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Angiotensin II (AII) is present in the mammalian ovary and has been correlated with atresia in follicles. Since the theca interna may be one site at which atresia is initiated, we wished to determine whether AII exerts an effect on theca interna from explanted ovarian follicles of hamsters. Hamsters were sacrified on the morning of proestrus, and ovaries were removed. Preovulatory follicles were excised from the ovaries, and cultured with one of the following components: medium alone (control); medium plus AII (1 x 10(-6) M); the AII-receptor antagonist [Sar1, Ile8] AII (1 x 10(-4) M); or AII plus antagonist. After 72 h, the follicles were processed for transmission electron microscopy (to determine quantities of theca interna organelles involved in the steroid synthetic pathway) or for protein determination (to normalize steroid production rates). The incubation medium was drawn off and analyzed by radioimmunoassay for progesterone, androstenedione, or estradiol-17 beta. There was a significant positive correlation (r = 0.92, P less than 0.01) between follicular androstenedione secretion and area comprising theca interna smooth endoplasmic reticulum. In the theca interna, AII induced a two-fold and 1.6-fold increase in lipid droplet number and area comprising smooth endoplasmic reticulum, respectively (P less than 0.05). Excess antagonist negated the increase in cell organelles and also reduced androstenedione secretion compared with AII alone (P less than 0.05). Most importantly, AII significantly augmented the ratio of androstenedione:estradiol-17 beta secretion by 44% over that of control. The ultrastructural changes observed in this study and the increase in the androstenedione:estradiol-17 beta production ratio are consistent with atresia-like changes in ovarian follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.

No structural changes could be observed in the stigma until 2 h before ovulation. Within a few minutes of ovulation, the stratum granulosum and theca interna disappear completely in the middle part of the stigma. By 30 min before ovulation, the rough endoplasmic reticulum in the fibroblasts of the theca externa develops conspicuously. Then a few minutes before ovulation, the theca externa becomes much thinner with a conspicuous disintegration of the collagenous fibers into individual fibrils. These structural changes may contribute to the fragility of the stigma tissue and induce rupture of the follicle.

The ultrastructural and biosynthetic characteristics of the steroid cells in the gonad of Monopterus albus have been studied. Ultrastructural features related to steroidogenesis have been identified in the interstitial Leydig cells, Sertoli cells, granulosa cells and thecal cells, and are especially abundant in the Leydig cells during the mid-intersexual phase. Steroidogenic ultrastructures in the Sertoli cells develop only during the maturation of the spermatogenic cysts, whereas in the granulosa and thecal cells, these features become obvious only during the maturation of the large oocytes. EM evidence also suggests a nutritive function for the Sertoli cells and the granulosa cells. Results of in vitro steroidogenic studies, using either testosterone or progesterone as a precursor, show a predominant conversion to androstenedione and 5 alpha-reduced compounds, and suggest a change in biosynthesis from 5 alpha-reduced products to androstenedione during sex reversal. 11-Ketotestosterone (11KT) has been identified, but not 11 beta-hydroxytestosterone. Production of 11KT is high in the late intersexual and the male phases, but a lack of a marked variation in 11KT production between the early and the mid-intersexual phase suggests that this steroid is not a trigger for natural sex reversal in Monopterus.

The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy. In follicles less than 3.0 mm in diameter, the theca interna consisted of about 8-12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure. In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages. It was concluded that: (1) the theca interna of 2.0-2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca interna to the membrana granulosa.

Plasma estradiol-17 beta 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 delta 5, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3 beta-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3 beta-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.

The autonomic nerve supply of the guinea-pig ovary was investigated by a combination of light- and electron microscopy. At the light-microscopic level, adrenergic fibres were identified due to their formaldehyde-induced fluorescence. In addition, the ovary contained acetylcholinesterase-positive fibres. In all parts of the ovary, the adrenergic fibres were most numerous. At the ultrastructural level it was possible to identify the adrenergic nerve terminals with the aid of the false adrenergic transmitter, 5-hydroxy-dopamine. Thus, large numbers of adrenergic terminals, characterized by their content of 50-60 nm, electron-dense synaptic vesicles, were seen within the interstitial gland, where they formed close contacts with the endocrine cells (membrane-to-membrane distance, 20-100 nm). The follicular theca externa was also richly supplied by adrenergic nerves. At this location, close contacts (50-100 nm) were identified between the nerve terminals and the smooth muscle-like cells. Very few adrenergic nerve fibres were present in the theca interna of follicles or in the corpus luteum. Non-adrenergic nerve terminals, characterized by electron-lucent synaptic vesicles of 50-60 nm diameter, were observed together with the adrenergic fibres. They were always present in much lower numbers than the latter. No "p-type" nerves were identified by electron microscopy.

Low concentrations of cytochalasin B (CCB) are known to inhibit ovulation in the frog, Hyla regilla. Examination of amphibian thecal cell ultrastructure reveals filaments (average diameter 71 A) arranged in bundles parallel to the surface of the oocyte. These filaments are often associated with hemidesmosome-like plaques on the basal plasmalemma, While individual filaments appear unaltered morphologically by CCB (1-5 microgram/ml), their organization into bundles, apparent relationship to the hemidesmosomes, and the highly contorted configuration of the thecal cells after oocyte expulsion, suggest that a nonmuscular contractile system residing within the follicle plays a fundamental role in ovulation. Our data suggest that the flattened epitheloid thecal cells shorten all axes that run parallel to the oocyte surface via filament bundle contractions, while they remain tightly bound together by macular attachment plaques. These cells thus increase in height to become cuboidal-low columnar in shape; the area covered by the base of each is greatly reduced. As this "thecal sac" decreases in size, the compression generated by the contractile mechanism forces the oocyte through the enzymatically weakened apex of the follicle and ovulation results.

Morphological alterations induced by dehydroepiandrosterone (DHA) were studied in polycystic mouse ovaries (PCO). Treated mice showed ovulatory failure and cystic changes; cysts and follicles in various stages of growth and atresia were present although corpora lutea were absent. The levels of testosterone, dihydrotestosterone, 3 alpha- and 3 beta-androstanediol, estrone and androstenedione increased, whereas estradiol was not detectable. The ultrastructure of granulosa cells in healthy and atretic follicles was similar to that of control animals, although the membrana granulosa in cysts was reduced to a monolayer of flattened cells. The theca interna of healthy and atretic follicles and ovarian cysts showed ultrastructural signs of abnormal steroidogenic stimulation. No significant differences (0.7 less than P less than 0.8) were found between the extensive surface area of gap junctions of healthy follicles of control and DHA-treated animals. On the P-face of granulosa cells of large healthy follicles, meandering strands of tight junctional particles were observed; their average length was significantly longer than those in healthy follicles of control animals (P less than 0.001). This increase was probably related to the large amounts of androgens present in the treated animals. Theca interna cells possessed small gap junctions; no significant differences (P greater than 0.9) in gap-junction surface area were observed between DHA-treated and control animals. These results suggest that the size of gap junctions is probably unrelated to the steroidogenic activities of theca cells.

Two types of filaments (microfilaments 4--6 nm in diameter, and intermediate filaments 7--10 nm in diameter) are common in the surface epithelial cells and theca fibroblasts of vitellogenic ovarian follicles of the lizard Anolis carolinensis. Heavy meromyosin (HMM), which forms complexes with actin filaments, interacts only with the microfilaments of theca fibroblasts. After myosin extraction of follicles no filaments disappeared, but when this treatment was followed by incubation with deoxyribonuclease I (DNA-ase I), which depolymerizes F-actin to G-actin, microfilaments disappeared from the theca fibroblasts. It is concluded that microfilaments in theca fibroblasts are actin-like and may contract to provide the mechanism of expulsion for the oocyte during ovulation. The intermediate filaments of the surface epithelial cells and theca fibroblasts may serve as a skeletal system for the large (up to 8 mm in diameter) vitellogenic follicle.

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.

Intercellular junctions in the young mouse ovary were examined by electron microscopy utilizing freeze-fracture and thin-sectioning techniques. Projections from the granulosa cells adjacent to the oocyte (GI) traverse the zona pellucida and form small gap junctions on the oocyte surface. On the P-face of these cells, the junctional aggregations are occasionally associated with linear strands of particles. In contrast, large gap junctional areas are frequently observed between the more peripherally located granulosa cells (GE) and are also present in the theca interna (TI) cell layer surrounding the follicles. Three types of tight junctional strands are discernible on the P-face of theca externa cells (TE): angularly zigzag strands consisting of intermittently distributed intramembranous particles on wide ridges, intermediate zigzag strands consisting of more continuously distributed particles, and wavy strands consisting of rather fused particles. Tight junctional strands are also present in the middle of grooves on the E-face of endothelial cells of blood vessels. In the germinal epithelial cell layer, tight junctional strands appear to be discrete and form a less anastomosing network.