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 confined 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.

The distribution of gonadotropin-releasing hormone (GnRH) was studied in the brain of adult female rats with three immunocytochemical techniques using antisera to unconjugated synthetic GnRH and to GnRH conjugated with limpet hemocyanin. GnRH was found in nervous tissue surrounding blood vessels of the organum vasculosum of the lamina terminalis. In the median eminence it occurred in nervous tissue associated primarily with the tuberoinfundibular sulci throughout their extent. Cephalic to the pars tuberalis GnRH often spread across the median eminence from sulcus to sulcus. Caudally, with widening of the median eminence, GnRH occurred dorsal to the tuberoinfundibular sulci, and especially in the external lamina medial to the sulci. A broad median zone of the median eminence was rather free of GnRH. GnRH was most concentrated in the region of continuity between the dorsolateral walls of the infundibulum and floor of the third ventricle where the tuberoinfundibular sulci are deep. Caudal to the infundibulum GnRH was disposed in a flat zone through the cephalic portion of the floor of the mammillary recess. In the median eminence GnRH appeared to be located in axons that terminated there. The amount of demonstrable GnRH varied significantly from rat to rat. The distributions of GnRH as revealed by use of antisera to unconjugated and conjugated GnRH were essentially the same. The apparent order of sensitivity of the immunocytochemical methods was: the peroxidase-antiperoxidase (PAP) (Sternberger et al.) procedure greater than the immunoglobulin-enzyme bridge (Mason et al.) procedure smaller than the conjugated antibody (Nakane and Pierce) procedure.

The direct action of 17 p-estradiol and cholesterol on cells of the pituitary pars distalis, as revealed by staining with peroxidase-labeled antibody, was studied in female rats. Pellets of pure cholesterol and of estradiol mixed with cholesterol were implanted into the left lobe of the pars distalis 14-32 days after ovariectomy and left in place for 7-16 days. Rabbit antisera to rat prolactin, human growth hormone, human chorionic gonadotropin (for gonadotropes -presumably luteinizing hormone cells), and porcine corticotropin were used. In no case were cells altered in the contralateral lobe of the pars distalis; cholesterol likewise had no significant effect on the ipsilateral lobe. However, in the ipsilateral lobe containing an estrogen pellet, prolactin cells were hypertrophied and hyperplastic; cells assumed to be responsible for luteinizing hormone secretion were reduced in size and stained more intensely; and growth hormone cells were reduced in size. Corticotropin cells remained unaffected. For the most part estrogenic effects were distributed ventrally, caudally and laterally from the pellet and not far medially, never reaching the midline. It was concluded that estrogen acts directly on the hypophysis, the spread of the effects reflecting the direction of blood flow within the gland. These observations support the hypothesis that the pituitary gland serves as a site for feedback action by estrogen.One of the most important current problems in neuroendocrinology is to ascertain whether the hypothalamus or hypophysis serves as the primary site for feedback action by target-organ hormones. Evidence available at present indicates that both sites are probably responsive to estrogen but the manner in which the functional roles of the hypothalamus and hypophysis are integrated remains unclear.Several investigators have implanted ovarian tissue or estrogen into the hypothalamus and, from the effects observed, concluded that estrogens act primarily on the hypothalamus. Thus, Flerko and Szentitgothai ('57) found that ovarian secretory activity was depressed in rats bearing small intrahypothalamic autotransplants of ovarian tissue, but not in rats bearing intrapituitary implants. Likewise, implantation of estrogen into the median eminence of the rabbit caused depression of ovarian function (Davidson and Sawyer, and elevated the prolactin content of the gland (Kanematsu and Sawyer, '63a). These workers found that estrogen placed into the hypophysis failed to duplicate these effects, the pituitary prolactin content actually being depressed (Kanematsu and Sawyer, 63a).Apparently the rat responds differently to these experimental procedures. After Lisk ('60) showed that estrogen implanted into the hypothalamic arcuate nucleus causes atrophy of the genital tract, McCann and his colleagues found that estrogen located in either the median eminence or hypophysis was effective; such implants prevented the postovariectomy rise in plasma luteinizing hormone (Ramirez Abrams and McCann, '64) and elicited peripheral e...

Since differential chemical staining has been unsatisfactory for demonstration of specific secretory cell types in the hypophyseal pars distalis of the mouse, the objective of this study was to determine whether the peroxidase-antiperoxidase immunocytochemical procedure might be more effective. Accordingly, representative sections from the hypophyses of 17 female and 15 male adult mice of the Swiss-Webster strain were immunostained, 16 antisera to 5 pituitary hormones or their subunits being utilized. Five secretory cell types were demonstrated. Somatotropes were ovoid to spheroidal and distributed quite generally in the gland except for the "sex zone" where they were scarce. Somatotropes were larger and more numerous in the male than in the female. Mammotropes were polyhedral and also generally distributed in the gland except for the "sex zone" where few were observed. Mammotropes were larger and more numerous in the female than in the male. Corticotropes were small, stellate and few. They were most common near the ventral surface of the gland and formed bilateral centromedial groups in the lateral wings. Thyrotropes were usually large and polyhedral. They were restricted almost solely to the ventral region of the pars distalis. Gonadotropes were polyhedral, and generally distributed, except for aggregation in the cephalomedian "sex zone." Most gonadotropes appeared to contain both luteinizing hormone and follicle-stimulating hormone. Thus, all secretory cell types recognized in other species can be demonstrated readily in the mouse hypophysis with immunocytochemistry.

s y n t h e t i c l u t e i n i z i n g hormone-releasing hormone (LRH), LRH was l o c a l i z e d i n t h e p e r i p h e r a l r e g i o n o f t h e median eminence i n t h e mouse and r a t , and more g e n e r a l l y i n t h e median eminence o f t h e guinea p i g . t u d i e s i n d i c a t e s t h a t l u t e i n i z i n g hormone-releasing hormone (LRH) i s elabor a t e d i n t h e hypothalamus, t r a n s p o r t e d t o t h e median eminence i n axons, andt h e r e secreted i n t o t h e primary plexus o f t h e hypothalamo-hypophyseal vascular system. G r e a t l y needed a t t h e moment i s a c l e a r demonstration o f what nerve c e l l s e l a b o r a t e LRH and where t h i s hormone i s released i n t o t h e blood stream. Recently Leonardell i e t a1 . ( ' 7 3 ) used immunofl uorescence t o d e s c r i b e t h e l o c a li z a t i o n o f LRH i n t h e median eminence o f t h e mouse and guinea p i g . Subsequently,they ( B a r r y e t a l . , ' 7 3 ) r e p o r t e d t h e l o c a l i z a t i o n o f LRH i n nerve c e l l bodies s c a t t e r e d through t h e a n t e r i o r hypothalamus from t h e p r e o p t i c and septa1 areas t o t h e caudal p o r t i o n of t h e t u b e r cinereum.The o b j e c t i v e o f our p r e l i m i n a r y paper i s t o r e p o r t t h e l o c a l i z a t i o n o f LRH i n hypothalami o f r a t , mouse and guinea p i g u t i l i z i n g immunohistochemistry.MATERIALS AND METHODS Four female r a t s o f t h e Sprague-Dawley s t r a i n , one female and one male o f s t r a i n 129 SvSl mouse, and one female guinea p i g were 129

The cytoplasm of the megakaryocyte in the rat spleen possesses three zones, the perinuclear, intermediate and marginal. The perinuclear zone is characterized by the presence of Golgi apparatus, ribosomes, endoplasmic reticulum, and mitochondria. These organelles are found also in the more voluminous intermediate zone which in addition exhibits platelet granules and an extensive development of vesicles from smooth-surfaced endoplasmic reticulum to form demarcation membranes by coalescence. The marginal zone is almost devoid of the organelles and inclusions present elsewhere. Shedding of platelets appears to occur by extension of a paired demarcation membrane from the intermediate zone to the cell membrane and subsequent separation of its lamellae so that all of the essential organelles and inclusions of the intermediate zone may be included within the platelet. In addition, platelets contain vesicles which are probably pinocytotic in nature. Platelets are sometimes engulfed by the cytoplasm o f phagocytic cells.

In order to verify the concept that growth hormone and prolactin are contained in two different populations of acidophils, sections of Bouin-fixed rat hypophyses were stained immunochemically. For this purpose the histochemical demonstration of peroxidase was utilized after sequential application to the tissue section of rabbit antiserum to human growth hormone (or antiserum to rat prolactin) followed by application of peroxidase-labeled sheep antiserum to rabbit gamma-globulin. It was found that growth hormone cells and prolactin cells, when revealed immunochemically, corresponded structurally to cell types that could be differentiated with reasonable certainty in sections stained with the Masson trichrome procedure. When delineated immunochemically, growth hormone cells were larger and more densely arranged in the adult male than in the intact female; thcy exhibited little change in the female after ovariectomy. In contrast, prolactin cells were large and frequent in the female hypophysis but were small and less frequent in the male and in the female after ovariectomy. By double-staining, growth hormone and prolactin cells were differentiated in the same section. It was concluded that ( a ) growth hormone and prolactin are contained in, and presumably secreted by, two different populations of acidophils; and ( b ) the Masson procedure permits a reasonably accurate differentiation of the two cell types.

The objective was to assess the effectiveness of antiserums to the B-subunit of bovine TSH (thyrotropin) and a-and p-subunits of LH (luteinizing hormone) for immunochemical staining of rat pituitary cells and in so doing to acquire more information about the properties of the subunits. For the following reasons it was concluded that antiserum to TSH-/3 is specific for demonstration of TSH-cells. The morphology and distribution of the presumptive TSH-cells so revealed conform to the descriptions presented by other investigators who have used histological techniques. When pituitary TSH content was reduced by thyroidectomy or administration of propylthiouracil or thyroxine, the stainability of TSH-cells was reduced; it was increased with elevation of TSH content during rebound after cessation of propylthiouracil treatment. The effectiveness of anti-TSH-p was lost after absorption with bovine TSH.The specificity of antiserum to LH-P for demonstration of LH-cells was indicated since the cells demonstrated with it correspond to the gonadotropic cells described by others with respect to morphology, distribution and response to gonadectomy. Antiserum to LH-a, the nonspecific subunit of LH, permitted staining of both TSH-and LH-cells. The individuality of TSH-and LH-cells, as revealed immunochemically with antiserums to the respective specific P-subunits, was demonstrated by double staining and by comparison of adjacent sections stained with one of the two antiserums.