Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Morphological studies of polycystic mouse ovaries induced by dehydroepiandrosterone

Summary 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- and 3-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<P<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<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>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.The following trivial names have been used: Dihydrotestosterone: 5-androstan 17 ol-13 one; 3-androstanediol: 5-androstan 3,17 diol; 3-androstanediol: 5-androstan 3,17 diol     

Localization of apoptotic cells in the cystic ovarian follicles of cows: a DNA-end labeling histochemical study

We examined the frequency of apoptosis in cystic follicular cells to investigate the cause of the delay in regression of cystic follicles. Paraffin sections of healthy antral follicles, early and late atretic ones, and early and late cystic ones were stained using the terminal deoxynucleotidyl transferase (Tdt)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labeling (TUNEL) method to detect apoptotic cells. In the granulosa layer of early cystic and atretic follicles, TUNEL-positive cells were evident. In the theca interna of both early and late atresia, high frequencies of TUNEL-positive cells were observed. In the theca interna, a high frequency of TUNEL-positive cells was noted in the early cystic follicles, whereas their frequency decreased in late cystic follicles. These results suggest that apoptosis occurs in the granulosa and theca interna cells of cystic as well as atretic follicles, but the frequency of apoptosis in theca interna cells decreases in late cystic follicles, which may be responsible for the delay of follicular regression.     

Comparison of glucose-6-phosphate dehydrogenase activity in healthy and atretic follicles in rat ovary

Changes in the glucose-6-phosphate dehydrogenase activity have been determined in relation to atresia of Graafian follicles in the rat ovary. Induction of atresia in follicles either due to absence of hCG in the hormonally stimulated immature ovaries or by repeated injections of pentobarbitone sodium to proestrous rats caused significant rise in the enzyme activity. Measurement of enzyme activity in isolated follicular compartments of healthy and atretic follicles revealed that it is significantly higher in the thecal tissue than the granulosa. Increase in enzyme activity in the atretic follicles than the healthy ones occurs due to its rise both in theca and granulosa cells. The significance of these changes in the enzyme activity in healthy and atretic follicles are discussed in relation to the precocious luteinization of cells in the follicular envelope with the onset of atresia.     

Das histochemische Verhalten der Azolesterasen sowie weiterer Oxydoreduktasen bei der Follikelatresie im Ovarium einiger Nager

Zusammenfassung Histochemische Untersuchungen am Ratten-, Mäuse- und Meerschweinchenovarium ergaben ein unterschiedliches Verhalten der Azolesterasen (= Cholinesterase und Acetylcholinesterase) in Granulosa, Theca und Thecaorgan. Die Differenzierung der genannten Fermente wurde mit Hilfe von DFP, iso-OMPA und 284 C 51 durchgeführt. Die -Hydroxybuttersäuredehydrogenase gleicht in der Verteilung weitgehend der NADH-Cytochrom-c-Reduktase, zeigt aber quantitative Speziesunterschiede. Bei der -Ketoglutaratoxydase ist im Ratten- und Mäuseovarium erst beim atresierenden Follikel eine deutliche Aktivität der Granulosa und eine Zunahme in der Theca externa feststellbar, während im Meerschweinchenovarium die Theca interna immer, die Granulosa aber nur beim Abbau reagiert.

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The histochemical behaviour of the azolesterases and other oxydoreductases during atresia of follicles in the ovaries of some rodents

Summary Histochemical investigations on the ovaries of rats, mice and guinea pigs revealed a variable behaviour of the azolesterases (= cholinesterase and acetylcholinesterase) in granulosa, theca and theca organ. The differentiation of these enzymes was carried out with DFP, iso-OMPA and 284 C 51. The distribution of -hydroxybutyrate dehydrogenase closely parallels that of NADH-cytochrome-c-reductase but shows quantitative variations in the different species. In the ovaries of rats and mice -ketoglutarate oxidase shows a marked activity only in atretic follicles and an increased activity in the theca externa. In guinea pig ovaries, however, the theca interna always reacts, but the granulosa only does so in process of decomposing.

     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Structural changes occurring during atresia in sheep ovarian follicles

Summary The structural changes that characterize primary, secondary and tertiary atresia in sheep Graafian follicles have been studied by means of histological, histochemical and ultrastructural techniques.In primary atresia vacuoles representing swollen endoplasmic reticulum are prominent along the antral border together with disorganized granulosa cells containing pyknotic nuclei. Phagocytic cells, which increase in number as atresia progresses, were seen within the membrana granulosa and are considered to be transformed granulosa cells. Even in follicles classified as nonatretic, a few antral vacuoles and occasional pyknotic nuclei are present.During secondary atresia there is a large increase in the number of cells with pyknotic nuclei; many of these nuclei had been extruded and had fused to form the characteristic Feulgen-positive atretic bodies found along the edge of the antral cavity. These bodies usually have a diameter of up to 15 m but occasionally reached as much as 400 m. A second area of degeneration is frequently present in the membrana granulosa, two or three cell layers from the basal lamina, and it is at this level that exfoliation of granulosa cells occurs in tertiary atresia. In contrast to the membrana granulosa, there are during secondary atresia, only slight indications of degeneration in the cumulus.In tertiary atresia the membrana granulosa is highly disorganized; the atretic bodies are often fewer in number than at earlier stages. The basal lamina remains essentially intact. It is at this stage that the first clear signs of degeneration occur in the theca interna. Despite some disintegration of the cumulus, the integrity of the oocyte is maintained and its nucleus remains vesicular.Changes in the thecal microcirculation may play a key role in atresia: adjacent to the basal lamina of non-atretic follicles, there is a well-developed capillary network which is significantly reduced as atresia progresses.The authors are greatly indebted to Dr. H.M. Dott and Mr. G.C. Foster for carrying out the analysis with the Quantimet image analysing computer. The skilled technical assistance of Mrs. Linda Collins is also gratefully acknowledged     

Follicle growth in the ovary of the rabbit after ovulation-inducing application of human chorionic gonadotropin

Summary The growth of tertiary follicles, i.e., the proliferation of cells in the stratum granulosum and in the capillary network of the theca interna, after injection of ovulation-inducing human chorionic gonadotropin (HCG), was investigated in the rabbit by means of autoradiographic and morphometric methods.Based on the frequency distribution of follicles with different sizes and on the labeling index (LI) of granulosa cells as a function of follicle size and of time prior to and after HCG stimulation, two groups of tertiary tollicles can be distinguished: growing (250–900 m in diameter) and mature (>900 m in diameter) elements. The growth of both groups is influenced by the release of gonadotropins.After HCG stimulation, follicles belonging to the first group grow rapidly. During, and a short time after ovulation, almost all non-ruptured follicles larger than 600 m in diameter become atretic. Within 35–50 h the ruptured and atretic mature follicles (>900 m in diameter) are replaced by follicles out of the group of growing follicles.From these results the following concept for regulation of follicle growth is derived: In principle, all growing follicles possess the potential to develop into mature follicles. When a sufficient number of mature follicles is generated, these mature follicles determine the number of succeeding growing follicles. Follicles that are not required for providing mature follicles become atretic as soon as they reach a diameter of 700 m. When the majority of mature follicles is lost during ovulation (by rupture or atresia), this inhibition regulated by mature follicles is abolished, and all of the growing follicles again are capable to develop into mature follicles.The relative amount of capillaries in the theca interna of growing and mature follicles remains constant with increasing follicle size. This means that the capillary network grows parallel to the increasing size of follicles. No differences are found between intact and atretic follicles; advanced atretic follicles were excluded from this study.The labeling index (LI) of granulosa cells in the stratum granulosum and of endothelial cells in the theca interna, as a function of follicle size and of time after HCG stimulation, are closely correlated. A change in the LI of granulosa cells is usually followed with a certain delay by a similar alteration of the LI of endothelial cells in the theca interna. This suggests that granulosa cells have a certain regulatory function on capillary growth.     

Effect of melengestrol acetate on ovarian follicles,interstitial gland and corpora lutea in the rat: a quantitative morphological study

Summary Mature virgin rats were injected daily with 50 g melengestrol acetate (17-acetoxy-6-methyl-16-methyIenepregna-4,6-diene-3,20-dione, or MGA) and 0.2 g estradiol benzoate for periods of 1, 2, 3, and 4 weeks. Ovarian structures were examined histologically, histochemically and in the electron microscope. Graafian follicles during treatment did not attain pre-ovulatory size, ovulation was inhibited, and corpora hemorrhagica were not formed. During treatment, the percentage volume of the ovary occupied by atretic Graafian follicles increased, and that occupied by developing Graafian follicles decreased. In atresia, the membrana granulosa degenerated, and the theca cells hypertrophied peripherally to contribute to the surrounding interstitial gland. The structure and the weak lipid reaction of the hypertrophied theca cells of test follicles, when compared with estrus control follicles, suggested a low level of steroid production. The strong lipid reaction and ultrastructure of the interstitial gland suggested a lipid storage rather than secretory function. Corpora lutea, containing cells with a light lipid reaction and organelles suggestive of steroid synthesis, persisted throughout treatment. On the basis of claims, which associate the theca cells with estrogen synthesis and the luteal cells with progesterone synthesis, the morphological features described are consistent with primarily progestational and low-level estrogenic activities of MGA in the rat.This investigation was supported by the Alexander von Humboldt-Stiftung. The author is on leave from the Department of Veterinary Anatomy, University of Minnesota, St. Paul, U.S.A. The melengestrol acetate was supplied by the Upjohn Company, Kalamazoo, U.S.A.     

Steroidogenesis in porcine atretic follicles: loss of aromatase activity in isolated granulosa and theca

To evaluate the mechanisms involved in the reduction of estrogen concentrations in porcine follicular fluid during atresia, nonatretic and atretic follicles ranging from 4 to 7 mm in diameter were selected. Follicular fluid estrogen concentrations were 7-16-fold less in the atretic follicles. Isolated granulosa cells from atretic follicles demonstrated a significant reduction in aromatase activity and in follicle-stimulating hormone (FSH)-induced progesterone production in vitro compared to granulosa cells from nonatretic follicles. Isolated theca from atretic follicles also demonstrated a reduction in estrogen production. However, androgen concentrations were equivalent in the follicular fluid of atretic and nonatretic follicles, and theca from atretic follicles maintained testosterone and androstenedione production in vitro. The loss of thecal aromatase activity with atresia is not secondary to a reduction in FSH responsiveness, since FSH did not increase thecal progesterone production in vitro. Cell degeneration also does not account for the reduction in thecal estrogen production, since both androgen output in vitro and follicular fluid androgen concentrations were maintained. These data thus demonstrate that a mechanism other than reduced FSH responsiveness must account for the selective loss of thecal aromatase activity in this stage of atresia.     

Histochemical demonstration of Δ5-3 ß-OHD activity in the granulosa cells of ovarian follicles of immature and mature mice correlated with some ultrastructural observations

Summary A systematic study of ⁵-3-hydroxysteroid dehydrogenase in the granulosa cells of immature and mature mice was made. The histochemical results were compared with the ultrastructural findings on the same cells in an attempt to determine whether the granulosa cells are capable of a steroidogenic role.In newborn and immature mice the granulosa cells of a great amount of follicles demonstrated a moderate or strong histochemical activity. In mature mice the granulosa cells demonstrated a weak or moderate activity normally only in preovulatory follicles and in some other atretic follicles. The granulosa cells of the normal developing follicles did not show such activity. In addition the histological control of numerous parallel sections demonstrated particularly in immature ovaries the presence of a great amount of atretic follicles.In the cytoplasm of the granulosa cells of the follicles in immature ovaries only clusters of lipid droplets with ribosomes were noted; while in the preovulatory follicles of mature animals there started to appear mitochondria with tubular cristae, smooth membranes of the endoplasmic reticulum and irregular lipid droplets. In the obviously atretic follicles several granulosa cells as well as theca interna cells showed numerous lipid droplets and ribosomes together with different degenerating organelles. The granulosa cells of the normal developing follicles showed a well developed Golgi complex, granular endoplasmic reticulum and free ribosomes.The histochemical and ultrastructural findings suggest a steroidogenic role of the granulosa cells only in the larger preovulatory follicles (probably related to an early luteinization of this layer) but this role was not demonstrated in the same cells in normal developing follicles.In addition, since an histochemical positivity was demonstrated also in the granulosa cells of some obviously atretic follicles, it is possible that many of the follicles having granulosa cells filled with lipid droplets and attached ribosomes and histochemically positive might be, in the immature ovaries, in a very precocious stage of atresia.It is to precise for these cells whether a cytoplasm with these two strictly correlated components (lipid droplets and attached ribosomes) and showing an histochemical positivity could carry-on all the biochemical steps involved in steroid biosynthesis or only has only a temporary capability to produce some precursors of steroids.The present results were partially presented to the 56^ème Congrès de l'Association des Anatomistes (Nantes, 4–8/4/1971) and to the 66° Verhandlungen der Anatomischen Gesellschaft (Zagreb, 2/4/1971).     

Biochemical assessment of limits to estrogen synthesis in porcine follicles

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17 alpha-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.     

Immunocytochemical study of cell proliferation in the cystic ovarian follicles in cows

We examined the frequency of proliferating cells in cystic, atretic and healthy antral follicles to determine whether a disorder of cell proliferation was responsible for the occurrence of bovine cystic follicles. Paraffin sections of healthy follicles and various stages of atretic and cystic follicles were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). The PCNA-positive cells were counted in 4 different regions of a follicle from the apical to the basal side. In the granulosa layer, a significantly higher frequency of PCNA-positive cells was observed in the healthy follicle in the basal region as compared with the apical region. A similar pattern of PCNA-positive cells population was observed in the granulosa layer of atretic follicles, although the frequency in the basal region was significantly lower in the atretic than the healthy follicle. The rate of cell proliferation in the granulosa layer of cystic follicles was markedly lower at the basal region than that of atretic follicles. In the theca interna, the frequency of PCNA-positive cells in atretic follicles at the early stages was higher than that in cystic follicles at the early stages. These results suggest that in the healthy follicle the proliferative activity of granulosa cells is higher in the basal than the apical region, and that the cell proliferation activity in the granulosa and theca interna may decrease in association with the induction of a follicular cyst.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Immunocytochemical and biochemical studies on the localization and changes of 17 α-hydroxylase/C17–C20 lyase activity in immature rat ovary treated with PMSG and hCG

Summary The localization of cytochrome P-450 of 17 -hydroxylase/C17–C20 lyase (P-450_{17 , lyase}) and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-450_{17 , lyase} was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-450_{17 , lyase} was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-450_{17 , lyase} again. The 17 -hydroxylating activity of P-450_{17 , lyase} was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG+hCG(3 h)-treated and PMSG+hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes. These immunocytochemical and biochemical findings suggest that 1) P-450_{17 , lyase} is localized in both the theca interna cell and interstitial gland cell, and these cells are the main site of the androstenedione production in the ovary, and that 2) the decreased production of estrogen occurring just before ovulation is not brought about by the decreased activity of P-450_{17 , lyase}, but done by the loss of the enzyme.Supported by grants from the Ministry of Education, Science and Culture, Japan     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Steroid synthesizing cellular sites in the ovaries of Calotes versicolor (Daud.), Hemidactylus flaviviridis (Ruppel) & Chamaeleon calcaratus (Boulenger): histochemical study.

A histochemical study of steroid synthesizing cellular sites in the ovaries of Calotes versicolor (Daud.), Hemidactylus flavivirdes (Ruppel) and Chamaeleon calcaratus (Boulenger) is discussed. THe distribution of delta 5-3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 11beta-hydroxysteroid dehydrogenase, glucose-6phosphate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase and reduced nicotinamide-adenine dinucleotide diaphorase enzyme activities was studied in ovaries of the 3 species of lizards. All the enzyme activities occurred in 1) patches of cells of theca interna; 2) granulosa cells of large preovulatory, postovulatory, and atretic follicles; 3) interstitial cells of the ovarian stroma; and in the 4) ooplasm of the growing oocyte, suggesting their steroidogenic capacity. It was observed that following completion of follicular atresia, the phagocytic granulosa cells degenerate and the remaining cells of theca interna contribute to the formation of interstitial gland cells.     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.