Maryanne C. McClellan

Luteinizing hormone, progesterone and the morphological development of normal and superovulated corpora lutea in sheep

SummaryThe development of granulosa-lutein cells was studied in 27 normal and 32 superovulated ewes between days 0–4 (day 0 began with the preovulatory LH peak in normal animals and the HCG injection in superovulated ewes). The pattern of differentiation was similar in both groups. Following initial hormonal stimulation (0–12 hours after LH or HCG), granulosa cells were approximately 100 μ2 and contained small, pleomorphic nuclei with large amounts of clumped chromatin. Elongate cells lining the basement membrane possessed large, heterogeneous dense bodies, and a well-developed Golgi apparatus. Mitotic figures were observed up to 6 hours prior to ovulation.Sixteen to 20 hours following the LH surge or HCG injection, hypertrophy of granulosa cells was evident. Nuclei contained definitive nucleoli. Blood vessels in the theca interna were abundant and highly dilated.Ovulation occurred approximately 24 hours after the LH peak or HCG injection. Visible signs of luteinization were evident 6–12 hours after ovulation. A slight increase in serum progesterone levels was detected.The second post-ovulatory day was characterized by continuing hypertrophy of granulosa cells and extensive proliferation of smooth endoplasmic reticulum and mitochondria. Nuclei of granulosa cells were larger and possessed extremely large nucleoli. Numerous mitotic figures were apparent within the corpus luteum. Serum progesterone concentrations began increasing at 60–72 hours after hormone stimulation.By the end of the third post-ovulatory day, the corpus luteum consisted of large, pleomorphic, parenchymal cells, interspersed between capillaries and connective tissue elements. Only an occasional mitotic figure was apparent within the corpus luteum at 100 hours.Light microscopic autoradiography of 5, 10, and 15 day corpora lutea taken from ewes pulsed with 3H thymidine at specific times before and after ovulation revealed that granulosa cells did not undergo secondary mitoses following ovulation. In contrast, thecal, mesenchymal and endothelial cells did mitose on day 3.

ESTROGEN RECEPTORS PROGESTIN RECEPTORS AND DNA SYNTHESIS IN THE MACAQUE ENDOMETRIUM DURING THE LUTEAL-FOLLICULAR TRANSITION

We have suggested that in the nonhuman primate endometrium, stromal cells might play a role in mediating the effects of estrogen on the epithelium, especially during the luteal-follicular transition (LFT) when target cells normally escape from the inhibitory influence of progesterone (P). We now report that like estrogen receptors (ER), endometrial progestin receptors (PR) are detectable only in stromal cells until the fifth day of the LFT. With a technique that combined immunocytochemistry and autoradiography on the same sections, we characterized the cellular distribution of ER or PR coincidentally with the localization of [3H]thymidine taken up in vitro by endometria from monkeys undergoing an LFT. DNA synthesis in the glands of the upper endometrium was E2-dependent, but the distribution of [3H]thymidine was not positively correlated with the presence of ER or PR. Readministration of P to animals on days 3 or 4 of the LFT significantly reduced the [3H]thymidine labeling index of the glandular epithelium and caused stromal ER to decline, but P did not block the eventual appearance of ER in epithelial cells on day 5 of the LFT. Thus, E2 stimulated DNA synthesis in epithelial cells that lacked ER, and P suppressed DNA synthesis in these cells even though PR was only detected in the stroma when P treatment began. These data are consistent with a role for endometrial stromal cells in mediating the effects of E2 and P on the epithelium during the LFT.

Subcellular compartmentalization of the luteal cell in the ovary of the dog

SummaryThe compartmentalization of the parenchyma of the corpus luteum in the dog was studied by both 100 and 1000 KV electron microscopy. The organelles within the luteal cell are oriented with a high degree of consistency towards the pericapillary space. Characteristically, the avascular pole and the lateral margins of the cell possess predominantly stacked and whorled cisternae of agranular ER. In the central medial portions of the cell, pleomorphic mitochondria with tubulo-vesicular cristae and anastomosing tubules of agranular ER predominate. However, the distribution of organelles in this compartment is graded. Mitochondria predominate in the central medial areas while tubular ER is more dominant peripherally. Microfilaments are ubiquitous in this compartment and run a longitudinal course between and around the subcellular components towards the pericapillary space. The Golgi apparatus is large and prominent and is positioned over the pole of the nucleus that faces the basal lamina. Coated vesicles are abundant in the Golgi regions and along the lateral surface of the cell. Three distinct regional specializations of the cell surface exist. The basal surface contains long pleomorphic cytoplasmic folds that fill the pericapillary space, are interonnected by small gap junctions and contain abundant multivesicular bodies. The lateral cell surface is covered with microvilli and is organized into tortuous intercellular channels and canaliculi. These are interrupted at intervals by cytoplasmic protrusions that extend from one cell well into the cytoplasm of the next. Large, well-developed gap junctions line the margins of the cells furthest removed from the pericapillary space. Finally, individual cells exhibit heterogeneity with respect to the amount one subcellular organelle or compartment is expressed relative to another. These observations are discussed in relation to the subcellular compartmentalization of progesterone synthesis and release.

Immunocytochemistry of Estrogen and Progestin Receptors in the Primate Reproductive Tract

The central focus of our research program, since its inception, has been on steroid receptors as components of regulatory mechanisms that control tissue structure and function in the reproductive tract in female primates. Our approach has been to correlate fluctuations in the levels of receptors with the morphological and physiological effects of the gonadal steroids. Our long-term goal is to deepen our understanding of how steroids bring about the various transformations that occur in the primate reproductive tract during the individual’s life history. Our current research depends heavily on immunocytochemical techniques that have come to maturity in our laboratory over the last few years (1–3). Our findings to date support the hypothesis (4) that there are significant stromal-epithelial interactions involved in steroid hormone action in the adult reproductive tract. Our findings also support the view that in vivo, the bulk of the estrogen receptor (ER) and progestin receptor (PR) are located in target cell nuclei, even in the absence of ligand (5–7).

Cellular Localization of Estrogen and Progestin Receptors in the Macaque Reproductive System

The past few years have seen a dramatic change in our understanding of the intracellular localization of steroid receptors. Previous lines of evidence had suggested that estrogen and progestin receptors were localized in the cytoplasm in the absence of ligand and that ligand binding to receptor provoked translocation of the steroid-receptor complex to the nucleus. Various techniques, including immunocytochemistry in cryosections, have revealed that most steroid receptors are nuclear proteins whether occupied by steroids or not. In addition, most studies of the hormonal regulation of receptors have been done on extracts of homogenates of whole tissues. Generally this work has indicated that estrogens increase the amount of both the estrogen and progestin receptors and that progestins suppress the level of both receptors. Immunocytochemistry has now revealed that these regulatory processes differ markedly among the different cell types of the reproductive tract. In addition there is new immunocytochemical evidence of the importance of stromal-epithelial interactions in steroid hormone action. Steroid-induced epithelial growth and/or differentiation can occur in tissues where steroid receptors are only detectable in the stromal cells. In this review, immunocytochemical evidence for the nuclear localization of estrogen and progestin receptors, for the variation in receptor regulation in different cell types and for the possible hormone action will be presented. All data will be based on experimental studies of the reproductive system of male and female nonhuman primates.

Localization of Human Chorionic Gonadotropin in Lysosomes of Ovine Luteal Cells

The first event in most current models regarding the hypothetical mechanism of gonadotropic hormone action is believed to involve binding to specific receptors located on the plasma membrane of target cells (Catt and Dufau, 1976; Cuatrecasas et al, 1975; Helmreich, 1976; Roth, 1973; Ryan and Lee, 1976). This binding initiates a series of acute biochemical responses, including activation of adenylate cyclase (Jungmann and Russell, 1977; Marsh, 1976), calcium-exchange mechanisms (Rasmussen and Goodman, 1975), and steroid secretion, probably as a consequence of the activation or synthesis of certain enzymes or functional proteins or both (Hermier et al, 1971; Abel et al, 1976). The interaction of hormone with receptors can also result in a number of sustained intracellular responses such as induction of synthesis of RNA and protein (Jungmann and Russell, 1977; Marsh, 1976). The signals that initiate all these regulatory events are believed to originate at the plasma membrane; however, evidence has been provided recently that protein hormones may also be capable of entering target cells and stimulating directly a variety of intracellular components (Braendle et al, 1973; Danzo et al, 1972; De Krester et al, 1971; Midgley and Beals, 1971; Petrusz and Uhlarik, 1973; Rajaniemi and Vanha-Perttula, 1972; Rao, Ch. V., et al.=, 1971; Sterling et al, 1977; Szego, 1975).