Morphological changes in the trophoblast, uterus and corpus luteum during delayed implantation and implantation in the western spotted skunk.

Morphological interactions of tropholbast and uterus from stages of preimplantation and implantation were studied in 14 western spotted skunks. In addition, the granulosa lutein cells and plasma progesterone levels were studied. In animals several days from implantation the height of epithelial cells decreased, but began to increase in animals approaching implantation. During the preimplantation period a few leucocytes infiltrated the epithelium, but in animals just prior to implantation many leucocytes infiltrated the epithelium.     

Structural characteristics of the corpus luteum during implantation in the rhesus monkey (Macaca mulatta)

Corpora lutea were obtained from ten pregnant rhesus monkeys during implantation, and the ultrastructure of granulosa and theca lutein cells was characterized. Specimens were individually staged with regard to the extent of implantation and the relationship to the rise in circulating progesterone and estrogen which is characteristic of early pregnancy. Structural changes characteristic of granulosa lutein cells occurring during implantation included: change in form of endoplasmic reticulum from predominantly agranular tubules to predominantly granular cisternae; reduction in size and number of lipid droplets; increase in area occupied by the Golgi and increase in length of the cisternae of the Golgi complex; development of numerous microvillus-lined intracellular spaces; increase in numbers of membrane-bound dense bodies including peroxisomelike bodies, multivesicular bodies within lobopodia, and other lysosomelike bodies; and alterations of mitochondrial cristae. These changes were suggestive of the production of a secretory protein, rapid utilization of existing steroid precursor reserves for the production of progesterone, and a reduction in capability for steroid precursor accumulation and processing by granulosa lutein cells. Structural changes characteristic of theca lutein cells occurring during implantation included an increase in size and number of lipid droplets, an increase in agranular endoplasmic reticulum, and an increase in area occupied by the Golgi complex. These changes were suggestive of an increased capability for steroid precursor accumulation and processing, perhaps for estrogen production, by the theca lutein cells.     

Fine structure of the corpus Luteum of the raccoon during pregnancy

Summary Ultrastructure of the granulosa lutein cells of the raccoon from throughout pregnancy has been described. The lutein cells often from epithelial cords which are separated by the connective tissues, capillaries and lymphatics. Based on the arrangements and modifications of the cytoplasmic organelles and inclusions, three types of lutein cells have been recognized. The type I lutein cells predominantly contain tubular, agranular endoplasmic reticulum, juxtanuclear Golgi complexes, a few round to rod-shaped mitochondria, some free ribosomes, and occasional lipid droplets. Occasionally the tubular cristae of mitochondria and tubular smooth endoplasmic reticulum appear contiguous. The type II cells contain abundant lace-like and/or stacked fenestrated endoplasmic reticulum cisternae that frequently form membranous whorls, some tubular, agranular endoplasmic reticulum, mitochondria, and lipid droplets. Mitochondria are usually small, but unusual large ones also occur. The small, rod-to round-shaped mitochondria usually have tubular cristae; but the large, oval, elongate, and cup shaped mitochondria possess tubular, lamellar, plate like, and whorl-like cristae. The plasma membranes of the cells are complexly elaborated and folded, especially when apposing each other. In favorable sections, strands of fenestrated cisternae appose the folds of the plasma membranes. In general, the amount of cytoplasmic organelles and inclusions vary greatly in the cells. The type III cells predominantly contain lipid droplets and sparse cytoplasmic organelles. The type I and II cells are found throughout pregnancy, but the type III cells are observed from mid gestation to term. The cytological features of type I and II cells suggest that they probably secrete most of the steroids, whereas the type III cells primarily store lipids.This research was supported by UPSHS grant AM-11376 and NIH contract 69-2136.     

Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells. Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

On the ultrastructure of granulosa lutein cells in porcine corpus luteum

Summary In young corpora lutea the endoplasmic reticulum membranes are sparse. A marked increase of smooth membranes then follows up to the peak of dioestrus. Continuities between smooth and rough endoplasmic reticulum are obvious during the same period. These observations suggest that the agranular membranes develop from the granular ones.During the most intense development of the endoplasmic reticulum the membranes show a tendency to be arranged in whorls. Since these are numerous only during the period of high progesterone secretion, a multitude of whorls constitutes a useful morphologic sign of high functional activity in the porcine granulosa lutein cells.During the first half of the oestrous cycle the increase in endoplasmic reticulum in general also parallels the increase in progesterone secretion. However, this secretion as well as ⁵-3-hydroxysteroid dehydrogenase activity declines earlier and more rapidly than the endoplasmic reticulum regresses. Steroid hormone synthesis may therefore be lacking although the agranular membranes appear morphologically normal.The mechanisms of induction of the endoplasmic reticulum membranes and enzymes active in steroid synthesis are discussed and it is suggested that luteinizing hormone (LH) may act as a trigger by increasing transport across membranes.Read at the Meeting of the Swedish Society for Pathology in Umeå, September 25, 1965 (Bjersing, 1966).This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 13 X-78-01, 12 X-78-02, and 12 X-78-03).     

Ultrastructural studies of the bovine Graafian Follicle and corpus luteum

Summary Ovaries were obtained from normal adult dairy cows at all days of the estrous cycle. The largest Graafian follicle and corpus luteum were excised and prepared for electron microscopic study.In the follicle wall, membrana granulosa cells contained granular endoplasmic reticulum and mitochondria with villous or lamellar cristae. The theca interna cells during proestrus and estrus contained ribosomes separated from endoplasmic reticulum. The latter during these periods assumed tubular and tortuous shapes. Mitochondria during these periods assumed rounded shapes, were occasionally cup-shaped, and developed tubular cristae.In the corpus luteum, the large luteal cells during metestrus and diestrus contained an abundance of agranular, tubular, branching membranes of endoplasmic reticulum and Golgi apparatus. Mitochondria were large, with tubular cristae, but smaller mitochondria, with irregular or villous cristae, were also present. Transitional bodies of the latter mitochondria to another form were observed. Cup-shaped and annular mitochondria were present during diestrus. In the small luteal cells, large vesicular membrane formations were present and often associated with lipid bodies. The cells were lipid-laden. Lysosomes and granular bodies were present during luteal regression.The observed features of the granulosa cells are related to protein synthesis, those of the pre-ovulatory theca interna cells and metestrus-diestrus large luteal cells to steroid synthesis, and those of the small luteal cells to lipid storage.This investigation was supported by a General Research Support Grant to the College of Veterinary Medicine, University of Minnesota, and Research Grant No. GM-07009, of the United States Public Health Service. Approved for publication as Scientific Journal Series Paper No. 6344, Minnesota Agricultural Experiment Station. The work reported is taken from the senior author's Ph. D. thesis.     

Ultrastructural analysis of the granulosa — Luteal cell transition in the ovary of the dog

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Partial development of the steroidogenic ultrastructural features in degenerative corpora lutea after a single injection of pituitary extract in the Western painted turtle (Chrysemys picta)

Pituitary glands were removed from sexually mature female turtles (Chrysemys picta) and they were injected intraperitoneally (i.p.) into other mature females of the same species (experimental). In addition mature females of the same species received saline injection only (controls). Initially all the turtles used in this study were steroidogenically inactive with corpora lutea already undergoing luteolysis (degeneration) as these turtles had ovioposited their eggs approximately 2 weeks earlier. Forty-eight hour post injection the corpora lutea were removed from the control and experimental turtles. In the experimental turtles, the lutein granulosa cells developed ultrastructural features such as tubular and cisternal smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae associated with lipid droplets. However, the controls maintained degenerative corpora lutea without steroidogenic ultrastructural features. The circulating progesterone (Pro) levels in the experimental turtles were significantly higher than the controls (P<0.049). Although the 48h development of steroidogenic ultrastructural features in the lutein granulosa cells was only partial in development, the effect of the pituitary taken from the inactive donor triggered an activating process within a short period, clear evidence of gonadotropic effect on the inactive corpora lutea. The present data offer interesting information on the short-term effect of gonadotropins during the non-reproductive period. This information may have useful implication under natural conditions particularly during the onset of a new reproductive cycle where the ovary is still inactive.     

Fine structure of the lutein cell in the house musk shrew,Suncus murinus

Summary The ultrastructure of lutein cells during pregnancy and the post partum period was examined by transmission electron microscopy in the house musk shrew, Suncus murinus. Lutein cells on day 13 of pregnancy contained an extensive system of anastomosing tubules or cisternae of smooth ER and many enlarged mitochondria with numerous tubulovesicular cristae. From day 13 on, the number of small granules, possibly lysosomes, increased gradually. Between day 20 and 25, the loss of smooth ER began and elongated, or flattened mitochondria increased. Regressing lutein cells observed after parturition were characterized by abundant large dense bodies, bizarre mitochondria and a decrease in the amount of smooth ER. Unusual forms of mitochondria were always observed after day 5 of pregnancy. Two types could be distinguished; one, found frequently in the second third of pregnancy, was ring-, disc-, cup-or dumb-bell-shaped with tubulovesicular cristae, and the other, found exclusively in the last third of pregnancy and after parturition, was elongated, flattened and sometimes twisted. The paucity of lipid droplets was a characteristic feature of the lutein cells of this species. The significance of these ultrastructural changes of cellular organelles is discussed in relation to the ovarian and plasma levels of progesterone.     

Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Summary Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells.Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

Spontaneous testicular neoplasm in mice with testicular feminization

Summary Testicular neoplasms occur spontaneously in androgen insensitive mice with testicular feminization (tfm/y); they are composed of Leydig cells, lipid-laden cells, fibroblastlike cells, and macrophages. The small Leydig cells in the periphery of the tumor are structurally similar to nontumorous tfm/y Leydig cells, whereas centrally located large Leydig cells contain numerous lipid droplets, mitochondria with tubular cristae, and abundant smooth endoplasmic reticulum. The lipid-laden cells exhibit a crescentic nucleus which is displaced toward the periphery of the cytoplasm by a large lipid vacuole. The fibroblastlike cells have a large amount of rough endoplasmic reticulum, lysosomes, free ribosomes, and lipid vacuoles. Macrophages are characterized by numerous layered and dense osmiophilic structures closely associated with crystalshaped bodies. An in vitro study shows that, in comparison with the normal testes, the tfm/y tumors produce significantly less testosterone but a larger quantity of androstenedione. Also, the tumors are capable of converting progesterone to estrone and estradiol-17. The plasma level of testosterone is significantly lower in tumor-bearing animals than in normal littermates, but slightly higher than in the nontumorous tfm/y animals. Since the abnormal steroid enzyme activity is found in both tumor-bearing and nontumorous tfm/y mice, the basic cause of aberrations in sex steroid production appears to be genetic rather than the direct result of alterations in their Leydig cells.     

Correlation of plasma estradiol-17β and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char,Salvelinus leucomaenis

Summary Plasma estradiol-17 and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis. Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation.In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed ⁵, 3-hydroxysteroid dehydrogenase (3-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.Nanae Fish Culture Experimental Station Contribution No 14     

The fine structure of the lutein cells in the mink (Mustela vison) with special reference to the secretory activity during pregnancy

Summary The ultrastructure of the lutein cells in the mink throughout pregnancy and the regression periodpost partum is described. To correlate the fine structure with the changes in the peripheral plasma progesterone levels, the concentrations of progesterone were measured by a rapid competitive protein-binding assay.Even during the delay period (e.g. as long the plasma progesterone levels remain at the basal level, <8ng/ml), the lutein cells in the mink exhibit structural criteria of functional activity. However, the increase in progesterone secretion is accompanied by some morphological transformations, characterized by the presence of more and more small dense homogenous bodies in the cytoplasm, which become irregular and scalloped during the stage with maximum release of progesterone. At this stage the agranular endoplasmic reticulum is often cisternal or vesicular.During the decline of the progesterone levels, typical and moderate electron-dense lipid droplets are found increasingly more within the lutein cells. The expanded agranular ER is now more sparse, while the granular ER becomes more pronounced, often forming parallel arrays. During this phase the mitochondria become elongated, dumb-bell, or cup shaped. After parturition the corpora lutea consist of cells in various stages of degeneration. At day 14post partum only a few lutein cells are still identifiable.Evidently the observed morphological changes take place in the lutein cells during the life span of corpora lutea. This feature lends further support to the concept that the mink lutein cells are steroid-producing cells and furthermore, that the corpora lutea may be the main sites of gestagen production during pregnancy.     

OBSERVATIONS ON THE FINE STRUCTURE OF LUTEIN CELLS

Corpora lutea from the period of delayed implantation and from early postimplantation stages of the armadillo, mink, and rat were fixed in buffered osmium tetroxide-sucrose or potassium permanganate. After rapid dehydration, the portions of the corpora lutea were embedded in either methacrylate or epoxy resin. Examination of the lutein cells by electron microscopy revealed the presence, in the better preserved material, of an extensive development of tubular agranular endoplasmic reticulum. Although the membranes of the endoplasmic reticulum are the most striking feature of the lutein cells of both stages of the three animals examined, very numerous large mitochondria with cristae that exhibit a variety of forms tending toward villiform, and protrusions and foldings of the lutein cell margins on the pericapillary space are also characteristic of these cells. Certain minor differences in the lutein cells of the species examined are also noted. No indications of conversion of mitochondria into lipid, of accumulation of lipid in the Golgi area, or of the protrusion of lutein cells into spaces between the endothelial cells, as suggested by other authors, were noted in these preparations. Some of the difficulties inherent in the visualization of the secretory activity of cells producing steroid hormones are briefly discussed.     

The ultrastructure of the corpus luteum of the guinea-pig

Summary An electron-microscopic investigation, based on the suggestion that differences seen in progesterone levels under differing hormonal conditions might be reflected in the ultrastructural organisation of the lutein cells of the guinea-pig was undertaken. Comparisons were made between corpora lutea taken from animals during the normal oestrous cycle, pregnancy and lactation, and after hysterectomy or hypophysectomy.The lutein cells from the oestrous cycle corpus luteum appeared to be of two types, light and dark. The former were more numerous. The main difference between them lay in the arrangement of the endoplasmic reticulum. Lutein cells from corpora lutea (with the exception of the old degenerating corpora lutea) all contained well-developed agranular endoplasmic reticulum, little granular endoplasmic reticulum, several electron-dense lipid granules, lysosomal bodies which ranged from small spherical bodies to large autophagic vesicles and mitochondria. The mitochondria were numerous, and in the corpus luteum of pregnancy, they were closely associated with the parallel arrays of granular endoplasmic reticulum.With minor exceptions, the lutein cells of the guinea-pig present a strikingly uniform picture despite their hormonal condition.The manner in which this uniformity of ultrastructure may be related to observed differences in progesterone levels in the corpus luteum of the guinea-pig is discussed.Meat and Livestock Commission (MLC) Scholar.The authors wish to thank Dr. J. S. Perry for doing the surgery involved in this work and for the specimens of corpora lutea of hysterectomy. They are also grateful to him for his helpful discussions and interest throughout.     

Structural changes in the granulosa lutein cells of pregnant cows between 60 and 245 days.

The structural changes in the granulosa lutein cells in the pregnant cows between 60 and 245 days, were observed. The polyhedral or pump cells has an acidophilic cytoplasm around the spherical nucleus. The glycogen granules increased in number up to 230 days of pregnancy. The lipid globules increased in size and amount during advanced pregnancy. The mitochondria showed a wide range of variations in shape and size. The matrix of many mitochondria contained dense inclusions which replaced the entire matrix in late pregnancy. The endoplasmic reticulum was chiefly of smooth type but a few stacks of the rough variety could be seen in the early days of pregnancy. Sometimes, whorls of smooth-surfaced endoplasmic reticulum enclosing mitochondria were seen. The Golgi complex was fully developed in the granulosa cells of all cases studied. The dense granules and lipid globules increased in size and number in the older corpora lutea. The cell surface showed numerous infolding of the plasma membrane.     

OBSERVATIONS ON THE FINE STRUCTURE OF LUTEIN CELLS : II. The Effects of Hypophysectomy and Mammotrophic Hormone in the Rat

Corpus luteum formation was induced in 26-day-old rats which were subsequently hypophysectomized and injected with mammotrophic hormone (MH, LTH). Sections of corpora lutea from these animals were examined with the electron microscope and compared with similarly prepared (Caulfield's fixed, Araldite embedded) corpora from normal pregnancy and from controls, the latter consisting of corpora prior to hypophysectomy and corpora from uninjected rats 7 to 14 days after hypophysectomy. Lutein cells from corpora lutea of injected animals and of normal pregnancy are characterized by abundant, tortuous, tubular agranular endoplasmic reticulum and by mitochondria, many of which are disc-shaped with dense matrices and both villiform and lamelliform cristae. The endoplasmic reticulum is most abundant in lutein cells from pregnant animals, in which cells it is in the form of thin, highly tortuous tubules. The form of the lipid droplets seen in cells of stimulated animals varies greatly. Marginal foldings of the lutein cells on the perivascular space were found in all instances. Lutein cells from hypophysectomized animals have a less highly developed agranular endoplasmic reticulum. The mitochondria have irregular outlines and a relatively lucid matrix. The lipid droplets in these cells show less tendency to be extracted, but are not so large or abundant as in the cells of onset controls. Granules believed to contain lipid pigments are common in the lutein cells of these control animals. It is suggested that lutein cells from corpora lutea which are actively secreting progesterone may be readily distinguished from lutein cells from non-active corpora by means of the multiple characteristics enumerated. It is further suggested that mammotrophic hormone has a general effect on the metabolism of lutein cells rather than solely affecting a specific organelle, the abundance or composition of which may be the limiting factor in the production of progesterone.     

Androgen-induced changes in rat ovarian granulosa cells in vitro

Ovarian granulosa cells collected from small antral follicles from immature rats were cultured in McCoy's 5A medium, for 1-6 days in the presence of delta 4-androstenedione, testosterone, dihydrotestosterone, and dehydroepiandrosterone (10(-5) M and 10(-7) M). Granulosa cells examined by electron microscopy demonstrated many lipid droplets, mitochondria with tubular cristae and profiles of smooth endoplasmic reticulum, all suggestive of active metabolism in the cell. Cells cultured in androstenedione, testosterone, dihydrotestosterone and dehydroepiandrosterone produced estrogen and progesterone as measured by radioimmunoassay. By day 4, cells cultured in androgen had almost completely degenerated. The control cells acquired none of the aforementioned characteristics and survived up to beyond 6 days, at which time the experiments were terminated. This study supports the hypothesis that high concentrations of androgens in cultured granulosa cells contribute to their degeneration through altered structure, which is associated with functional change.     

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

The 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 mu2 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.