Theca Cell Layer Research Articles

Development of antral follicles in human cryopreserved ovarian tissue following xenografting.

This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites > or = 20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.

Estrogen receptor-beta expression in relation to the expression of luteinizing hormone receptor and cytochrome P450 enzymes in rat ovarian follicles.

Changes in mRNA expression for estrogen receptor (ER beta) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ER beta, LHr, cytochrome P450 side-chain cleavage enzyme (P450(scc)), 17 alpha-hydroxylase (P450(c17)), aromatase (P450(arom)), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40-100 microm), small (101-275 microm), medium (276-450 microm), and large (451-850 microm). Atretic follicles were divided into medium (276-450 microm) or large follicles (451-850 microm). A low level of ER beta mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ER beta mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450(scc), and P450(arom) were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450(scc), P450(c17), and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450(scc), P450(c17), P450(arom), and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ER beta mRNA was observed in granulosa cells during follicular development. The increased expression of ER beta and a concomitant initiation of LHr, P450(scc), and P450(arom) expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ER beta mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450(scc), P450(c17), and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.

Gene expression of endothelin-1 in the porcine ovary: follicular development.

We have investigated which follicular compartment and stage of follicular development are associated with endothelin-1 (ET-1) gene expression in the porcine ovary. The localization of mature ET-1 peptide and of its mRNA was determined by immunohistochemistry and by in situ hybridization. Stage of follicular development associated with ET-1 expression was investigated in terms of follicular class and occurrence of atresia. The latter was investigated by determining the occurrence of DNA fragmentation in apoptotic cells on adjacent sections to those used for ET-1 gene expression. Fifteen ovaries from 10 prepubertal pigs stimulated with gonadotropin were collected; a total of 1050 follicles were examined. Specific ET-1 immunoreactivity was restricted to the ovarian vasculature and to the granulosa cell compartment of antral follicles. The pattern of ET-1 mRNA expression was similar to that found for ET-1 immunoreactivity. Primordial, primary, and most secondary follicles did not express ET-1. The theca cell layer did not express ET-1 regardless of follicle developmental stage. ET-1 expression occurred with a significantly greater probability (P < 0.001 by the likelihood ratio test) in the granulosa cell compartment of antral follicles than in any other follicle class. Furthermore, in antral follicles, ET-1 expression occurred with a greater likelihood in large antral follicles than in small antral follicles (P < 0.001 by the likelihood ratio test). In small antral follicles, only 16.8% expressed ET-1; in contrast, 66.7% of large antral follicles exhibited ET-1 expression. It is interesting that in follicles in which ovulation had already occurred, intense ET-1 expression was found only in the prominent developing vasculature, the other cells present in the luteinized follicle did not display any ET-1 expression. The pattern of ET-1 gene expression observed in this study would be in agreement with our previous suggestion of a plausible physiological role for ET-1 in preventing premature progesterone production by granulosa cells of an antral follicle. The occurrence of atresia and expression of ET-1 in the same follicle was rare. Small and large antral follicles constituted 5.1% and 5.6%, respectively, of the examined follicles in this category. The majority of atretic follicles did not express ET-1 and, conversely, follicles that expressed ET-1 were not atretic. To the best of our knowledge, this is the first report in which large, nonatretic follicles are clearly identified as the population of follicles expressing ET-1. The results of this study delineate the follicular developmental stage and the compartment of when and where ET-1 may be physiologically meaningful.

The Major Chicken Egg Envelope Protein ZP1 Is Different from ZPB and Is Synthesized in the Liver

The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen. In contrast, it is absent in rooster liver but can be efficiently induced by estrogen treatment of the animal. An immunoscreen of a liver lambda-ZAP library yielded a cDNA coding for a protein of 934 amino acids. It displayed significant homology to members of the ZP1/ZPB family from other species, notably to mouse and rat ZP1, and was therefore designated chkZP1. It is clearly different from a protein designated chkZPB that had been deposited in the data base previously. Alignment of the known members of the ZP1/ZPB family demonstrated the existence of at least three subgroups, with representatives of both the ZP1 and the ZPB sequence homology group occurring in vertebrates. Northern blot analysis of liver extracts revealed the presence of a single 3. 2-kilobase mRNA coding for chkZP1, distinct from the chkZPB transcript detectable in follicles. Immunohistochemical analysis of follicle sections demonstrates that chkZP1 can be found in the blood vessels of the theca cell layer as well as in the pvm surrounding the oocyte. Thus, in the chicken, at least one of the major pvm components is synthesized in the liver and is transported via the bloodstream to the follicle.

The Expression of the Sex Steroid-Synthesizing Enzymes CYP11A1, 3β-HSD, CYP17, and CYP19 in Gonads and Adrenals of Adult and Developing Zebra Finches

Songbirds have emerged as important animal models for understanding how sex steroids influence brain and behavior, particularly how they direct the sexually dimorphic development of the neural circuits controlling song and then activate adult song behavior. Presumably, sex steroids synthesized in the gonads are responsible for these actions on brain. However, experiments do not always reveal a direct relationship between gonadal function, circulating sex steroids, and activation and/or organization of song. Thus, it is critical that we understand more about the sites and mechanisms of sex steroid synthesis in this group of birds. Toward this end, we have established the use in zebra finches of chicken cDNA probes to the principal androgen synthetic enzymes, CYP11A1, 3β-HSD, and CYP17. On Northern blots, these probes recognized bands of the appropriate size and in tissues similar to those seen in chickens. With these probes, and a probe to CYP19 specific to the zebra finch, we used in situ hybridization to examine the cellular expression of these enzymes in gonads and adrenals of adult and developing zebra finches (1 to 20 days posthatching). In adults, we identified significant expression of CYP11A1 and CYP17 in large ovarian follicles, particularly the thecal cell layer and over the testicular interstitial area. 3β-HSD was expressed by both theca and granulosa and in testicular interstitial and seminiferous tubular cells. In adrenals, CYP11A1 and 3β-HSD are abundant with lesser amounts of CYP17. Developmentally, we identified high expression of CYP11A1 and 3β-HSD in the adrenals, CYP17 in both testes and ovaries, and CYP19 in ovaries only. These results suggest that the ovaries but not the testes may secrete estrogen developmentally and the adrenals may contribute precursors for gonadal steroidogensis.

Type VI collagen expression during growth of human ovarian follicles

Objective: To identify type VI collagen expression in human ovarian follicles during follicular growth. Design: In vitro experiment. Setting: Department of Obstetrics and Gynecology, Wakayama Medical College, Japan. Patient(s): Regularly cycling women who underwent adnexectomy. Intervention(s): Immunohistochemistry and in situ hybridization for human type VI collagen. Main Outcome Measure(s): Expression of type VI collagen. Result(s): Expression of type VI collagen was observed in the theca cell layers during folliculogenesis, whereas no expression of type VI collagen was observed in the granulosa cell layers at the mRNA and protein levels. As the follicles grew, immunostaining for type VI collagen became intense in the theca cell layers, especially the theca externa. In preovulatory follicles, however, weak, fragmented, or discontinuous immunostaining of the theca cell layers was observed. This fragmented or discontinuous immunostaining was evident predominantly in the apical area of preovulatory follicles rather than in the basal area. Conclusion(s): Type VI collagen is present in the theca cell layers of follicles during folliculogenesis and plays an important role in interactions between the theca cells and extracellular matrix. These interactions may lead to changes in the shape, proliferation, migration, or differentiation of follicular cells during follicular development, maturation, and ovulation.

Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle.

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.

Revelations of ovarian follicle biology from gene knockout mice

Autocrine, paracrine and endocrine factors are necessary for normal ovarian folliculogenesis. A number of studies have demonstrated that the pituitary hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as granulosa cell-derived growth factors such as activins, inhibins, and kit ligand are necessary for follicle development. Recent knockout studies from our laboratory have demonstrated that mice lacking the pituitary proteins FSHβ and activin receptor type II are infertile due to blocks at the pre-antral and antral follicle stages, respectively. Although the somatic cells of the ovary (the granulosa and theca cells) have long been implicated in ovarian function, only recently have we shown that the oocyte plays an essential role in controlling its own fate by influencing somatic cell functions. Mice lacking the oocyte-secreted growth factor, growth differentiation factor-9 (GDF-9), are infertile due to a block at the one-layer primary follicle stage, leading to secondary defects in thecal cell layer formation, oocyte growth and meiotic competence, and granulosa cell differentiation. Furthermore, using recombinant GDF-9, we demonstrate that GDF-9 also regulates cumulus expansion and expression of several key granulosa cell-specific genes. Thus, GDF-9 functions as an oocyte-secreted growth and differentiation factor during early and late folliculogenesis and at ovulation to regulate several key somatic cell functions essential for female reproduction.

Function of the polycystic ovary

The mechanism of the increase in the number of follicles in polycystic ovaries (PCO) is still not understood, but most data indicate that folliculogenesis is abnormal at all stages. This increase is probably due to prolonged follicle growth in both ovulatory (ov) and anovulatory PCO (anovPCO) rather than over-recruitment from the primordial stage. There appear to be fewer follicles in ovPCO than in anovPCO and this may be due to slow growth combined with arrested development in the anovPCO, preventing the normal process of atresia. This idea is supported by data indicating that the preponderance of structures in anovPCO are functional follicles rather than atretic cysts. In contrast, follicles in ovPCO are primarily atretic, analogous to the situation in the normal ovary. Closer analysis of the function of these follicles reveals an intrinsic defect in the theca cell layer in the expression of the genes encoding steroidogenic enzymes. Although androgen production is increased in all PCOs, the serum concentrations of androgen tended to be higher in anovulatory women indicating that there may be a link between the ovarian concentration and anovulation. Granulosa cells in small follicles from anovPCO behave in a similar fashion to those in preovulatory follicles from normal ovaries in terms of steridogenic responses. This, in combination with the finding of LH-responsiveness in small follicles from anovPCO, has led to the suggestion that these follicles are prematurely luteinized. The recent finding that follistatin gene regulation may be abnormal in women with polycystic ovary syndrome (PCOS) provides a new insight into the possible defect in the function of these ovaries. Elucidation of the precise nature of the defect may provide the key to understanding the apparent contradiction of increased steroidogenesis in the presence of abnormal and arrested follicle growth.

Immunolocalization of transforming growth factor-beta1 during follicular development and atresia in the mouse ovary.

In each estrous cycle not all follicles recruited grow to ovulate, as some degenerate through the process of atresia. Apoptosis is the mechanism underlying follicle atresia. TGF-beta1 has been implicated in the induction and promotion of apoptosis in many cell types and it is expressed in the ovary. In this study immunohistochemistry was used to localize TGF-beta1 in order to correlate the growth factor expression with morphological follicular atresia during diestrus in mice. Small and medium sized follicles had no staining for TGF-beta1 in the granulosa or theca cell layer. Weak to moderate TGF-beta1 expression was present in the theca cells of both large healthy and atretic antral follicles. Large healthy antral follicles showed weak granulosa staining but this was not observed in atretic follicles. Strong TGF-beta1 staining was present in the interstitial, corpus luteum and oocytes of all follicle stages. These results suggest that in the mouse TGF-beta1 promote follicular growth and differentiation rather than follicular atresia.

Immunohistochemical detection of plasminogen activator inhibitor-1 in polycystic ovaries

Anovulation in women with polycystic ovary syndrome (PCOS) is incompletely understood. The concentration of the glycoprotein plasminogen activator inhibitor-1 (PAI-1) is raised in insulin resistance. This has been described in the granulosa and theca cell layers of the animal but not the human ovary. This study was performed to investigate the location of PAI-1 in the human ovary and investigate whether it may contribute to anovulation in PCOS. PAPI-1 was localized immunohistochemically and quantitated using computer image analysis in 17 ovarian follicles from fwe women with a diagnosis of PCOS and compared with 15 follicles from six normal ovaries. PAI-1 was predominantly found in the granulosa and theca cells in both polycystic and normal ovaries. Image analysis did not reveal a difference in the PAI-1 signal from polycystic compared with normal ovaries. This study shows that PAI-1 plays a role in human ovulation, but its role in PCOS requires further research.

Kit ligand actions on ovarian stromal cells: effects on theca cell recruitment and steroid production.

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.

The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy.

In human ovaries, angiogenesis is known to be associated with the development of follicles and the formation of the corpus luteum (CL). A complex vascular network is formed within the thecal cell layer during follicular growth, and rapid neovascularization occurs toward the granulosa cell layer after ovulation. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine, stimulating endothelial cell growth and enhancing microvascular permeability. A specific receptor for VEGF, fms-like tyrosine kinase (Flt-1), is expressed in vascular endothelial cells that mediates the action of VEGF. We examined the localization and expression of VEGF and Flt-1, using an immunohistochemical technique and RT-PCR analysis, in human follicles and corpora lutea during the normal menstrual cycle and early pregnancy. We measured concentrations of VEGF in extracts of human CL using an enzyme-linked immunosorbent assay during the luteal phase and early pregnancy. Immunostaining for VEGF was observed in granulosa cells from small antral follicles to preovulatory follicles. The staining was detected in thecal cells from medium-sized to preovulatory follicles. The intensity of the staining was gradually increased as a follicle grew. Flt-1 was localized in granulosa and thecal cells of preovulatory follicles as well as in endothelial cells. In the human CL, the intense staining for VEGF was observed in granulosa and thecal lutein cells, especially in the midluteal phase. The immunostaining for Flt-1 was faint in endothelial cells in the CL, whereas it was distinct in granulosa and thecal lutein cells. The concentrations of VEGF in lutein extracts were high in the early and midluteal phases and tended to decrease toward the late luteal phase. During early pregnancy, a measurable amount of VEGF was detected. RT-PCR analysis demonstrated that messenger ribonucleic acids encoding VEGF121, VEGF165, and Flt-1 were expressed in the CL. These results suggest that VEGF might have an autocrine role in the ovulatory process and luteal function as well as a paracrine role in angiogenesis.

Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary.

Growth differentiation factor-9 (GDF-9), a secreted member of the transforming growth factor-beta superfamily, is expressed at high levels in the mammalian oocyte beginning at the type 3a primary follicle stage. We have previously demonstrated that GDF-9-deficient female mice are infertile because of an early block in folliculogenesis at the type 3b primary follicle stage. To address the molecular defects that result from the absence of GDF-9, we have analyzed the expression of several important ovarian marker genes. The major findings of our studies are as follows: 1) There are no detectable signals around GDF-9-deficient follicles for several theca cell layer markers [i.e. 17alpha-hydroxylase, LH receptor (LHR), and c-kit, the receptor for kit ligand]. This demonstrates that in the absence of GDF-9, the follicles are incompetent to emit a signal that recruits theca cell precursors to surround the follicle; 2) The primary follicles of GDF-9-deficient mice demonstrate an up-regulation of kit ligand and inhibin-alpha. This suggests that these two important secreted growth factors, expressed in the granulosa cells, may be directly regulated in a paracrine fashion by GDF-9. Up-regulation of kit ligand, via signaling through c-kit on the oocyte, may be directly involved in the increased size of GDF-9-deficient oocytes and the eventual demise of the oocyte; 3) After loss of the oocyte, the cells of the GDF-9-deficient follicles remain in a steroidogenic cluster that histologically resembles small corpora lutea. However, at the molecular level, these cells are positive for both luteal markers (e.g. LHR and P-450 side chain cleavage) and nonluteal markers (e.g. inhibin alpha and P-450 aromatase). This demonstrates that initially the presence of the oocyte prevents the expression of luteinized markers, but that the absence of GDF-9 at an early timepoint alters the differentiation program of the granulosa cells; and 4) As demonstrated by staining with either proliferating cell nuclear antigen (PCNA) or Ki-67 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) labeling, the granulosa cells of GDF-9-deficient type 3b primary follicles fail to proliferate but also fail to undergo cell death. This suggests that granulosa cells of type 3b follicles require GDF-9 for continued growth and also to become competent to undergo apoptosis, possibly through a differentiation event Thus, these studies have enlightened us as to the paracrine roles of GDF-9 as well as the normal steps of granulosa cell and theca cell growth and differentiation within ovarian follicles.

Immunolocalization and expression of the steroidogenic acute regulatory protein during the transitional stages of rat follicular differentiation.

This study was designed to determine the pattern of expression and cellular distribution of the steroidogenic acute regulatory protein (StAR) during the transitional stages of follicular differentiation in rat ovary. Using specific antisera against the StAR, immunohistochemistry, Western blotting, and immunoprecipitation analyses provide evidence confirming the localization and expression of StAR in granulosa cells (GCs) of juvenile rat ovaries before and after PMSG treatment. The results also show that StAR expression occurs in theca intersitial cells surrounding preantral, antral, and larger antral follicles in adult diestrous ovaries. Furthermore, we have demonstrated heterogenous StAR immunoreactivity in the granulosa cell layers and cells of the corpora lutea. A novel finding presented here is that, during ongoing growth and differentiation of the follicle, the immunoreactivity of StAR tends to shift from the GC of early antral follicles to the theca cell layers in the adult. The spatiotemporal changes or shifts in StAR expression and cellular localization also coincide with the appearance of more acidic isoforms of the 30-kD protein, as determined by two-dimensional gel electrophoresis. Although the functional implications of these observations remain unclear, the acute temporal changes in StAR expression and localization may not only reflect the dynamic steroidogenic capacity of follicular cells but may also support a possible role for FSH in the induction of follicular maturation.

Expression of gap junctional proteins connexin 43, 32, and 26 throughout follicular development and atresia in cows.

Detection of connexin (Cx) proteins has been used as an indicator of the presence of structural and functional gap junctions in tissues. To examine the role of gap junctions during follicular growth and atresia, the presence of three major connexins, Cx43, Cx32, and Cx26, was evaluated in bovine ovaries by using immunohistochemistry and Western immunoblot analysis. Cx43 was not present in primordial follicles, but was present in granulosa cells of primary/secondary and antral follicles. Cx43 also was present on the borders between granulosa cells and the oocyte. Expression of Cx43 increased in healthy developing antral follicles, but decreased during follicular atresia. Cx32 was not present in healthy follicles but was present in granulosa cells of atretic antral, and especially small antral follicles. Cx26 was present in the oocyte of primordial and primary/secondary follicles, and in the granulosa and/or thecal cell layers of healthy antral follicles. The percentage of healthy antral follicles that expressed Cx26 also increased during follicular development, but decreased during atresia. Cx32 and Cx26 also were detected in ovarian blood vessels and in stromal tissues adjacent to the tunica albuginea in some ovaries. The pattern of expression of these Cx indicates that gap junctional proteins may be involved in the control of follicular growth and atresia in cows.

Theca cells and theca-cell conditioned medium inhibit the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH-mediated meiosis in oocytes of COCGs.

Distribution of F4/80-positive cells in developing ovaries in the mouse.

The mature ovary contains a large number of macrophages. In the present study, the distribution of macrophages in murine ovaries at various developmental stages was immunohistochemically studied using a monoclonal antibody against F4/80, a highly specific antigen of murine macrophages. The results showed that definite F4/80-positive stains were hardly detectable in ovaries on day 0 after birth. On day 7, a few F4/ 80-positive cells could be identified between the developing follicles. The positive stains were irregular in shape and showed little physical contact with the primordial or primary follicles. By days 14 and 21, when the theca cell layers of growing follicles were developing, the positive cells had extended or elongated to surround the cell layer. On day 28, besides the presence of elongating positive cells surrounding the growing follicles, irregularly shaped F4/80-positive cells became apparent in the interstitium between the growing follicles and also in the capsular tissues. Thereafter, positive cells with stellate appearance were detected in the corpora lutea, which first developed around 6 weeks of age. Although the positive cells were homogenously distributed in the corpora lutea in virgin adults, only a few sporadic positive cells were found there in pregnant mice. However, the positive cells infiltrated into the corpora lutea again in the postpartum period. These results show that ovarian macrophages exhibit dramatical changes in their distribution from neonatal to postpartum periods.

Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary.

Nitric oxide (NO) has emerged as a novel regulator of several ovarian events, such as ovulation, steroidogenesis, and apoptotic cell death. The NO synthases (NOS) are a family of enzymes that catalyze the oxidation of L-arginine to NO and L-citrulline. The purpose of the present study was to localize NOS isoforms in the rat ovary and to examine their hormonal regulation. We conducted immunohistochemistry and Western blot analysis using isoform-specific antibodies against brain NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Immature rats were superovulated by injecting PMSG (10 I.U. s.c.) followed by an injection of human CG (hCG; 10 I.U. s.c.) 48 h later. Ovaries were obtained from control rats (no PMSG), 24 h and 48 h after PMSG treatment and 2 h, 8 h, 12 h, 20 h or 6 days and 10 days after hCG injection (n = 3-5 rats/group). Rat ovaries were clearly devoid of brain NOS staining at any of the time points studied. In control ovaries, eNOS was detected in the theca cell layer, ovarian stroma, and on the surface of oocytes. During follicular development, eNOS staining was still expressed in the theca cell layer and was also present in mural granulosa cells. After ovulation, homogenous eNOS staining was observed within cells of the corpus luteum (CL). Western blots of ovarian homogenates demonstrated that during PMSG-induced follicle growth, eNOS levels increased by 2.5-fold relative to control rats (P < 0.05). eNOS levels were further increased 12 h and 20 h after hCG injection (5-fold and 7-fold, respectively, relative to control; P < 0.05). The greatest amount of eNOS was observed in ovaries 10 days after hCG injection (15-fold relative to control; P < 0.05). We also detected expression of iNOS in the ovary, but the pattern and cell-specific staining differed from that observed for eNOS. In immature ovaries and during follicular development, iNOS staining was found within the theca cell layer and stroma. After ovulation, iNOS staining was present only in the external layers of the developing CL, but in the degenerating CL (10 days post-hCG), strong staining in nonparenchymal cells was observed within the entire CL. Western blots showed no changes in levels of ovarian iNOS protein during follicular development, but a significant increase (6-fold relative to control; P < 0.05) was observed after an ovulatory dose of hCG. The highest level of iNOS was observed in ovaries 10 days after hCG injection (10-fold relative to control; P < 0.05). Our data demonstrate that ovarian eNOS and iNOS show distinct cell-specific expression patterns and are differentially regulated during follicular and luteal development.

17α,20β-Dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: Mechanisms of synthesis and action

Meiotic maturation of fish oocytes is induced by the action of maturation-inducing hormone (MIH). 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DP) was identified as the MIH of several fish species, including salmonid fishes. The interaction of two ovarian follicle cell layers, the thecal and granulosa cell layers, is required for the synthesis of 17α,20β-DP; the thecal layer produces 17α-hydroxyprogesterone that is converted to 17α,20β-DP in granulosa cells by the action of 20β-hydroxysteroid dehydrogenase (20β-HSD). The preovulatory surge of LH-like gonadotropin (GTH II) is responsible for rapid expression of 20β-HSD mRNA transcripts in granulosa cells. 17α,20β-DP acts via a receptor on the plasma membrane of oocytes. A specific 17α,20β-DP receptor has been identified and characterized from defolliculated oocytes of several fish species. The concentrations of 17α,20β-DP membrane receptor increase immediately prior to oocyte maturation. The pertussis toxin-sensitive inhibitory G protein is involved in the signal transduction pathway of 17α,20β-DP. The early steps following 17α,20β-DP action involve the formation of the major mediator of this steroid, maturation-promoting factor, which consists of cdc2 kinase (34 kDa) and cyclin B (46–48 kDa). Immature oocytes contain only monomeric 35 kDa cdc2 and do not stockpile cyclin B, although immature oocytes contain mRNA for cyclin B. 17α,20β-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting 35 kDa cdc2 through its threonine 161 phosphorylation by a threonine kinase (MO15), producing the 34-kDa active cdc2. 17α,20β-DP-induced oocyte maturation is blocked by cordycepin, a polyadenylation inhibitor. Furthermore, cyclin B mRNA was polyadenylated during 17α,20β-DP-induced oocyte maturation. These findings suggest that 17α,20β-DP initiates translation of cyclin B mRNA through cytoplasmic 3′ poly(A) elongation.