Granulosa Cells Of Mature Follicles Research Articles

Dynamic changes in mitochondrial 3D structure during folliculogenesis and luteal formation in the goat large luteal cell lineage

In mammalian ovaries, mitochondria are integral sites of energy production and steroidogenesis. While shifts in cellular activities and steroidogenesis are well characterized during the differentiation of large luteal cells in folliculogenesis and luteal formation, mitochondrial dynamics during this process have not been previously evaluated. In this study, we collected ovaries containing primordial follicles, mature follicles, corpus hemorrhagicum, or corpus luteum from goats at specific times in the estrous cycle. Enzyme histochemistry, ultrastructural observations, and 3D structural analysis of serial sections of mitochondria revealed that branched mitochondrial networks were predominant in follicles, while spherical and tubular mitochondria were typical in large luteal cells. Furthermore, the average mitochondrial diameter and volume increased from folliculogenesis to luteal formation. In primordial follicles, the signals of cytochrome c oxidase and ATP synthase were undetectable in most cells, and the large luteal cells from the corpus hemorrhagicum also showed low enzyme signals and content when compared with granulosa cells in mature follicles or large luteal cells from the corpus luteum. Our findings suggest that the mitochondrial enlargement could be an event during folliculogenesis and luteal formation, while the modulation of mitochondrial morphology and respiratory enzyme expressions may be related to tissue remodeling during luteal formation.

Endogenous and Synthetic Regulators of the Peripheral Components of the Hypothalamo-Hypophyseal-Gonadal and -Thyroid Axes

Activity in the peripheral components of the hypothalamo-hypophyseal-gonadal and -thyroid axes is regulated by hypophyseal hormones – gonadotropins and thyrotropic hormones (thyroid-stimulating hormone, TSH), which are secreted by specialized cells in the adenohypophysis. Luteinizing hormone (LH) and its homolog chorionic gonadotropin (CG) realize their steroidal effects by binding to LH/CG receptors on the surfaces of Leydig cells in the testes and theca cells and granulosa cells in mature follicles in the ovaries. Follicle-stimulating hormone (FSH) binds FSH receptors on Sertoli cells in the testes and granulosa cells in primordial and maturing follicles in the ovaries, controlling the processes of folliculogenesis, spermatogenesis, and steroidogenesis. TSH, via activation of TSH receptors, stimulates the synthesis of thyroid hormones by thyrocytes in the thyroid gland. Gonadotropins (LH, CG, and FSH) and TSH, which bind with high affinity to the extracellular domains of specific G protein-coupled receptors, directly activate various signal cascades operating via different types of G-proteins and β-arrestins. Recombinant gonadotropins and gonadotropins extracted from natural sources and used for the treatment of reproductive dysfunction and as assisted reproduction technologies have a number of drawbacks, which has led to the development of peptide and low molecular weight regulators of LH/CG and FSH receptors which interact with allosteric sites on the transmembrane or cytoplasmic domains of the receptors. Wide perspectives in the regulation of reproductive functions and the control of fertility are opened up by the use of adipokines, peptides of the insulin and relaxin families, and the antidiabetic drug metformin, which not only regulate and modify the responses of the gonads to gonadotropins, but also themselves influence steroidogenesis and gamete maturation. In the case of TSH receptors, the most acute problem is that of reversing increases in their activity in autoimmune and oncological diseases of the thyroid gland and in endocrine ophthalmopathy. The greatest potential in this direction lies in the ongoing development of low molecular weight inverse agonists and neutral antagonists, which interact with an allosteric site located in the transmembrane domain of the TSH receptor. The present review addresses contemporary advances in the development and study of endogenous and synthetic regulators and modulators of gonadotrophic and TSH receptors, along with their influences on the peripheral components of the hypothalamo-hypophyseal-gonadal and -thyroid axes.

Graft-Versus-Host Disease Targets Ovary and Causes Infertility After Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for various hematologic malignancies. Infertility associated with ovarian failure is a serious late complication for female survivors of allo-HSCT. While the role of pretransplant conditioning regimen has been well appreciated, it remains to be elucidated whether GVHD could be causally related to female infertility. We have addressed this issue in a mouse model of non-irradiated bone marrow transplantation (BMT) to avoid devastating effects of irradiation on the ovary. Method Female B6D2F1 (H-2b/d) mice (10 week old) were injected with 80 × 106 splenocytes from allogeneic B6 (H-2b) or syngeneic B6D2F1 donors on day 0. To track migration of donor T cells, CAG-EGFP B6 mice were utilized. Morphometric and immunohistochemical assessments were performed on ovarian sections harvested from recipients on day +21 to evaluate the integrity of ovarian architecture and inflammatory changes. To examine ovary functions, recipients were injected with pregnant mare’s serum gonadotropin (PMSG) on day +19 and human chorionic gonadotropin (hCG) on day +21 to induce ovulation and then oocytes were collected. To evaluate fertility of the recipients, recipients were mated with B6D2F1 males after BMT and mating was repeated 6 times until day +150, and newborns were enumerated. Result In this model, GVHD occurred early after BMT at a peak around day +21 particularly in the gut and liver. Histological examination of the ovaries from allogeneic recipients on day +21 demonstrated GFP+ donor T cells infiltrating in the layer of granulosa cells of mature follicles beyond the basement membrane, cleaved-caspase 3+ apoptotic granulose cells surrounded by lymphocytes (satellitosis), and the destruction of PAS+ basal membrane, whereas there was neither donor T cell infiltration nor pathological changes of the ovaries in syngeneic animals (p = 0.002, Table). Majority of T cells infiltrating in the ovary was CD8+ T cells. In experiments of forced ovulation, numbers of oocytes were significantly less in allogeneic animals than in syngeneic animals (p = 0.043, Table), indicating an impairment of ovary functions. When allogeneic recipients were mated to healthy male mice, numbers of both delivery and newborns per litter decreased, whereas syngeneic recipients remain to be fully fertile. As a result, total numbers of newborns delivered by day +150 after BMT were significantly less in allogeneic animals than in syngeneic animals (p< 0.001, Table). Administration of 10 mg/kg of prednisolone from day 0 to day +20 significantly reduced ovarian GVHD and restored numbers of oocytes in allogeneic animals to the levels of syngeneic animals. Conclusion Our results demonstrate for the first time that GVHD targets ovary and induces impairment of ovary functions and infertility; and therefore, control of GVHD as well as protection of the ovary from the conditioning is important to prevent female fertility after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Expression of GDF-9, BMP-15 and their receptors in mammalian ovary follicles

The synergetic process of folliculogenesis is mainly regulated by GDF-9 and BMP-15 as well as their receptors, such as BMPR2, TβR1 and BMPR1B. Expressions of these factors and the receptors are significant different among species. This study was designed to detect expression of GDF-9, BMP-15 and their receptors in mouse, porcine and human healthy follicles by immunohistochemistry. Three ages of human ovary were studied according to ovarian developmental schedule, i.e. gestational week (GW) 16, puberty (14 year-old) and adult (40 year-old). The results showed that both GDF-9 and BMP-15 were detectable in oocytes from primary follicles onward, besides, BMP-15 also presented in granulosa cells (GCs) and follicular follicle of mature follicles in mouse. However, they were maintained in oocytes and GCs from primordial to mature follicles in porcine except that GDF-9 was undetectable in GCs of mature follicles. For human ovary, GDF-9 presented in oocytes of primordial follicles in all samples, whereas BMP-15 was only observed in primordial follicle of adult ovary. Receptors, BMPR2, TβR1 and BMPR1B were found in oocytes and GCs of all follicles in mouse and porcine. In human, they were stained in oocytes from primordial follices but BMPR1B was not expressed in pubertal primordial follicles. Furthermore, we found that GDF-9, BMP-15 and three receptors distributed in adult corpus lutea. Collectively, our studies suggested that GDF-9, BMP-15 and their receptors might correlate with primordial follicular recruitment in pig and human. Positive expression of the receptors (BMPR2, TβR1 and BMPR1B)in primordial follicles of mouse ovaries indicated that these receptors might interact with others ligands besides GDF-9 and BMP-15 to regulate primordial follicular activity in mouse. Moreover, presence of GDF-9 in oocytes and BMP-15 in oocytes and GCs of mature follicles from mice and porcine elucidated coordinated roles of GDF-9 and BMP-15 in cumulus oophorus expansion. Additionally, expression of these factors in adult human corpus lutea suggested they play roles in corpus luteum activity.

Immunohistochemical Demonstration of the Mineralocorticoid Receptor, 11β-Hydroxysteroid Dehydrogenase-1 and −2, and Hexose-6-phosphate Dehydrogenase in Rat Ovary

An IHC survey using several monoclonal antibodies against different portions of the rat mineralocorticoid receptor (MR) molecule demonstrated significant specific MR immunoreactivity in the ovary, prompting further study of the localization of MR and of determinants of extrinsic MR ligand specificity, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2, and hexose-6-phosphate dehydrogenase (H6PDH). MR expression (real-time RT-PCR and Western blot) did not differ significantly in whole rat ovaries at early diestrus, late diestrus, estrus, and a few hours after ovulation. MR immunostaining was most intense in corporal lutea cells, light to moderate in oocytes and granulosa cells, and least intense in theca cells. Light immunoreactivity for 11beta-HSD2 occurred in most cells, with some mural granulosa cells of mature follicles staining more strongly. The distribution of immunoreactivity for 11beta-HSD1 and H6PDH required to generate NADPH, the cofactor required for reductase activity of 11beta-HSD1, was similar, with the most-intense staining in the cytoplasm of corporal lutea and theca cells and light or no staining in the granulosa and oocytes. MR function in the ovary is as yet unclear, but distinct patterns of distribution of 11beta-HSD1 and -2 and H6PDH suggest that the ligand for MR activation in different cells of the ovary may be differentially regulated.

Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis.

Ovarian granulosa cells are coupled via gap junctions containing connexin43 (Cx43 or alpha-1 connexin). In the absence of Cx43, granulosa cells stop growing in an early preantral stage. However, the fact that granulosa cells of mature follicles express multiple connexins complicated interpretation of this finding. The present experiments were designed to clarify the role of Cx43 vs. these other connexins in the earliest stages of folliculogenesis. Dye injection experiments revealed that granulosa cells from Cx43 knockout follicles are not coupled, and this was confirmed by ionic current injections. Furthermore, electron microscopy revealed that gap junctions are extremely rare in mutant granulosa cells. In contrast, mutant granulosa cells were able to form gap junctions with wild-type granulosa cells in a dye preloading assay. It was concluded that mutant granulosa cells contain a population of connexons, composed of an unidentified connexin, that do not normally contribute to gap junctions. Therefore, although Cx43 is not the only gap junction protein present in granulosa cells of early preantral follicles, it is the only one that makes a significant contribution to intercellular coupling.

Intercellular Communication via Connexin43 Gap Junctions Is Required for Ovarian Folliculogenesis in the Mouse

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1−/Gja1−) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1−/Gja1− ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.

The effect of periovulatory imbalance of prolactin on the expression of prolactin receptors in rat ovary cells

Using the technique of immunohistochemistry in combination with cytophotometry, we have studied the effect of periovulatory hyper- and hypoprolactinemia on the expression of prolactin receptors in various cell types of rat ovaries during early estrus. It has been shown that intense specific staining of oocytes is positively controlled by prolactin. The maximal intensity of specific staining was found in cells of the cumulus and the inner layer of granulosa cells in mature follicles; staining intensity gradually diminished towards the outer boundary cell layer. Postovulatory follicles are distinct from those mature follicles in which there was no ovulation in their more intense manifestation of prolactin receptors in cells of the inner layer and cumulus, as well as in increased positive staining (after prolactin administration) only in the granulosa layer cells closest to theca. In follicles which did not ovulate by the time of the early estrus, prolactin administration leads to a proportional growth of specific immunoreactivity in all cell layers of the granulosa. The administration of bromocryptin, an inhibitor of prolactin secretion, leading to a 10-fold decrease in the prolactin level in the blood, results in a twofold decrease in the intensity of specific staining of all cell layers of the granulosa in either type of follicle. Corpora lutea of the previous cycle have irregularly positioned luteocytes with weak and strong specific staining, the intensity of which is not changed in response to prolactin and diminishes slightly after the administration of bromocryptin. We conclude that the most intense changes in the content of prolactin receptors under the conditions of imbalance of this hormone during the periovulatory period are observed in those follicles where the oocyte did not ovulate by the time of early estrus.

Direct Ovarian Effects of a GnRH Agonist in Hypophysectomized Immature Rats: Inhibition of Theca-Interstitial Luteinizing Hormone Receptor mRNA Expression and Induction of Interstitial Cell Differentiation

The effect of a gonadotropin-releasing hormone (GnRH) agonist on luteinizing hormone (LH) receptor mRNA expression was examined histologically in the ovaries of immature hypophysectomized (HPX) rats by in situ hybridization. In the ovaries of HPX rats treated with diethylstilbestrol (DES) and pregnant mare serum gonadotropin (PMSG), LH receptor mRNA was expressed in the granulosa cells of mature follicles as well as the theca-interstitial cells. In DES-primed ovaries of rats treated with both GnRH agonist plus PMSG, many follicles were luteinized without ovulation, and the signal of LH receptor mRNA disappeared completely in the theca-interstitial cells as well as the luteinized cells, but remained in the granulosa cells of unaffected mature follicles. The complete suppression of the theca-interstitial LH receptor expression by GnRH agonist was also observed in HPX rats that received no other treatment. On the other hand, the coadministration of a GnRH antagonist with PMSG resulted in the hyperstimulation of follicular growth, accompanied by very strong expression of LH receptor mRNA in the granulosa cells as well as the thecainterstitial cells. In addition, morphological changes in the ovarian interstitial cells were also induced by the administration of GnRH agonist in HPX rats: loose connective tissue decreased and the interstitial cell mass markedly increased. The increase of the interstitial cells became more prominent when rats were treated with GnRH agonist and testosterone simultaneously. These results suggest that GnRH may be an important factor for modulating the interstitial cell function and differentiation in the rat ovary.

Gonadotropin-Releasing Hormone Receptor mRNA Expression in the Ovaries of Neonatal and Adult Rats

Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on the rat ovary by acting through its specific receptors. To determine the cell types responsive to the action of GnRH under physiological conditions in the ovary, distribution of the GnRH receptor mRNA was studied histologically by in situ hybridization in neonatal and adult rats. Expression of the luteinizing hormone receptor mRNA was also examined to judge the growing state of follicles and the corpora lutea. In neonatal rat ovaries, no significant GnRH receptor mRNA signal was detected until 5 days after birth. The expression was first observed at 10 days in the interstitial cells. At 15 days of age, the receptor mRNA was expressed in the granulosa cells of most preantral and early antral follicles, while no hybridization signal was detected in oocyte and theca cells. In adult cycling rats, GnRH receptor mRNA was detected mainly in the granulosa cells of most follicles and luteal cells. The granulosa cells of atretic follicles showed a very high level of the mRNA expression throughout their degenerating process. A strong hybridization signal was also detected in the mural granulosa cells of mature follicles. Newly formed (developing) corpora lutea exhibited signals with moderate intensity in the luteal cells, and the older ones showed weaker signals. The finding that the initial expression of GnRH receptor mRNA was seen in the interstitial cells of neonatal ovaries implies an unknown function of the ovarian GnRH receptor in ovarian development. The high level expression of GnRH receptor mRNA in atretic and mature follicles supports the putative roles of GnRH in the induction of follicular atresia and ovulation in rat ovaries.

Cyclic and maturation-dependent regulation of follicle-stimulating hormone receptor and luteinizing hormone receptor messenger ribonucleic acid expression in the porcine ovary.

We sought to characterize the ovarian expression of mRNAs for the receptors for FSH (FSHr) and LH (LHr) at the different stages of the estrous cycle of the pig and to localize the receptor mRNAs to individual cell types in follicles and corpora lutea as a function of their developmental status. Northern blot analyses indicated that multiple FSHr and LHr mRNA transcripts occur on Days 0, 4, 7, 12, and 16 of the estrous cycle. In situ hybridization analyses revealed considerable cyclic variation in expression among small, medium, and large follicles. In small follicles of both immature prepubertal ovaries and mature adult ovaries at all days of the cycle studied, FSHr mRNA expression was strongly positive and was spatially restricted to granulosa cells. FSHr mRNA expression was strongly positive in granulosa cells of medium follicles but had declined in granulosa cells of mature follicles on Day 0. LHr expression in small follicles, in contrast, was spatially restricted to theca cells and was estrous cycle-dependent. LHr expression in theca cells was weakly positive in small follicles on Days 12, 0, and 4, was strongly positive in small follicles on Day 7 and in medium follicles on Days 12 and 16, and had declined in large mature follicles on Day 0. LHr expression in granulosa cells was negative in small follicles on Days 4 and 7, weakly positive in medium follicles on Days 12 and 16, and positive in large follicles on Day 0. In degenerating follicles, LHr mRNA was expressed weakly in the theca cells, and FSHr mRNA expression in granulosa cells was absent. In the corpus luteum, FSHr mRNA was not expressed, but LHr mRNA was expressed in some cells on Days 4 and 7, was expressed maximally on Day 12, and had declined partially by Day 16. These studies indicate that there is maturation-dependent regulation of FSHr and LHr expression in the adult porcine ovary. FSHr mRNA content decreases in parallel with FSHr protein content as follicles increase in size.

Variable expression of cellular retinol- and cellular retinoic acid-binding proteins in the rat uterus and ovary during the estrous cycle.

Ovaries and uteri from normal adult female rats at known stages of the estrous cycle were analyzed for the presence of cellular retinol-binding protein (CRBP) and both types of cellular retinoic acid-binding protein (CRABP and CRABP II). Northern and Western blot analysis of the uteri revealed a peak of CRBP during diestrus and a peak of CRABP during proestrus, whereas CRABP II peaked sharply during estrus. Immunohistochemical studies showed CRABP II localized to the luminal epithelium, while both CRBP and CRABP were observed only in the smooth muscle layers of the uterus. In the ovary, CRABP was not detected, while CRBP levels remained relatively constant throughout the cycle and CRABP II peaked slightly during metestrus. CRBP in the ovary was localized to the oocytes, nearby granulosa cells, and some regions of stroma. CRABP II was found predominantly in the granulosa cells of mature follicles and early corpora lutea, as well as some regions of the stroma. These results suggest a need for further studies to assess the role of retinol and its metabolites in normal uterine function and ovarian follicular development.

Plasminogen activator activity and thymidine incorporation in avian granulosa cells during follicular development and the periovulatory period.

The hormonal and second messenger regulation of plasminogen activator (PA) activities in avian granulosa and theca cells has been documented. However, the physiological role(s) of PAs in the avian ovary remains poorly understood. The present studies were designed to evaluate PA activity in hen granulosa cells collected from the most mature (F1) preovulatory follicle at three discrete time points relative to a spontaneous ovulation and from follicles collected at various stages of follicular development. Levels of PA activity in the granulosa layer of the F1 follicle declined by greater than 90% as follicles were collected closer to their anticipated time of ovulation (e.g., from 17-16 h to 0.75-0.15 h; p less than 0.05). Timing of tissue collection was confirmed by evaluation of serum progesterone levels, which peaked as expected at the 6-5-h time point. During follicular development, PA activity was several times greater in rapidly growing follicles (6-12 mm, 1-3 wk prior to ovulation) than in slowly growing (1-5 mm) or preovulatory (F3 and F1) follicles (p less than 0.05). Granulosa cells of these rapidly growing follicles also incorporated significantly higher levels of 3H-thymidine than did granulosa cells of mature follicles (p less than 0.05), suggesting a higher level of DNA synthesis. Similarly, granulosa cells of the mitotically active germinal disc region of the F1 granulosa layer were found to possess at least 3-fold higher (p less than 0.05) levels of PA activity and a 2-fold greater level of 3H-thymidine incorporation than the more mature granulosa cells isolated from the remaining F1 granulosa layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of basic fibroblast growth factor in bovine adrenal gland, ovary, and pituitary.

We studied the distribution of basic fibroblast growth factor (bFGF) immunoreactivity in bovine adrenal gland, ovary, and pituitary, using a polyclonal anti-bFGF antibody. In the adrenal gland, the inner layers of the capsule, the zona glomerulosa of the cortex, and the chromaffin cells of the adrenal medulla were intensely stained. In the ovary, follicular epithelial cells of growing follicles and granulosa cells of mature follicles showed strong bFGF-like immunoreactivity. Endocrine cells of the pituitary anterior and intermediate lobes displayed a positive immunoreaction. Blood vessels, including endothelial and smooth muscle cells, as well as stromal cells in all three organs studied, were not stained. This distribution pattern of bFGF immunoreactivity is only partially compatible with the established mitogenic role of this protein, and suggests a wider spectrum of bFGF functions.

Rapid changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat estrous cycle.

Distributions of inhibin alpha-, beta A-, and beta B-subunits in different ovarian compartments were studied in cycling female rats by in situ hybridization with complementary RNA probes and using immunohistochemical localization with antibodies selective for each inhibin subunit. Consistent with earlier studies showing inhibin production by granulosa cells of maturing follicles, we also detected mRNAs for inhibin alpha-, beta A-, and beta B-subunits in granulosa cells of these follicles. However, based on immunohistochemistry and in situ hybridization, we found that inhibin alpha- is not only expressed in granulosa cells of mature follicles but in follicles at all stages of maturation, including primary to tertiary follicles. A number of primordial follicles also contained alpha mRNA and immunodetectable alpha-subunit. Interestingly, theca interna and interstitial gland cells contained inhibin alpha mRNA and alpha-subunit. Low levels of inhibin alpha immunoreactivity as well as specific hybridization to the complementary inhibin alpha mRNA probe were observed in newly formed luteal tissue. beta-Subunits, on the other hand, were detected exclusively in granulosa cells of healthy tertiary follicles. The changes in expression of inhibin alpha-, beta A-, and beta B-subunits were more pronounced during the follicular phase of the cycle: inhibin alpha reached its highest level in granulosa cells, theca interna, and interstitial gland cells a few hours after the LH/FSH surge, while at the same time the beta-subunits decreased dramatically in granulosa cells of mature follicles. Immediately before ovulation (estrus 0200 h), the alpha-subunit sharply declined in preovulatory follicles and was present mainly in granulosa cells from nonovulatory follicles at various stages of maturation. At that time, the beta A- and beta B-subunits could not be detected in preovulatory follicles but were localized mainly in small tertiary follicles (less than 300 microns). Unlike for the alpha- and beta B-subunits, beta A mRNA and immunoreactivity was present in large tertiary follicles (approximately 600 microns) immediately before ovulation. The present findings support the hypothesis that a decrease in inhibin production could be responsible for the secondary FSH surge observed early on estrus. This could be initiated by a change in the ratios of activin-inhibin production by decreasing first, the levels of beta-subunits, second, the levels of alpha-subunit, and third, by a resurgence of activin A produced mainly by granulosa cells from large tertiary follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Demonstration of Immunoreactive LHRH in the Human Ovaries by Peroxidase Immunocytochemistry

AbstractThe distribution of immunoreactive LHRH in the human ovary was examined using paraffin sections by peroxidase immunocytochemistry. Immunoreactive LHRH is seen in the granulosa cells of mature follicles, and in the theca interna cells of atretic follicles. But the immunoreactive LHRH is not found in the theca interna cells or stromal components of mature follicles, and in the granulosa cells or stromal components of atretic follicles. There may be a possibility that immunoreactive LHRH serves as a paracrine modulator in follicular maturation.

Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells.

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.

Quantitation of nexus junctions in the granulosa cell layer of rabbit ovarian follicles during ovulation.

Electron microscopy was used in a semi-quantitative study to determine changes in the abundance and size of surface nexuses and changes in the abundance of interiorized nexuses in growing and mature ovarian follicles during the ovulatory process. Mature follicles contain larger granulosa cells than follicles in the early stage of antral formation. Also, the granulosa cells of mature follicles have a slightly greater number of surface nexuses (without a change in nexus length), and more interiorized nexuses, compared to immature follicles. As a mature follicle approaches rupture, there is an appreciable decrease in the number of surface nexuses per granulosa cell. There is also a slight reduction in the number of interiorized nexuses at this time. It is concluded that this decrease in both surface nexuses and interiorized nexuses may be a consequence of ovulatory changes during which the rate of granulosa cell division is greater than the rate of formation of new nexuses. Additionally, the disruption to cell-to-cell cohesion during the ovulatory process appears to be independent of the interiorization of surface nexuses.