bioassay; IF, immunofluorescence; IE, immunoelectrophoresis; PH, passive hemagglutination; TC, tissue culture; RIA, radioimmunoassay. 1 Only one specimen was studied. 2 Only a small amount of hormone was detected at seven weeks while much more was found at eight weeks. thyrotrophs and gonadotrophs in the fetus the most propitious areas for immunocyto- is inconclusive. Special attention will be chemical analysis with the peroxidase- given also to the development of zonation labeled antibody method of Nakane and in the pars distalis and to cytogenesis in Pierce ('67) or the immunoglobulin-en- the partes intermedia and tuberalis, as zyme bridge method of Mason et d. these subjects have received minimal at- ('69). Almost all of the description of re- tention to date. sults is based on use of the Nakane-Pierce procedure. The Mason method, because MATERIALS AND METHODS of its greater theoretical sensitivity, was Twenty-one human fetuses were ob- employed when the results were unsatis- tained by therapeutic and spontaneous factory or maximal sensitivity was desired. abortion (table 2).5 Crown-rump (CR) Thus the Mason procedure was used on length, crown-heel length and body weight the youngest specimens with antisera to all were recorded. Gestational (fertilization) hormones and in attempting to clarify the age for this report was determined from incidence of gonadotrophs in all specimens. the crown-rump length, using the plot of Sections adjacent to those stained irn- Patten ('68, fig. VII-3). The fetuses ranged munocytochemically were prepared with from 13 to 217 mm in CR length, i.e., from either the Masson ('28) acid fuchsin- 6 to 23 weeks in gestational age. Sex was ponceau de xylidene-aniline blue, Dawson- ascertained in ten cases, but no variations Friedgood ('38) azocarmine-orange G, or in pituitary cytology could be related to Adams and Swettenham ('58) periodic this variable. acid-Schiff-Alcian blue-orange G procedure The hypophysis and adjoining tissues to permit correlation of immunocytochemi- were excised within 20 minutes after de- cal findings with the cell types as revealed livery of the fetus and fixed in Bouin's by older histological methods. fluid. After dehydration and embedding in Antisera 6,7 to the following hormones paraffin, blocks were sectioned serially at were employed for the demonstration of 3 with some specimens being cut sagit- specific cell types as indicated : human tally and others transversely. Every twentieth section was stained with the Masson 5The collection of human fetuses used for this study was completed prior to enactment into law of ('28) procedure to permit identification of the National Research Act of 1974. IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS 139

TABLE 2 Summary of data pertaining to the fetuses studied and to the presence of secretory cell types in the hypophyseal pars distalis

Cell types identified in the pars distalis Speci- Fertil- men ization Menstrual Fetal Somato- Mammo- Cortico- Melano- Thyro- Gonado- CRLI age age weight troph troph troph troph troph troph (LH)

mm wk2 wk gm 1 13 6 0 0 0 0 0 2 23 7- 0 + 0 0 0 3 60 10.5 20 + + 0 0 + 4 72 11.5 12 20 + 0 + 0 0 + 5 80 12 13 35 + 0 + 0 0 + 6 90 13 15 + 0 + 0 + + 7 105 14 14 70 + 0 + + + + 8 105 14 73 + 0 + + + + 9 105 14 16 63 + 0 + + 0 + 10 107 14 14 85 + 0 + + + + 11 110 14 116 + 0 + + + + 12 117 14.5 118 + + + + + 13 125 15.5 21 129 + 0 + + + + 14 126 15.5 18 137 + 0 + + + + 15 128 15.5 15 116 + c! + + + + 16 131 15.5 150 + 0 + + + + 17 133 16 15 156 + 0 + + + + 18 137 16.5 14-16 155 + 0 + + + + 19 138 16.5 14-16 153 + 0 + + + + 20 144 16.5 16 186 + +? + + + + 21 217 23 28 779 + + + + + + 1 Crown-rump length. 2 Estimated from the crown-rump length. 3 Crown-tail length.

and bovine growth hormones for the somat- lower mammals to demonstrate LH-con- otroph; ovine and bovine prolactins for the taining cells (Baker et al., '72). mammotroph; human thyrotrophin, bovine Control procedures were carried out to TSH-p, and human TSH-b for the thyro- verify the specificity of these antisera for troph; human chorionic gonadotrophin and demonstration of cell types in the human bovine LH-p for the LH-gonadotroph; p'-"- fetus. Thus, when anti-hGH, corticotrophin and p'7-39-~~rti~~tr~phinfor corticotrophin, anti-p,''-39-~orti~otrophin, the corticotroph; and human p-melano- and anti-p-MSH were absorbed with the trophin for the melanotroph. Specificity of respective human hormonal antigen prior these antisera for delineating cell types has to use in the immunocytochemical proce- been verified for the rat as follows: anti- 6 Abbreviations -:ill be employed as follows: soma- totrophin (growth hormone), GH; thyrotrophin, TSH; hTSH (Baker and Yu, '71a,b) ; anti-bTSH-p luteinizing hormone, LH; melanotrophin (melanocyte- stimulating hormone), MSH; and human chorionic and anti-hCG (Baker et al., '72); and gonadotrophin, hCG. Antiserum will be designated by anti-p'-^-corticotrophin, anti-p,'7-3P-~~rti-the prefix "anti-" before the name of the hormone. A small letter prefix will indicate the species of origin cotrophin, and anti-p-MSH (Baker et al., for a hormone, e.g., anti-oLH signifies antiserum to '70; Baker and Drummond, '72). Our pres- ovine luteinizing hormone. 7 We thank the National Pituitary Agency of the ent antiserum to human growth hormone National Institute for Arthritis and Metabolic Diseases for providing human growth hormone (NIH-GH-HS shows the same properties for demonstra- 1395), ,bovine growth hormone (NIH-GH-B5), bovine tion of somatotrophs in the rat as did the prolactin (NIH-P-B3) and ovine prolactin (NIH-P-SG). Appreciation is extended also to Professor H. J. Bein antiserum to a different human growth and Dr. R. W. Rittel, Ciba-Geigy Ltd., Basle, for gl-2'- corticotrophin, &17-39.corticotrophin and human 8- hormone preparation that was shown pre- melanotrophin, and to the following individuals for viously to be specific (Baker et al., '69). the antisera indicated: Dr. W. D. Odell, anti-hTSH, Dr. J. G. Pierce, Jr., anti-bTSH-,!I anti-hTSH-@ and Anti-hCG permits immunocytochemical anti-bLH-8; and Dr. A. R. Midgley,' Jr., anti-hCG and staining of gonadotrophs in the adult hu- anti-oLH. Antisera to human and bovine growth hormone, bovine and ovine prolactins, ,91-24-corticotrophin, man hypophysis (Baker, '74) and may be @,17-39-corticotrophin, and 8-melanotrophin were prepared by B. L. Baker following the immunization used interchangeably with anti-oLH in schedule of Midgley el al. ('71). 140 BURTON L. BAKER AND ROBERT B. JAFFE dure, no staining was obtained. To acquire Cytogenesis in the pars distalis additional evidence regarding the individ- Somatotrophs. Growth hormone cells uality of the various cell types, immuno- were absent from the hypophysis at six cytochemical double staining with antisera and seven weeks of gestation (table 2, to two hormones was used, with 3,3’- specimens S-l,2) but were quite numerous diaminobenzidine and a-naphthol being at 10.5 weeks (S-3). They are illustrated at used as substrates. If two populations of 11.5 weeks (figs. 1, 4). It follows that cells of different color were delineated with somatotrophs must appear in the pars no cells showing a blend of the two colors, distalis between 7 and 10.5 weeks. The it could be inferred that two distinctly dif- ovoid to polyhedral somatotrophs were ferent cell types were present. Verification small but stained intensely (fig. 1) with of the specifiicity of immunocytochemistry anti-hGH in the immunocytochemical pro- for differentiating thyrotrophs and gonado- cedure. They occurred in all parts of pa- trophs proved difficult in the youngest rcnchymal cell cords and bore no special fetuses. This matter will be discussed later. positional relationship to the capillaries, As OBSERVATIONS early as 10.5 weeks (S-3), and more clearly at 14 weeks (S-8, figs. 5-7), the intra- Pituitary cytogenesis and intraglandular glandular distribution of somatotrophs was distribution of the cell types can be under- gy-nerally indicative of the adult pattern. stood more fully if some aspects of the At this time they were most numerous in general embryology of the hypophysis are the lateral wings; in the median zone some kept in mind. Early in its development, the somatotrophs were located in the caudal hypophyseal (Rathke’s) pouch is flattened portion near the residual cleft but only a dorsoventrally. At six to seven weeks a few appeared in the anterior area. Somato- midline protuberance termed the “anterior trophs occurred in increasing numbers in chamber” by Atwell (’26) or the “median all specimens obtained from successively diverticulum” by Rouvihre et al. (’47) later times in gestation (fig. 8). In the forms on the anterodorsal face. The lateral 23-week specimen (S-21) somatotrophs borders of the flattened hypophyseal pouch were large and well granulated (fig. 2). proliferate to form marginal cushions, cre- Anti-bovine growth hormone was effec- ating a fossa that faces the floor of the tive for the immunocytochemical demon- brain. The enlarging median diverticulum stration of some somatotrophs in the hu- divides this fossa into bilateral portions man fetus. However, the number seen was and each marginal cushion is divided into much smaller than when antiserum to medial and lateral parts by an incisure. human growth hormone was used. Ultimately, the median diverticulum and Somatotrophs are acidophils and it is the medial portions of the marginal cush- important to consider whether their early ions become the anteromedian zone of the history could be followed with histologi- pars distalis while the lateral portions of cal staining. Although somatotrophs were the marginal cushions become the lateral observed immunocytochemically at 10.5 part of the pars distalis. From the median weeks, acidophils were not demonstrable diverticulum, and probably the marginal until 11.5 weeks with the Dawson-Fried- cushions (lateral lobes of Atwell, ’26), good (’38) azocarmine-orange G method; epithelium grows cephalically ventral to with Masson staining they were not ob- the infundibular stem and tuber cinereum served until 15.5 weeks (S-16; fig. 3). In to form the pars tuberalis (fig. 15). the eight specimens ranging from 15.5 to Mesenchyme fills the fossa (fig. 14) and 16.5 weeks in gestational age, acidophils eventually the bilateral connective tissue were recognized in all but one. In every trabeculae of the adult gland arise from it case they were scarce and usually poorly (Pietsch, ’30). By proliferation of epithe- granulated. On the other hand, in all speci- lial tissue, the amount of connective tissue mens the immunocytochemical procedure in the fossa becomes reduced relative to regularly showed that many more cells the mass of parenchyma (Rouviere et al., actually contained growth hormone. ’47; Conklin, ’68). At 23 weeks (S-21) acidophils were quite IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS 141 numerous and well granulated. At this time marily alongside the vascularized develop- two types were distinguishable. One type ing trabeculae and their extensions, this contained large, densely packed, bright red relationship existing as far as the incisure cytoplasmic granules and may be the mam- (Atwell, ’26) where the dorsal mesen- motroph. This type occurred in clusters in chyme enters the hypophyseal fossa (fig. the median zone. More numerous were 14). Corticotrophs were also numerous at acidophils that contained much finer gran- the lateral and anterolateral borders of the ules of lesser affinity for the red dyes of pars distalis. As the gland enlarged with the Masson combination. Comparison with advancing pregnancy, the distribution of immunocytochemically stained prepara- corticotrophs along the trabeculae and at tions showed that these cells contained the lateral borders of the pars distalis be- growth hormone. came even more distinctive (S-19, 16.5 Mammotrophs (prolactin cells). For weeks; fig, 11). Superiorly in the postero- demonstration of mammotrophs we were median zone corticotrophs formed a bridge dependent upon antisera to ovine and across the midline, and in the oldest speci- bovine prolactins, because human prolac- men (S-21, table 2) corticotrophs located tin was not available for immunization. in the posteromedian zone were more With these antisera no specific cells were prominent than those in the lateral wings, demonstrable in specimens 1-19. How- where growth hormone cells had become ever, a few moderately well stained “mam- the dominant cell type. Beginning at 10.5 motrophs” were revealed in S-20 (16.5 weeks (S-3) anti-p’-“- and anti-ppt7-”- weeks) with anti-bovine prolactin, while corticotrophin proved to be comparably anti-ovine prolactin was ineffective. At 23 effective for demonstration of cortico- weeks (S-21, fig. 9) more “mammotrophs” trophs. appeared in the lateral wings and in the Comparison of immunocytochemically median zone near the residual cleft but stained and histologically stained sections they were still much less plentiful than showed that corticotrophs are basophils. As somatotrophs. At this time similar results early as 10.5 weeks (S-3) the cortico- were obtained with antisera to both ovine trophs were large, green basophils after and bovine prolactins. Double immuno- application of the Masson procedure. From cytochemical staining using anti-hGH and 10.5 to 14 weeks these cells stained in- anti-ovine prolactin indicated that somato- tensely with both immunocytochemical and trophs (fig. 10) and “mammotrophs” were histological techniques (figs. 18, 19). From distinctly different cells. However, a defini- 14 weeks on, basophils of many specimens tive conclusion cannot be drawn because were stained faintly with the Masson pro- of the small number of “mammotrophs” cedure, but this feature was not often that could be studied. indicative of light immunocytochemical Corticotrophs. Corticotrophs were ab- staining for corticotrophin. Corticotrophs sent from the hypophysis at six weeks (S-1, were periodic acid-Schiff (PAS)-positive table 2). However, at seven weeks (S-2) and in some of the older specimens this cells appeared along the ventrolateral reaction was quite intense. border of the hypophyseal pouch after Melanotrophs (MSH-cells). In contrast immunocytochemical staining with anti- to the presence of corticotrophs at seven pp17-39-~~rti~~tr~phin(fig. 13) but not with weeks of pregnancy, no immunocytochemi- p’”‘-corticotrophin. However, the stain- cal staining with anti-hMSH was obtained ing was not eliminated by prior absorp- until 14 weeks; this hormone was present tion of the antiserum with pp’7-39-~~rti~~-in hypophyses of all fetuses 14 weeks or trophin. Cytologically these cells differed older (fig. 20). In most 14-week specimens little from others composing the early melanotrophs were quite rare. In one fetus anlage of the pars distalis except that they (table 2, S-8; figs. 21-23) with more mel- were slightly larger. By 10.5 weeks (S-3) anotrophs than the others, their regional corticotrophs were present in considerable distribution was somewhat different from numbers and are illustrated at 11.5 weeks that of corticotrophs in that they were (figs. 12, 14). At 11.5 weeks the large, located mainly in the median zone and often ovoid corticotrophs were located pri- along capillaries in the lateral areas. The 142 BURTON L. BAKER AND ROBERT B. JAFFE parenchyma bordering the trabeculae was Although no part of the pars distalis was almost devoid of MSH staining, as was the totally devoid of thyrotrophs, from 13 most inferior portion of the gland (fig. 23). weeks on they were situated primarly in At 15.5 weeks (S-14; fig. 20) melano- the anteromedian zone (fig. 24), especially trophs were ovoid, densely granulated, and near the midline in the region of junction intensely stained. They were still most nu- with the pars tuberalis (fig. 27). Thyro- merous in the median zone, especially be- trophs were exceedingly rare in the pars tween the middle and junction with pars distalis lateral and inferior to the trabecu- tuberalis; also they appeared laterally, lae (fig. 24), especially in specimens 15.5 particularly along the border of the gland. weeks or older. At later ages, up to and including 23 weeks In the fetus, thyrotrophs did not stain (S-21), melanotrophs continued to be readily with Alcian blue of the Adams- scarce. Swettenham ('58) procedure as they do Several procedures were carried out to in adult man. clarify the relationship of corticotrophs Gonadotrophs (luteinizing-hormone cells). and melanotrophs as demonstrated im- The earliest identification of presumptive munocytochemically. Because the amino gonadotrophs was made at 10.5 weeks acid sequence of hMSH occurs in (S-3, table 2). They occurred in all older corticotrophin, immunological cross-reac- specimens, generally in rather large num- tion between the two hormones might be bers. From 10.5 through 13 weeks, most expected. However, prior absorption of gonadotrophs were small, usually stellate, anti-p'-"-corticotrophin with hMSH or of and lightly stained; some were as large anti-hMSH with p'-"- corticotrophin did as gonadotrophs of the older specimens not interfere with immunocytochemical (fig. 31). Upon first appearing in the staining with either antiserum. These ob- pars distalis (S-3) gonadotrophs were servations suggest that immunological common to the anterosuperior region, with cross-reaction between the two hormones very few being found inferior to the level did not result in staining of more than one of the trabeculae (fig. 28). With advancing hormone with each antiserum. Second, sec- age gonadotrophs became more widely dis- tions were doubly-stained immunocyto- tributed in the pars distalis, being most chemically with an ti-p' -*"-corticotrophin numerous at the posterolateral borders, be- and anti-pMSH. The results, as illustrated tween the residual cleft and a plane ex- by S-21 (table 2), were only partially con- tending between the trabeculae, and in the clusive. Some cells contained only cortico- anteromedian zone (fig. 29) that con- trophin while others contained both cor- tinued into the pars tuberalis. They were ticotrophin and melanotrophin. In these often especially scarce in the immediate preparations we could not be sure that any vicinity of the trabeculae and in the antero- cells contained only melanotrophin. lateral region of the pars distalis. Double Thyrotroph. With the use of anti-hTSH, immunocytochemical staining was utilized presumptive thyrotrophs were first en- with the oldest fetus (S-21, table 2) to countered at 13 weeks (S-6; table 2) and examine the individuality of gonadotrophs appeared in all older specimens (fig. 27) and thyrotrophs. With this procedure they except one at 14 weeks. In fetuses 16.5 appeared to be distinctly different cell weeks or younger, thyrotrophs were usu- types (fig. 26) although being similar in ally polyhedral and small with little cyto- size and shape. plasm, but the cytoplasm stained intensely. Anti-bLH-p was tried in the immunocyto- However, by 23 weeks (S-21) the cytochemical procedure on all hypophyses that plasmic volume of some cells had in- gave the strongest staining with anti-hCG. creased considerably (fig. 25). Antiserum In only the 23-week old fetus (S-21) was to the specific subunit of thyrotrophin it effective, Anti-oLH was used similarly (hTSH-p) was also useful for delineation and was ineffective except in two cases of this cell type. Anti-bTSH-p was ineffec- (S-11, 20), when some staining was ob- tive. tained. Thyrotrophs constituted a small propor- In contrast to the ease with which one tion of the parenchymal cell population. can demonstrate gonadotrophs in the fe- IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS 143 tus with immunocytochemistry, difficulty stem (S-14). In neither the pars inter- was experienced with histological staining. media nor its associated tubules was With the acid trichrome procedure (Mas- growth hormone, prolactin, or thyrotrophin son, '28), or with Alcian blue-PAS (Adams detected, and Swettenham, '58) (table 2: S-7, 8, 11, 15, 19) it was usually difficult or impos- DISCUSSION sible to identify any basophils other than It can be concluded from this study those previously shown to be corticotrophs. that ( 1 ) specific secretory cell types appear in the adenohypophysis nearly as soon as Cytogenesis in the pars tuberalis the embryonic anlage begins to differenti- LH was first detected in the pars tuber- ate, and (2) one of the glycoprotein hor- alis of two fetuses at 15.5 weeks (table 2, mones (gonadotrophin-LH) is synthesized S-15, 16; fig. 33) when lightly stained almost as early as the polypeptide hor- LH-cells8 were identified in the anterior mones. portion of the lobe. In the five older fetuses, Somatotrqhs. Our results indicate that the pars tuberalis of two contained LH- the production of growth hormone begins cells. LH-cells appeared throughout the between 7 and 10.5 weeks. The large num- pars tuberalis (fig. 32) of $19 (16.5 ber of intensely stained somatotrophs ob- weeks). In the pars tuberalis covering the served at 10.5 weeks suggests that initia- infundibular stem we did not detect cells tion of hormone synthesis must have that contained growth hormone, cortico- occurred considerably earlier. Unfortun- trophin (figs. 15, 16), melanotrophin or ately, none of our specimens represented thyrotrophin (fig. 27), except that a few the 7 to 10.5 week period. Most previous corticotrophs were located in the inferior investigators (table 1) have found that portion of the pars tuberalis of two fetuses growth hormone is demonstrable in the (S-16, 21). human hypophysis at eight to nine weeks. Cytogenesis in the pars intermedia Thus, Gitlin and Biasucci ('69) observed that pituitary glands from fetuses at eight Early in pituitary organogenesis the and one-half weeks could synthesize growth caudal wall of the hypophyseal pouch hormone when grown in culture, but those could be identified as the pars intermedia. from younger specimens could not. Tubular or follicular arrangements of epi- It seems likely that technical problems thelioid cells grew from the pars inter- may have accounted for the failure of Ellis media toward the pars nervosa (fig. 27). et al. ('66) to detect somatotrophs con- Similar tubules also partially encased the sistently by immunofluorescence before 17 pars nervosa and surrounded the inferior weeks. Even at 14 to 16 weeks they found portion of the infundibular stem (fig. 15). somatotrophs to be infrequent, which dif- Rare, lightly stained corticotrophic celIs fers markedly from our observations. Simi- were found in the pars intermedia of all larly, insensitivity of the bioassay proce- specimens. The tubules associated with dure may explain why Levina ('68) de- the pars intermedia, whether located be- tected no growth hormone in the gland tween the partes intermedia and nervosa before 15 weeks. or encompassing the pars nervosa, con- Our successful demonstration of some tained numerous intensely stained cortico- somatotrophs in the fetal hypophysis with trophic cells (fig. 15); this was especially antiserum to bovine growth hormone was true in the more anterosuperior region unexpected. Previously Nayak et al. ('69) (figs. 16, 17). More irregularly from speci- reported that somatotrophs could not be de- men to specimen, staining was obtained tected in hypophyses of human adults also with anti-hp-MSH, this also being when anti-bGH was used with immuno- more impressive in the tubules than in the fluorescence. Furthermore, they observed pars intermedia itself (fig. 30). The tubu- no cross-reaction between antiserum to bo- lar epithelium of two older specimens (S-16, 19) included a few LH-cells; they 8Cells in the partes tuberalis and intermedia that contain a hormone are designated by the name of the were especially prominent in some tubules hormone only because the functional role of these cells has not been demonstrated conclusively. Exam- near the inferior part of the infundibular ples: LH-cells, corticotrophic cells, MSH-cells. 144 BURTON L. BAKER AND ROBERT B. JAFFE vine growth hormone and human growth (S-21). Not only are the somatotrophic hormone in a hemagglutination test. This acidophils the dominant cell type during conflict in experimental observations could the last half of pregnancy, but the pitui- result from a difference in the antigenic tary content. of growth hormone also in- properties of growth hormone in the fetal creases with advancing gestation (Kaplan hypophysis as compared with the adult. et al., '72; Gitlin and Biasucci, '69; Pavlova Thus, there may be incomplete "matura- et al., '68). tion" of the hormone in the fetus or it may Mammotroph. Although histological respond differently to the fixing fluid. staining revealed the presence of two types Some disagreement exists concerning the time when acidophils appear in the of acidophils at 23 weeks, our designation hypophysis and whether synthesis of of one of them as a "mammotroph" can- growth hormone precedes the formation of not be considered conclusive for reasons storage granules that enable the cytologist previously stated. Indeed, Goluboff and to recognize acidophils. In our series, intra- Ezrin ('69) did not observe prolactin cells cellular growth hormone was demonstra- in the hypophyses of three fetuses 31 to ble before acidophils could be identified. 38 weeks old after staining with the Although immunocytochemistry revealed Brookes carmoisine S-orange G method. abundant somatotrophs at 10.5 weeks, Nevertheless, in agreement with our ob- acidophils as stained with orange G of the servation is the finding of Levina ('68) Dawson-Friedgood procedure were first ob- that prolactin is first detectable in the fetal served in small numbers at 11.5 weeks. hypophysis at about 19 weeks. Application of either the Dawson-Friedgood Corticotroph/meZanotroph. Dubois et al. or Masson technique resulted in irregular ('73) recently reported the presence of demonstration of acidophils until 14.5 corticotrophs in the median "posterior weeks after which time they appeared in lamina" of the hypophysis of a 7-week variable numbers in all specimens, How- human fetus as demonstrated by means ever, in all specimens younger than 23 of immunofluorescence. Anti-pp"-3y-~~rti~~- weeks, immunocytochemistry revealed far trophin, but not anti-pl-"-corticotrophin, more somatotrophs than did histological was effective. With our one 7-week speci- staining. men (S-2), we also observed staining with Many investigators have reported acido- anti-p,"-3y-~orti~otrophinonly, but it could phils to be present much earlier: Daikoku not be prevented by prior absorption of ('58) at eight weeks (30-mm CR length), the antiserum with pp1'-3y-~~rti~~tr~phin. Falin ('61) at nine weeks, Pearse ('53) Thus, some question exists concerning the and Conklin ('68) at 11 weeks, Ellis et al. presence of corticotrophin at this time. The ('66) at 12 to 16 weeks, and Romeis ('40) adequacy of anti-pp17-39-~orti~otrophinfor at 13 weeks. The discrepancies in these ob- immunocytochemical demonstration of servations probably result from variations corticotrophin in man is supported by the in technical procedures and in criteria for discovery that the amino acid sequence identification of an acidophil. In contrast of porcine corticotrophin is identical to that to several fixatives used by others which of man except for substitution of serine for preserve mitochondria well, e.g., Zenker- leucine at the 31 position (Riniker et al., formol or formalin, the acetic acid of '72). We do concur with the conclusion of Bouin's fluid used in our study makes mito- Dubois et al. that subsequently during chondria unstainable and thus eliminates human gestation anti-pp17-39-~~rti~~trophin confusion of these organelles with cyto- and anti-p'-"-corticotrophin are equally plasmic granules. Thus we may have been effective for the immunological study of able to make a more certain identification corticotrophs. The demonstration with bio- of acidophils than some other investigators. assay of corticotrophic activity in the A great increase in number and size of hypophysis of 9-week old fetus by Pavlova acidophils occurs by 16 to 21 weeks et al. ('68) also supports the contention (Pearse, '53; Levina, '68; Levina and that elaboration of this hormone begins Ivanova, '64); in our series this develop- early. Evidence presently available in- ment was especially significant at 23 weeks dicates that corticotrophin is the first hor- IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS 145 mone to be synthesized by the pars distalis quently did not exhibit a distinctive re- in man. gional distribution in the gland. However, We have shown that corticotrophs are in some specimens melanotrophs occurred basophils. These basophils are demon- in the anteromedian zone, a location not strated easily by histological staining and frequented by corticotrophs. This observa- can be identified between the eighth and tion suggests that some cells may contain eleventh weeks along the borders of the melanotrophin in the absence of cortico- vascularized mesenchyme that invades trophin, even though this was not demon- the pituitary fossa (Pearse, '53; Daikoku, strated with double immunocytochemical '58; Falin, '61; Pavlova et al., '68). They staining. are the p-cells of Romeis ('40) and Type I Thyrotrophs. When anti-hTSH was basophils of Andersen et al. ('70) which used with the immunocytochemical pro- were observed at eight weeks (28 to 30 mm cedure, thyrotrophs were observed first at CR length) by these investigators. Conklin 13 weeks and in all older specimens ex- ('68) reported such basophils to be pres- cept one. This finding agrees closely with ent at seven weeks and, like Pavlova et al. the observations of all previous investiga- ('68), correctly ascribed to them the secre- tors who have sought thyrotrophin in the tion of corticotrophin. hypophysis (table 1); they reported its The conclusion that corticotrophin ap- presence at 12 to 14 weeks. However, the pears between the seventh and tenth weeks status of the fetal thyroid suggests that is supported strongly by the secretory pituitary control may be effective some- status of the adrenal cortex. In an 8.5 what earlier. Thus at 10 to 11 weeks, the week fetus Murphy and Diez dAux ('72) ratio of thyroid weight to body weight found no cortisol, but total adrenal cor- reaches the adult level (Shepard et al., ticoids were exceedingly high, as was '64), follicles begin to accumulate colloid cortisol, at 11 weeks. They concluded that (Norris, '16), and the capacity to iodinate the adrenal cortex functions from the first organic substance begins (Shepard, '67). trimester on. Our study shows that the Thyrotrophin and thyroxine are detectable hypophysis is competent to stimulate the in fetal umbilical cord blood as early as 12 adrenal cortex at this time. weeks (Fisher et al., '70). Because the p basophil of the adult Undoubtedly previous investigators have hypophysis contains both corticotrophin successfully differentiated thyrotrophs in and melanotrophin (Phifer et al., '70; the human fetus by means of histological Baker, '74), it is of particular interest to staining. The Type I1 alcianophilic baso- know whether the same situation exists phi1 which Andersen et al. ('70) observed in fetal life. Dubois et al. ('73) concluded first in 11 to 12 week embryos may be the that it does. With double immunocyto- thyrotroph. Conklin ('68) also concluded chemical staining we have shown that that thyrotrophs (Alcian blue-positive) some corticotrophs contain melanotrophin arise at 12 weeks but he viewed them as while others do not. Different cells of origin being characteristic of the lateral wings might be indicated by the fact that the rather than the median zone. In our study hypophysis initiates its production of the they constituted only a small percentage of two hormones at somewhat different times. the parenchymal cell population; they were Although the evidence indicates that cor- located primarily in the median zone, ticotrophin may appear as early as seven especially in its superior portion. Few thy- weeks, we did not detect melanotrophin rotrophs could be found in the lateral until 14 weeks; it was present in all of wings. Prior to 23 weeks thyrotrophs were our specimens older than 14 weeks. Dubois small with little cytoplasm and possessed et al. ('73) agree that melanotrophin long thin processes. At 23 weeks they were arises later than corticotrophin. Also sug- larger and contained denser cytoplasm gestive of different cells of origin was a than was true of the younger fetuses. disparity in regional distribution of cor- Gonadotrophs. The status of knowledge ticotrophs and melanotrophs. In contrast regarding gonadotrophs in the fetal hypo- to corticotrophs, the cells containing mel- physis, and their secretory activity, has anotrophin were fewer in number and fre- heen__.__ unsatisfactorv. In view of the ease 146 BURTON L. BAKER AND ROBERT B. JAFFE with which gonadotrophs can be demon- man. Third, the loss in effectiveness of strated immunocytochemically in the fetal anti-hCG after absorption with human hypophysis, the previous difficulty experi- thyrotrophin was probably due to con- enced by cytologists in delineating them is tamination with LH, a condition widely of special interest. With histological meth- recognized to occur in such preparations. ods, few investigators of the human fetal This outcome does not necessarily indicate hypophysis have attempted to subdivide that anti-hCG is ineffective for differentiat- the basophils into categories that might ing gonadotrophs from thyrotrophs, al- produce corticotrophin (melanotrophin) , though the possibility of direct cross-reac- thyrotrophin, or gonadotrophic hormones. tion must be considered since Nisula et al. When such attempts have been made there ('74) believe that human chorionic gonad- was minimal justification for attributing otrophin possesses intrinsic thyrotrophic functions to the various cell types. As re- activity. Fourth, the regional distributions viewed previously, corticotrophic basophils, of gonadotrophs (fig. 29) and thyrotrophs because of their prominence, have been (fig. 24) are vastly different. noted by most workers. Few workers have In some of the younger specimens (13- indicated a source for gonadotrophin. 15.5 weeks) differentiation of the two cell Romeis ('40) first found y-cells at the types was less conclusive. At least in me- nineteenth to twentieth week (180 mm dian sagittal sections the distributions of CRL) and &-cellsat the forty-second week cells containing thyrotrophin (fig. 27) and (230 mm CRL). Similarly the basophils gonadotrophin (fig. 28) were similar. Be- that Conklin (68) considered to be the cause human TSH, FSH and LH possess a source of LH and FSH were observed first common a-subunit (Nisula et al., '73 ; at 22 and 23 weeks, respectively. Vaitukaitis et al., '73), possible immuno- The weight of the evidence shows that logic cross-reaction between these hor- gonadotrophins are present in the hypo- mones must be considered. On the other physis much earlier than this. In addition hand, it is conceivable that during early to our observations with immunocyto- genesis of thyrotrophs and gonadotrophs, chemistry, it is generally recognized that two or more of the glycoprotein hormones both FSH and LH are demonstrable in the might occur in a single cell. Investigation fetal hypophysis at 13 weeks and subse- of this difficult problem continues. quently (table 1). Using a hemagglutina- In conclusion, the times when specific tion test for LH and bioassay for FSH, secretory cell types appear in the pars Levina ('72) reported the presence of LH distalis of the human fetal hypophysis activity in serum beginning at the 18th agree rather well with evidence regarding week with a peak occurring at 20 to 26 the first occurrence of the various hor- weeks; the level of FSH followed a similar mones within the blood. pattern. Subsequently there was a sharp It may be significant that LH-containing fall, with the concentration of both hor- cells represent the only cell type identifi- mones being exceedingly low during weeks able in the pars tuberalis. They occur in 27 to 32. the pars tuberalis of the human adult as Specificity of the immunocytochemical shown by immunofluorescence (Midgley, methods for differentiation of thyrotrophs '66). Similarly, only LH-cells were found and gonadotrophs warrants further consid- with immunocytochemistry in the pars eration, In specimens older than 15.5 tuberalis of adult rats (Baker, unpublished) weeks the achivement of specificity was although electron microscopy indicates indicated by the following observations. that more than one secretory type may be First, double immunocytochemical staining present (Klein et al., '70). revealed that different cells contained thyrotrophin and gonadotrophin. Second, LITERATURE CITED the antiserum to human thyrotrophin, Adams, C. W. M., and K. V. Swettenham 1958 The histochemical identification of two types which contained antibodies to LH, was of basophil cell in the normal human adeno- absorbed with hCG before use. This treat- hypophysis, J. Path. Bact., 75: 95-103. ment eliminates immunocytochemical la- Andersen, H., K. Mollgard and F. A. Von Biilow beling of gonadotrophs in adult rat and 1970 On the specificity of staining by Alcian IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS 147

blue in the study of human foetal adenohy- Fukuchi, M., T. Inoue, H. Abe and Y. Kumahara pophysis. Histochemie, 22: 362-375. 1970 Thyrotropin in human fetal pituitaries. Atwell, W. J. 1926 The development of the J. Clin. Endocr. Metab., 31: 565-569. hypophysis cerebri in man, with special refer- Gailani, S. D., A. Nussbaum, W. J. McDougall ence to the pars tuberalis. Am. J. Anat., 37: and W. F. McLimans 1970 Studies on hor- 159-193. mone production by human fetal pituitary cell Baker, B. L. 1974 Functional cytology of the cultures. Proc. SOC.Exp. Biol. Med., 134: 27-32. hypophysial pars distalis and pars intermedia. Gitlin, D., and A. Biasucci 1969 Ontogenesis In: The Pituitary Gland and Its Control- of immunoreactive growth hormone, follicle- Adenohypophysis. Handbook of Physiology, stimulating hormone, thyroid-stimulating hor- Endocrinology IV, Part 1. Am. Physiol. SOC., mone, luteinizing hormone, chorionic prolactin Bethesda, pp. 45-80. and chorionic gonadotropin in the human con- Baker, B. L., and T. Drummond 1972 The cellu- ceptus. J. Clin. Endocr., 29: 926-935. lar origins of corticotropin and melanotropin Goluboff, L. G., and C. Ezrin 1969 Effect of as revealed by immunochemical staining. Anat. pregnancy on the somatotroph and the prolactin Rec., 134: 395409. cell of the human adenohypophysis. J. Clin. Baker, B. L., A. R. Midgley, Jr., B. E. Gersten and Endocr., 29: 1533-1538. Y.-Y. Yu 1969 Differentiation of growth hormone- and prolactin-containing acidophils Groom, G. V., M. A. Groom, I. D. Cooke and with peroxidase-labeled antibody. Anat. Rec., A. R. Boyns 1971 The secretion of immuno- 164: 163-171. reactive luteinizing hormone and follicle-stimu- Baker, B. L., S. Pek, A. R. Midgley, Jr. and B. E. lating hormone by the human foetal pituitary Gersten 1970 Identification of the cortico- in organ culture. J. Endocr., 49: 335-344. tropin cell in rat hypophyses with peroxidase- Hartemann, Ph., D. Malaprade, D. Lemoine, labeled antibody. Anat. Rec., 166: 557-567. G. Grignon, P. Nabet and M. Pierson 1973 Baker, B. L., J. G. Pierce and J. S. Cornell 1972 Mise en evidence des e1Cments morphologiques The utility of antiserums to subunits of TSH et des activites secrbtoires STH et LH dans and LH for immunochemical staining of the I’hypophyse de foetus humain au cows du rat hypophysis. Am. J. Anat., 135: 251-267. dCveloppement. C. R. SOC.Biol., 167: 105-110. Baker, B. L., and Y.-Y. Yu 1971a The thyro- Ivanova, E. A., and S. E. Levina 1966 Biologi- tropic cell of the rat hypophysis as studied cal estimation of thyrotrophin in the pituitary with peroxidase-labeled antibody. Am. J. Anat., of the human embryo and the placenta. DOH. 131: 55-71. Akad. Nauk., SSSR, 167: 1423-1426. 1971b Hypophyseal changes induced by Kaplan, S. L., M. M. Grumbach and T. H. Shepard thyroid deficiency and thyroxine administration 1972 The ontogenesis of human fetal hor- as revealed by immunochemical staining. Endo- mones. I. Growth hormone and insulin. J. Clin. crinology, 89: 996-1004. Invest, 51: 3080-3093. Brookes, L. D. 1968 A stain for differentiating Keene, M. F. L., and E. E. Hewer 1924 Glandu- two types of acidophil cells in the rat pituitary. lar activity in the human foetus. Lancet, 2: Stain Techn., 43: 41-42. 111-1 12. Conklin, J. L. 1968 The development of the Klein, M.-J., M.-E. Stoeckel, A. Porte and human fetal adenohypophysis. Anat. Rec., 160: F. Stutinsky 1970 Arguments ultrastruc- 79-91. turaux en faveur de l’existence de cellules Daikoku, S. 1958 Studies on the human foetal corticotropes (?I ACTH) dans la pars inter- pituitary. 2. On the form and histological de- media et dans la pars tuberalis de l’hypophyse velopment, especially that of the anterior pitui- du rat. C. R. Acad. Sci. (Paris), Ser. D, 271: tary. Tokushima J. Exp. Med., 5: 214-231. 2 159-2162. Dawson, A. B., and H. B. Friedgood 1938 Dif- Levina, S. E. 1968 Endocrine features in de- ferentiation of two classes of acidophiles in the velopment of human hypothalamus, hypo- anterior pituitary of the female rabbit and cat. physis, and placenta. Gen. Comp. Endocr., 11: Stain Tech., 13: 17-21. 151-159. Dubois, P., H. Vargues-Regairaz and M. P. Dubois 1973 Human foetal anterior pituitary immuno- 1972 Times of appearance of LH and fluorescent evidence for corticotropin and FSH activities in human fetal circulation. Gen. melanotropin activities. 2. Zellforsch., 145: Comp. Endocr., 19: 242-246. 131-143. Levina, S. E., and E. A. Ivanova 1964 Biologi- Ellis, S. T., J. S. Beck and A. R. Currie 1966 cal determination on pituitary prolactin in the The cellular localisation of growth hormone in human embryo. Dokl. Akad. Nauk, SSSR, 155: the human foetal adenohypophysis. J. Path. 988-99 1. Bact., 92: 179-183. Mason, T. E., R. F. Phifer, S. S. Spicer, R. A. Falin, L. I. 1961 The development of human Swallow and R. B. Dreskin 1969 An im- hypophysis and differentiation of cells of its munoglobulin-enzyme bridge method for local- anterior lobe during embryonic life. Acta Anat., izing tissue antigens. J. Histochem. Cytochem., 44: 188-205. 17: 563-569. Fisher, D. A., C. J. Hobel, R. Garza and C. A. Masson, P. 1928 Carcinoids (argentaffin-cell tu- Pierce 1970 Thyroid function in the preterm mors) and nerve hyperplasia of the appendicu- fetus. Pediatrics, 46: 208-216. lar mucosa. Am. J. Path., 4: 181-212. 148 BURTON L. BAKER AND ROBERT B. JAFFE

Midgley, A. R., Jr. 1966 Human pituitary Pearse, A. G. E. 1953 Cytological and cyto- luteinizing hormone: an immunohistochemical chemical investigations on the foetal and adult study. J. Histochem. Cytochem., 14: 159-166. hypophysis in various physiological and patho- Midgley, A. R., Jr., G. D. Niswender, V. L. Gay logical states. J. Path. Bact., 65: 355-370. and L. E. Reichert, Jr. 1971 Use of antibodies Phifer, R. F., S. S. S. Spicer and D. N. Orth for characterization of gonadotropins and 1970 Specific demonstration of the human steroids. Rec. Progr. Horm. Res., 27: 235-301. hypophyseal cells which produce adrenocortico- Murphy, B. E. P., and R. C. Diez d’Aux 1972 tropic hormone. J. Clin. Endocr. Metab., 31: Steroid levels in the human fetus: cortisol and 347-361. cortisone. J. Clin. Endocr. Metab., 35: 678-683. Pietsch, K. 1930 Aufbau and Entwicklung der Nakane, P. K., and G. B. Pierce, Jr. 1967 En- Pars tuberalis des menschlichen Hirnanhangs zyme-labeled antibodies for the light and elec- in ihren Beziehungen zu den iibrigen Hypo- tron microscopic localization of tissue antigens. physenteilen. 2. mikro. Anat. Forsch., 22: 227- J. Cell Biol., 33: 307-318. 258. Nayak, R., E. E. McGarry and J. C. Beck 1969 Riniker, B., P.Sieber, W. Rittel and H. Zuber Studies with fluorescein-conjugated antisera to 1972 Revised amino-acid sequences for por- growth hormones. I. Localization of antisera in cine and human adrenocorticotrophic hormone. the pituitary in various species. Canad. J. Nature New Biol., 235: 114-115. Physiol. Pharmacol., 47: 15-19. Romeis, B. 1940 Innersekretorische Driisen. 11. Hypophyse. Handb. mikro. Anat. Menschen. Nisula, B. C., P. 0. Kohler, J. L. Vaitukaitis, J. M. Hershman, and G. T. Rose 1973 Neutraliza- Vol. 6. Part 3. W. von Mollendorff, ed. Julius tion of human thyrotropin by antisera to sub- Springer, Berlin. units of glycoprotein hormones. J. Clin. Endocr. RouviBre, H., A. Giraud, P. Desclaux and P. Roux Metab., 37: 664-669. 1947 Le developpement du lobe antbrieur de l’hypophyse et ses consCquences physiepath- Nisula, B. C., F. J. Morgan and R. E. Canfield ologiques. Ann. Anat. Path., 17: 113-124. 1974 Evidence that chorionic gonadotropin Shepard, T. H. 1967 Onset of function in the has intrinsic thyrotropic activity. Biochem. human fetal thyroid: biochemical and radio- Biophys. Res. Commun., 59: 86-91. autographic studies from organ culture. J. Clin. Norris, E. H. 1916 The morphogenesis of the Endocr., 27: 945-958. follicles in the human thyroid gland. Am. J. Shepard, T. H., N. J. Andersen and H. Andersen Anat., 20: 411-448. 1964 The human fetal thyroid. I. Its weight Patten, B. M. 1968 Human Embryology. Third in relation to body weight, crown-rump length, ed. The Blakiston Division, McGraw-Hill Book foot length and estimated gestation age. Anat. Company, New York. Rec., 148: 123-128. Pavlova, E. B., T. S. Pronina and Y. B. Vaitukaitis, J. L., G. T. Ross, J. G. Pierce, J. S. Skebelskaya 1968 Histostructure of adeno- Cornell and L. E. Reichert, Jr. 1973 Genera- hypophysis of human fetuses and contents of tion of specific antisera with the hormone- somatotropic and adrenocorticotropic hormones. specific p-subunit of hTSH or hFSH. J. Clin. Gen. Comp. Endocr., 10: 269-276. Endocr. Metab., 37: 653-659. PLATES All preparations illustrated were stained with the Nakane-Pierce ('67) conjugated antibody procedure except those for figures 13a,b, 28 and 30, which were prepared with the immunoglobulin-enzyme bridge technique of Mason et al. ('69).

Abbreviations C, residual cleft N, pars nervosa D, pars distalis P, hypophyseal pouch I, infundibulum PI, pars intermedia IP, infundibular process s, somatotroph M, medianzone T, pars tuberalis m, mammotroph tr, trabecula ME, median eminence tu, tubule

PLATE 1

EXPLANATION OF FIGURES

In all illustrations immunocytochemical staining was used unless stated to the contrary.

3 Somatotrophs in the pars distalis of S-4 (table 2) (11.5 weeks). This is an enlargement of an area shown in figure 4. The cells are small, polyhedral and in most instances have few cytoplasmic granules. Anti-hGH. x 1000. 2 Somatotrophs in the pars distalis of 5-21 (23 weeks). As compared with those at 11.5 weeks (fig. 1) somatotrophs are larger and more ovoid. Anti-hGH. x 1000. 3 Acidophils (arrows) in the pars distalis of S-14 (15.5 weeks). This is the youngest specimen in which acidophils could be identified after staining with the Masson procedure. x 1000. 4 Somatotrophs (dark cells) in the region of the pars distalis lateral to one of the paired trabeculae in S-4 (11.5 weeks). Sagittal section, immunocytochemical staining with anti-hGH. x 100. 5,6,'7 Transverse sections through the hypophysis of S-8 (14 weeks) to show the distribution of somatotrophs. Figure 5 is superior and includes portions of the pars nervosa, junction of the pars nervosa with the infundibular stem, pars intermedia, residual cleft and one wing of the pars distalis. Somatotrophs are distributed laterally and posteriorly in the pars distalis, and are absent from the zone of junction with the pars tuberalis. In figure 6 the residual cleft, pars intermedia with its accompany- ing tubules, and one trabecula (tr) in the lateral zone of the pars distalis are shown. Somatotrorhs are most numerous posteriorly and laterally, except in the immediate vicinity of the trabecula. They are scarce in the anteromedian (M) zone. The section shown in figure 7 is inferior to that for figure 6. Most somatotrophs are located posteriorly and few are present anteriorly. Immunocytochemical staining with anti-hGH. x 42.

150 IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS PLATE 1 Burton L. Baker and Robert B. Jaffe

151 PLATE 2

EXPLANATION OF FIGURES

All sections were prepared with immunocytochemistry.

8 Transverse section through the hypophysis of S-21 (23 weeks) showing the distribution of somatotrophs. They are most numerous in the posterior portion of the median zone (M) and laterally except in the immediate vicinity of the trabecula (tr). They are absent from the anterior portion of the median zone and from the pars intermedia (PI) with its tubules. Anti-hGH. x 34. 9 A “mammotroph” (m) from the pars distalis of S-21 (23 weeks). Anti-o prolactin. x 1000. 10 Differentiation of somatotrophs (arrows) from a mammotroph (m) by double-immunocytochemical staining. Anti-hGH with diaminobenzidine as substrate was used for the somatotroph and anti-o prolactin with a-naphthol-pyronin for the mammotroph. x 1000. 11 Transverse section through the hypophysis of S-19 (16.5 weeks) to show the distribution of corticotrophs. They are most frequent adjacent to the trabecula (tr) and its extensions, as well as along the anterolateral border of the pars distalis. Some staining is evident in the pars intermedia (PI). Almost no corticotrophs appear in the anterior part of the median zone. Anti-,91-24-~orti~otrophin.x 42. 12 Corticotrophs border a trabecula in the pars distalis of S-4 (11.5 weeks). They vary in size and may be ovoid or polyhedral. Anti- &1”-39-c~rtic~tr~phin.x 1000.

152 IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS PLATE 2 Burton L. Baker and Robert B. Jaffe

153 PLATE 3

EXPLANATION OF FIGURES

All sections were prepared with immunocytochemistry.

13 a. Sagittal section through the lateral portion of the hypophyseal pouch of S-2 (7 weeks) stained with the Mason et al. ('69) technique utilizing anti-pP"-3~-corticotrophin. Stain- ing has occurred along the ventro-caudal border of the pouch (arrows). x 100. b. The area of staining indicated in figure 13a is illustrated at higher magnification. The cells containing corticotrophin show little structural modification from those constituting the remainder of the pouch. x 325. 14 A lateral sagittal section through the hypophysis of S-4 (11.5 weeks) showing the distribution of corticotrophs. Mesenchyme has invaded the pars distalis at the incisure (X) to form a trabecula (tr). Corticotrophs are located chiefly along the invading mesenchyme and near the inferior border of the pars distalis. Some corticotrophic cells are associated with the pars intermedia (PI). x 52. 15 Sagittal section of S-14 (15.5 weeks) illustrating all subdivisions of the hypophysis. Corticotrophs occur along vascular connective tissue of the pars distalis and at the inferior border of this lobe. A few positive cells appear in the pars intermedia (PI) and more in the tubules (tu) adjacent to the pars nervosa and superior to the infundibulum. No corticotrophs are found in the pars tuberalis (T) and few in the anteromedian zone of the pars distalis. Anti-P1-24-~orti~otrophin.X 42. 16 Corticotrophin-containing cells in an oblique frontal section through the median eminence (ME), infundibular stem, and superior part of the pars distalis of S-19 (16.5 weeks). Corticotrophs are scattered in the pars distalis and corticotrophic cells are abundant in the tubules (tu) derived from the pars intermedia. None occur through the full extent of the pars tuberalis (T) or the zone of junction between it and the pars distalis. Anti-P,,17-39-corticotrophin.X 42. 17 An enlargement of the area indicated by the rectangle in figure 10. Many corticotrophic cells occur in the pars intermedia (PI) and the associated tubules (tu). x 400.

154 IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS PLATE 3 Burton L. Baker and Robert B. Jaffe

155 PLATE 4

EXPLANATION OF FIGURES

18 Peripheral border of the pars distalis of S-4 (11.5 weeks) stained with the Masson procedure to show the basophils (arrows) that are corticotrophs. x 600. 19 Portion of the pars distalis of S-8 (14 weeks) bordering a trabecula (tr) and stained with the Masson procedure to show corticotrophic basophils (arrows). x 600. 20 Melanotrophs in the pars distalis of S-14 (15.5 weeks) Like the corticotrophs, melanotrophs (arrows) frequent the borders of vascularized connective tissue strands. Immunocytochemical staining with anti-hp-MSH. x 1000. 21,22, 23 As figures 5-7, these are transverse sections through the hypophysis of s-8 (14 weeks). They are stained to show the distribution of melanotrophs. In the superior section (fig. 21) they are few in the pars distalis but more numerous in tubules associated with the pars intermedia. In a deeper section (fig. 22) melanotrophs populate the median zone (M) and some occur laterally (arrow); in this case the peritrabecular region (tr) is almost devoid of melanotrophs. The inferior section (fig. 23) has very few melanotrophs. x 42.

156 IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS PLATE 4 Burton L. Baker and Robert B. Jaffe

157 PLATE 5

EXPLANATION OF FIGURES

All illustrated sections were stained immunocytochemically.

24 Portion of a transverse section through the hypophysis of S-21 (23 weeks) showing that thyrotrophs (arrows) are located primarily in the anterior portion of the median zone (M). Anti-hTSH. x 34. 25 Thyrotrophs in the median zone of S-21 illustrated in figure 24. Anti- hTSH. x 1000. 26 A portion of the pars distalis from S-21 (23 weeks) showing the differentiation of gonadotrophs and thyrotrophs by double immunocytochemical staining - anti-hCG with diaminobenzidine, and anti-hTSH with a-naphthol followed by pyronin. Gonadotrophs (arrows) appeared gold while thyrotrophs (ts) appeared lavender. x 1000. 27 Median sagittal section of S-14 (15.5 weeks) showing the distribution of thyrotrophs in the anterior median zone (arrows). They are absent from the pars tuberalis (T), pars intermedia (PI), and the tubules (tu) associated with the pars intermedia. x 42. 28 Sagittal section through the hypophysis of S-4 (11.5 weeks) showing the distribution of gonadotrophs (arrows). Compare with the distribution of thyrotrophs (fig. 27) at 15.5 weeks. At this time they appear chiefly in the anterior and superior portion of the median zone (arrows) of the pars distalis (D). Gonadotrophs are absent from the pars intermedia and tubules associated with it. Anti-hCG. X 42.

158 IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS PLATE 5 Burton L. Baker and Robert B. Jaffe

159 PLATE 6

EXPLANATION OF FIGURES

All illustrated sections were stained immunocytochemically.

29 A transverse section through the hypophysis of S-20 (16.5 weeks) at the level of the trabeculae (tr) to show the distribution of gonadotrophs. They are common throughout the pars distalis but are less numerous in the immediate vicinity of the trabeculae (tr) and in the anterolateral region (between the arrows). Gonadotrophs are absent from the pars intermedia (PI) and its associated tubules. Mason et al. ('69) technique. Anti-hCG. x 42. 30 Section through the median pars intermedia and associated tubules of S-14 (15.5 weeks). Scattered cells (arrows) in the tubules and fewer in the pars intermedia contain melanotrophin. Anti-hp-MSH. X 400. 31 A portion of the pars distalis of S-11 (14 weeks) showing gonadotrophs. Anti-hCG. x 400. 32 The pars tuberalis (T) of S-19 (16.5 weeks), extending far anteriorly beneath the median eminence, contains numerous gonadotrophic cells throughout its extent. The dotted line represents the boundary between the infundibulum and pars tuberalis. Anti-hCG. X 120. 33 A section through the rostral portion of the pars tuberalis in S-15 ( 15.5 weeks) containing at least three gonadotrophic cells. Anti-hCG. x 1000.

160 IMMUNOCYTOCHEMISTRY OF HUMAN FETAL HYPOPHYSIS PLATE 6 Burton L. Baker and Robert B. Jaffe