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 through- out 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, tis- sue 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 twen- tieth 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.