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The interleukin-1 system and female reproduction.

Interleukins (ILs) are known best for their involvement in the immune system and their role during inflammation. In the ovary, a growing body of evidence suggests that the ovarian follicle is a site of inflammatory reactions. Thus ovarian cells could represent sources and targets of ILs. Since then, the IL-1 system components (IL-1alpha, IL-1beta, IL-1 receptor antagonist, IL-1 receptors) have been demonstrated to have several sites of synthesis in the ovary. These factors have been localized in the various ovarian cell types, such as the oocyte, granulosa and theca cells, in several mammalian species. IL-1-like bioactivity has been reported in human and porcine follicular fluid at the time of ovulation. The role of IL-1 in local processes is still poorly known, although there is evidence for involvement in the ovulation process, and in oocyte maturation. More precisely, IL-1 may be involved in several ovulation-associated events such as the synthesis of proteases, regulation of plasminogen activator activity, prostaglandin and nitric oxide production. IL-1 also regulates ovarian steroidogenesis. These different aspects of the involvement of the IL-1 system in important aspects of female reproduction are discussed.

Animals↗

Plasminogen activator/plasminogen activator inhibitors in ovarian physiology.

The target extracellular matrix (ECM) degradation generated by plasminogen activator (PA) and regulated by plasminogen activator inhibitor (PAI) is an event that affects a wide variety of physiological and pathological processes in the ovary. Studies carried out over the past 25 years in a number of laboratories have elucidated some of the biochemical events related to the function and regulation of the PA system in the ovary. Hormone-induced coordinated expression of tissue-type PA (tPA) produced mainly by granulosa cells and its inhibitor PAI-1 secreted by theca cells in the preovulatory follicles is responsible for a controlled and directed proteolysis leading to the rupture of selected follicles in the rat, monkey and other mammals. Increase in tPA and PAI-1 expression in corpus luteum (CL) of rat and monkey at a later stage is well correlated with a sharp decrease in CL progesterone production, indicating its important role in the initiation of luteal regression. In contrast, the urokinase-type PA (uPA) may play an essential role in the early growing follicles during cell proliferation and migration, and in the early CL formation related to ECM degradation and angiogenesis. Ovarian function is also modulated by endogenously-produced local factors that regulate expression of the PA activator and inhibitor, and the MMP system. Thus, the next challenge is to identify the interrelationship between multiple paracrine and autocrine factors and the PA system, and to know how they regulate the protease and the protease inhibitor in the ovary.

Animals↗

Interactions between nutrition and ovarian activity in cattle: physiological, cellular and molecular mechanisms.

The effects of acute changes in dietary intake on ovarian activity can be correlated with changes in circulating concentrations of metabolic hormones including insulin, insulin-like growth factor I (IGF-I), growth hormone and leptin. There is no corresponding change in circulating gonadotrophin concentrations and it is proposed that the dietary induced changes in ovarian activity, resulting from acute changes in dietary intake, are a result of direct actions of these metabolic hormones on the ovary. Changes in the peripheral concentrations of insulin, IGF-I and leptin were also associated with the initiation of a synchronized wave of follicle growth and it is hypothesized that oestrogen secreted by the developing follicle is involved in regulating the secretion of these metabolic hormones. At the cellular level, physiological concentrations of insulin and IGF-I interact to stimulate oestradiol production by granulosa cells. In contrast, leptin inhibits FSH-stimulated oestradiol production by granulosa cells and LH-stimulated androstenedione production by theca cells. At the molecular level, dietary energy intake affects the expression of mRNA encoding components of the ovarian IGF system and these changes can directly influence the bioavailability of intrafollicular IGF. This, in turn, can increase the sensitivity or response of follicles to FSH and is one mechanism through which nutrition can directly affect follicle recruitment. Dietary induced increases in intrafollicular IGF bioavailability also have a negative effect on oocyte quality, and diets that are optimal for follicle growth may not necessarily be optimal for oocyte maturation.

Animal Nutritional Physiological Phenomena↗

Luteotropic and luteolytic effects of oxytocin in the porcine corpus luteum.

The presence and the release of oxytocin (OT) by corpora lutea (CL) of a number of species (Wathes et al. 1986, Watkins and Choy 1988) including ruminants (Ivell and Richter 1984, Hirst et al. 1986, Rodgers et al. 1983, Sawyer et al. 1986), primates (Dawood and Khan-Dawood 1986, Khan-Dawood 1987, Maas et al. 1992, Khan-Dawood et al. 1993), and the pig (Pitzel et al. 1984, Einspanier et al. 1991, Jarry et al. 1992) have been amply verified. Conflicting results concerning the effects of OT on steroidogenesis have been published; the peptide has been shown to be luteotrophic (Sawyer et al. 1986, Maas et al. 1992, Jarry et al. 1990), to have no effects (Rodgers et al. 1985) or to be luteolytic (Auletta et al. 1984, Auletta et al. 1988, Pitzel et al. 1988) and it appears that this confusion is only in part due to species differences but also the age of the luteal tissue seems to be of crucial importance for the understanding of the effects of OT (Schams et al. 1983, Wuttke et al. 1993, 1994). In the present contribution we will focus largely on our results obtained in the pig and where applicable, compare them with those obtained in other species. We will thus demonstrate that OT is released by luteal cells (Jarry et al. 1990, Einspanier et al. 1991, Jarry et al. 1992) and that luteal cells have OT receptors (Sernia et al. 1989, Pitzel et al. 1993a) which mediate the effects of the peptide on steroidogenesis. Finally, we will address the question whether OT is inhibitory or stimulatory to progesterone (P) and estradiol (E2) release, and we will come to the conclusion that OT is both luteotropic and luteolytic (Wuttke et al. 1993, 1994). The CL of all species investigated so far consists of two steroidogenic cell types. The so-called large luteal cells stem from follicular granulosa cells and they appear to be barely responsive to luteinizing hormone (LH)/human chorionic gonadotrophin (hCG) but they are highly receptive to prostaglandin F2 alpha (PGF2 alpha) (Hansel and Dowd 1986, Pitzel et al. 1990). Furthermore, they appear to produce OT (Rodgers et al. 1983, Theodosis et al. 1986). The small luteal cells are believed to derive from the follicular theca cells (Hansel and Dowd 1986, Pitzel et al. 1990). They are LH-receptive but synthesize few, if any, regulatory peptides. In the last few years it has become increasingly evident that cells deriving from the white blood cell line are involved in processes such as ovulation and luteolysis. Of crucial importance for the understanding of luteolysis is the morphological observation that macrophages invade the CL at the time of luteal regression (Adashi 1990, Paavola 1977, Kirsch et al. 1981).

Animals↗

Inhibition of the activities of P450 cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase and the amount of P450 cholesterol side-chain cleavage by testosterone and estradiol-17 beta in hen granulosa cells.

We have found that androgens and estradiol-17 beta (E2) produced by theca cells suppress progesterone (P4) secretion by granulosa cells of the domestic hen in a dose-dependent manner. Furthermore, testosterone (T) and E2 inhibited the conversion of cholesterol to pregnenolone (P5) and of P5 to P4, respectively. The aim of this study was to determine if T and E2 suppress P4 biosynthesis by changing activities of the cytochrome P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (Exp I) and the amount of P450scc (Exp II). Granulosa layers of the largest follicle of two to four hens were obtained 22 h before ovulation, pooled, and isolated granulosa cells were prepared. In Exp I, the specific activities of the P450scc and 3 beta-HSD were measured in mitochondrial and microsomal proteins of granulosa cells, respectively, in the presence of T or E2 (0-10 microM). Addition of T to mitochondrial proteins increased the Michaelis-Menten constant (Km) with no change in the maximum velocity (Vmax) of the P450scc, which suggests competitive inhibition (Ki = 30.9 microM), whereas E2 had no effect on Km and Vmax of the P450scc. Likewise, addition of E2 to microsomal proteins increased the Km with no change in the Vmax of the 3 beta-HSD, which suggests competitive inhibition (Ki = 15.1 microM), whereas T had no effect on Km and Vmax of the 3 beta-HSD. In Exp II, granulosa cells (3 x 10(5)/3 ml.tube) were incubated for 0-12 h in triplicate for each combined treatments of 25-OH-cholesterol (8 microM) and cyanoketone (10 microM), T, or E2 (0-10 microM) in the presence or absence of LH (25 ng). Protein content and P5 secretion were measured and the amount of P450scc was determined by Western blot analysis. Incubation of granulosa cells with T decreased the amount of the P450scc in granulosa cells cultured for 12 h and P5 secretion in granulosa cells cultured for 3 h or longer (P less than 0.05), without a change in protein content and cell viability. Our results suggest that P4 production by granulosa cells is suppressed by T and E2 acting as competitive inhibitors of the P450scc and 3 beta-HSD, respectively, and by T decreasing the amount of the P450scc. We conclude that steroidogenesis in the follicle of the chicken is regulated through the interaction of theca and granulosa layers.

3-Hydroxysteroid Dehydrogenases↗

Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature gilts.

Perifollicular angiogenesis is closely associated with ovarian follicular development. To investigate whether additional induction of perifollicular angiogenesis would support subsequent follicular development, we directly injected vascular endothelial growth factor (VEGF) gene fragments into the ovaries of miniature gilts, followed by gonadotroph treatment to stimulate follicle growth. In addition, to confirm extraexpression of the VEGF gene after injection, we assessed the expression of two isoforms of VEGF (VEGF 120 and VEGF 164) in granulosa cells and expression of fms-like tyrosine kinase (Flt-1), expression of fetal liver kinase (Flk-1), and density of capillary networks in theca cells. Direct injection of VEGF gene fragments into the ovaries was performed 7 days before eCG treatment. The ovaries in miniature gilts were removed 72 h after eCG treatment for histological examination. Granulosa cells and thecal tissues in the antral follicles (diameter, >4 mm) were collected to detect the mRNA expression of VEGF isoforms in the granulosa cells and of Flt-1 and Flk-1 in the thecal tissues by semiquantitative reverse transcription-polymerase chain reaction. The VEGF levels were measured in the follicular fluid by enzyme immunoassay. Injection of VEGF gene fragments increased the level of mRNA expression of VEGF 120 and 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene fragments compared with those treated with eCG alone. The Flt-1, but not the Flk-1, mRNA expression show a tendency toward increasing in the thecal tissues of antral follicles in the ovaries injected with VEGF gene fragments. These results demonstrate that Flt-1 may be predominantly involved in the regulation of the capillary network in the theca interna during follicular development. Our data suggest that the regulation of perifollicular angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. Our findings may offer an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.

Animals↗

Early follicular development and atretic changes in the ovary of the lamb-- fine structure and histochemistry.

The fine structure of follicles was studied in ovaries from lambs up to 32 days old. Some animals received pregnant mare serum gonadotrophin prior to sampling. Follicles examined were in the primordial to early antral stages. The oocyte is described and changes associated with its development and the formation of the zona pellucida are recorded. Characteristics in which the lamb oocyte differed from those of other mammals include a greater prominence of smooth endoplasmic reticulum, the presence of microtubules, fine filaments in the peripheral parts and a large number of almost empty vacuoles. The latter were more numerous in oocytes which appeared to be degenerating, including some of those in primordial follicles. Granulosa cells had characteristics usually associated with protein secretion while some theca cells had large amounts of endoplasmic reticulum bearing few ribosomes. In early atresia changes were evident successively in the oocytes, granulosa and theca. There was a weak positive reaction for 3 beta-hydroxy-delta 5-steroid dehydrogenase in the theca of some vesicular follicles from both control lambs and lambs treated with pregnant mare serum gonadotrophin. Follicles showing sings of atresia were negative for this enzyme.

Animals↗

Reptilian and avian follicular hierarchies: models for the study of ovarian development.

The presence of an ovarian follicular hierarchy is a characteristic feature of reptiles and birds. The hierarchy contains follicles at all stages of maturation and therefore, varying degrees of sensitivity to the ovulation-inducing effects of the gonadotropins. In the hen, ovulability is gained as the ability of the follicle to produce androgens and estrogens declines and the ability to produce progesterone increases. In the mature follicle, the granulosa cells are the site of progesterone production whereas the theca cells produce androgens and estrogens. Small follicles that have not yet been (and may never be) recruited into the yolk-filled hierarchy are the major producers of androgens and estrogens within the ovary. In reptiles the ovarian follicular hierarchy includes non-vitellogenic follicles and in some species includes follicles destined to become atretic. These two features distinguish the reptiles from the birds and provide experimental biologists with a unique model to investigate the physiological events that regulate the most common fate of ovarian follicles, atresia.

Animals↗

Ovarian expression of melatonin Mel(1a) receptor mRNA during mouse development.

Melatonin, secreted by the pineal gland, is involved in the regulation of many physiological functions of various species of animals. In the present study, the expression of gene for melatonin Mel(1a) receptor (MelR) was evaluated in the ovary, hypothalamus, and pituitary according to the developmental stages in female mice. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) and in situ PCR techniques were applied. According to the developmental stages, gene for MelR was differently expressed on ovary, hypothalamus, and pituitary. MelR gene was first expressed on pituitary prior to the expression in hypothalamus and ovary. Ovarian MelR gene started to express at birth. Unlike hypothalamic expression of MelR gene which was identified after birth, in pituitary, it was expressed at 16 days post coitum. In the ovary, the expression signal of MelR gene was identified on granulosa cells. However, the signal was not detected in the theca cells. It was weak in the primordial and atretic follicles. Taken together, it can be considered that melatonin has a pivotal role in the folliculogenesis.

Animals↗

Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries.

The distribution of the tyrosine kinase receptor c-Kit and its ligand stem cell factor (SCF) was evaluated by immunohistochemistry in primordial germ cells (PGCs) and human embryonic gonads during weeks 5-8 of prenatal life, and fetal ovaries during weeks 9-36 of prenatal life. Distinct c-Kit and SCF staining was present in primordial germ cells in the wall of the hindgut and in the dorsal mesentery, particularly on level with the 10th thoracic columnar segment. Several PGCs were in close contact with c-Kit-negative but SCF-positive autonomic nerve fibers of the dorsal mesentery. Many fibroblasts and mesothelial cells of the dorsal mesentery were clearly stained for SCF, but not for c-Kit. Prominent c-Kit and SCF staining was present in germ cells of the embryonic gonadal anlage and in oogonia during further ovarian development. However, oocytes were either unstained or faintly stained for SCF. Oocytes not yet enclosed in follicles or present in primordial follicles were either unstained or exhibited faint cytoplasmic c-Kit staining, whereas oocytes of growing preantral follicles again showed distinct cell membrane staining which decreased during further follicular growth. Theca cells did not stain for c-Kit. Some pregranulosa cells and the first formed granulosa cells of primordial follicles were c-Kit stained. Granulosa cells of other follicles were not c-Kit stained. In the inner part of the cortex, SCF immunolabeling was detected in some pregranulosa cells surrounding cords containing germ cells and involved in formation of primordial follicles. Granulosa cells of primordial and growing follicles, including medium-sized antral follicles also revealed SCF staining. In conclusion, this first report on SCF in human PGCs and embryonic and fetal ovaries together with the c-Kit data lend substantial countenance to the notion that c-Kit and SCF play important roles during ascent of primordial germ cells towards the gonadal anlage, and during oogenesis and folliculogenesis in the human fetal ovary. We suggest that both autocrine and paracrine mechanisms are involved in the proposed anti-apoptotic effect of the c-Kit/SCF duet while PGCs are present in the dorsal mesentery. The SCF-positive autonomic nerve fibers of the dorsal mesentery, mesothelial cells and fibroblasts may nurse and perhaps guide PGCs during their ascent.

Female↗

Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.

Animals↗

Granulosa cell apoptosis induced at the penultimate stage of follicular development is associated with increased levels of Fas and Fas ligand in the rat ovary.

Apoptosis of granulosa cells is the cellular mechanism of ovarian follicular atresia, and cytokines have been implicated as potential atretogenic factors. We therefore investigated the possible role of the cytokine Fas ligand (FasL) and its receptor Fas in apoptosis during ovarian follicular atresia induced by gonadotropin withdrawal. Immature rats pretreated with eCG were injected 24 h later with an antiserum generated against eCG (antibody group) or preimmune rabbit serum (control), and ovaries were removed 1 and 24 h after treatment. The eCG antiserum caused a dose-dependent inhibition of the significant increase in ovarian weight observed between 24 and 48 h after eCG treatment. In situ detection of fragmented DNA in histological sections identified cell death in atretic but not healthy small and medium-sized antral follicles of the antibody group. Cell death was distributed in a scattered pattern throughout the granulosa cell layer of small atretic follicles but was localized primarily in granulosa cells lining the antral cavity of atretic medium antral follicles. Immunohistochemistry of adjacent histological sections revealed intense positive immunostaining for Fas and FasL in granulosa cells of atretic small and medium antral follicles in a pattern coincidental to the localization of cell death. Intense FasL staining was evident in the theca cells of healthy small antral follicles. An increase in low molecular weight DNA (DNA "ladders") indicative of apoptosis was evident in granulosa cells of the antibody group. Western analysis demonstrated increased levels of both Fas and FasL in the granulosa cells of the antibody group. These results demonstrate that both Fas and FasL are present in ovarian granulosa cells and that FasL may be the signal that induces granulosa cell apoptosis during atresia at the penultimate stage of ovarian follicular development.

Animals↗

Composite mucinous and granulosa cell tumor of the ovary.

A composite mucinous and granulosa cell tumor of the ovary in a 76-year-old woman is herein reported. At laparotomy this tumor proved to be a solid and cystic mass measuring 10 cm in greatest diameter. Many of the cysts were lined with a benign mucinous epithelium of the endocervical type, and solid areas contained a proliferation of granulosa cells. These two disparate components were intimately mixed. A theca cell component was also present in areas adjacent to the mucinous epithelium. The coexistence of mucinous and granulosa cell tumor is extremely rare and only four such cases have previously been reported in the literature, and the histogenesis of this tumor has not yet been elucidated. In the present case it is suggested that the granulosa cell element commenced as a reactive stromal hyperplasia in the wall of the pre-existing mucinous neoplasm and thereafter progressed to the point of producing a tumor-like mass or neoplastic changes.

Adenocarcinoma, Mucinous↗

Cellular distribution of endothelin-converting enzyme-1 in human tissues.

Endothelin-converting enzyme-1 (ECE-1) is the key enzyme of endothelin biosynthesis, catalyzing the final processing step. As shown by the targeted disruption of the ECE-1 gene, mature endothelins must be produced at specific sites for normal embryonic development. Therefore, it is important to know the exact pattern of ECE-1 gene expression. In this study we investigated the cellular distribution of ECE-1 in a variety of human tissues by in situ hybridization and immunohistochemistry. Widespread expression of the ECE-1 gene was noted, with a similar distribution pattern for mRNA and protein in normal human tissues, suggesting a major biological role for ECE-1. ECE-1 levels were particularly high in the cardiovascular, reproductive, and endocrine systems. There was strong and consistent labeling for ECE-1 in the vascular endothelial cells of all organs examined and in various nonvascular cells, especially some glandular cells. A large amount of ECE-1 protein and mRNA was detected in the Leydig cells of the testis and in the granulosa and theca cells of the ovary. In the adrenal gland, ECE-1 was detected in the cortex and medulla, with the strongest labeling in the zona glomerulosa. Therefore, ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. These results point out the potential side effects of ECE-1 inhibitors that are currently under development for treatment of cardiovascular diseases. (J Histochem Cytochem 47:447-461, 1999)

Adolescent↗

Adenosine 5'-monophosphate-activated protein kinase regulates progesterone secretion in rat granulosa cells.

The AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism involved in fatty acid and cholesterol synthesis. In the ovary, cholesterol plays a key role in steroid production. We report the presence of AMPK in rat ovaries, and we have investigated its role in granulosa cells. We show using RT-PCR and Western blot that the mRNAs for the alpha1/2 and beta1/2 subunits and the proteins are found in the ovaries. Immunohistochemistry localized the alpha1 AMPK subunit in granulosa cells, corpus luteum, and oocyte and less abundantly in theca cells. Treatment with 1 mm 5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKalpha1 on Thr172 in primary granulosa cells. Simultaneously, phosphorylation of acetyl-coenzyme A carboxylase at Ser79 was also increased. AICAR treatment for 48 h halved progesterone secretion, 3beta-HSD protein and mRNA levels, and phosphorylation of both basal MAPK ERK1/2 and p38 and in response to IGF-I and/or FSH in granulosa cells. AICAR treatment (1 mM) had no detectable effect on basal and FSH- and/or IGF-I-induced estradiol production and on granulosa cell proliferation or viability. Adenovirus-mediated expression of dominant negative AMPK totally abolished the effects of AICAR on progesterone secretion, 3beta-HSD protein production, and MAPK ERK1/2 and p38 phosphorylation. Moreover, we showed using specific in- hibitors of ERK1/2 and p38 MAPK that the MAPK ERK1/2 and not p38 is involved in progesterone secretion and 3beta-HSD expression, strongly suggesting that the activation of AMPK in response to AICAR reduces progesterone production through the MAPK ERK1/2 signaling pathway in rat granulosa cells.

AMP-Activated Protein Kinases↗

Targeted disruption of the Insl3 gene causes bilateral cryptorchidism.

The sexual dimorphic position of the gonads in mammals is dependent on differential development of two ligaments, the cranial suspensory ligament (CSL) and the gubernaculum. During male embryogenesis, outgrowth of the gubernaculum and regression of the CSL result in transabdominal descent of the testes, whereas in the female, development of the CSL in conjunction with failure of the gubernaculum development holds the ovaries in a position lateral to the kidneys. Several lines of evidence suggest that regression of the CSL and induction of gubernaculum development are mediated by testosterone and a yet unidentified testicular factor, respectively. The Insl3 gene (originally designated Ley I-L), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the postnatal ovary. Here we show that male mice homozygous for a targeted deletion of the Insl3 locus exhibit bilateral cryptorchidism with free moving testes and genital ducts. These malformations are due to failure of gubernaculum development during embryogenesis. In double-mutant male mice for Insl3 and androgen receptor genes, testes are positioned adjacent to the kidneys and steadied in the abdomen by the CSL. These findings demonstrate, that the Insl3 induces gubernaculum development in an androgen-independent way, while androgen-mediated regression of the CSL occurs independently from Insl3.

Androgens↗

Regulation of gene expression in endothelial cells: the role of human follicular fluid.

A precise regulation of angiogenesis is a prerequisite for an adequate maturation of ovarian follicles. Despite the production of vascular endothelial growth factor (VEGF) by granulosa cells in antral follicles, angiogenesis is restricted to the theca cell layer. The maturing follicle remains avascular before ovulation, implying regulatory mechanisms which prevent premature follicular vascularization. In order to investigate the role of follicular fluid and of granulosa cells in the regulation of endothelial gene expression, human umbilical vein endothelial cells (HUVECs) were incubated in vitro with media conditioned with human follicular fluid obtained from individual patients undergoing oocyte retrieval for in vitro fertilization procedures or with culture medium conditioned by human granulosa cells respectively. Using microarray technology, the gene expression pattern was compared between untreated monolayers of HUVECs and HUVECs treated either with follicular fluid or with granulosa cell conditioned media. We identified a total of 15 genes that were significantly up-regulated and 11 genes that were significantly down-regulated in endothelial cells treated with follicular fluid at least 2.5-fold in more than 70% of comparisons. Up-regulated genes involved in angiogenesis were the anti-angiogenic factors gro-beta (16.5-fold), angiopoietin-2 (3.9-fold), alpha-2-macroglobulin (24.3-fold) and the pro-angiogenic factors E-selectin (5.3-fold) and vascular cell adhesion molecule-1 (VCAM-1) (4.4-fold), whereas a significant down-regulation of the pro-angiogenic genes fibulin-5 (3.5-fold) and elastin (14.9-fold) could be observed. Culturing of HUVECs with conditioned medium from cultured human luteinized granulosa cells demonstrated a similar regulatory pattern of gene expression for fibulin-5, elastin, gro-beta, and E-selectin. The gene regulation in endothelial cells by follicular fluid could be confirmed by RT-PCR for gro-beta, angiopoietin-2, elastin, fibulin-5, and E-selectin. The present work reveals that compounds secreted by granulosa cells lead to the expression of anti-angiogenic factors on the transcript level in endothelial cells and thus could help to explain the temporal and spatial discrepancy between the high expression of VEGF and the restricted angiogenesis in the preovulatory follicle.

Down-Regulation↗

Induced luteal regression: differential effects on follicular and luteal inhibin/activin subunit mRNAs in the marmoset monkey.

During the luteal phase of the primate ovulatory cycle the predominant inhibin/activin subunit mRNAs produced by the corpus luteum and antral follicles are those for the alpha- and beta B-subunits respectively. The control of expression of these mRNAs and the resultant nature of the endocrine and paracrine signals which they may potentially generate has yet to be elucidated. Inhibin/activin subunit mRNAs may have a role in both the paracrine regulation of follicular and luteal function and modulation of FSH secretion. The aim of this study was to investigate the expression of inhibin/activin subunit mRNAs following luteal regression induced by either withdrawal of LH support (GnRH antagonist treatment), or by a direct inhibitory action (prostaglandin administration). Marmoset monkeys with regular ovulatory cycles were treated on day 8 and 9 of the luteal phase with either GnRH antagonist, prostaglandin or vehicle (n = 3 per group). Ovaries were studied 48 h after onset of treatment (on day 10 of the luteal phase) by hybridizing frozen tissue sections with radiolabelled riboprobes specific to the inhibin/activin alpha-, beta A- and beta B-subunit mRNAs. After autoradiographic exposure, grain concentrations were quantified by image analysis. In corpora lutea from control marmosets there was high expression of alpha-mRNA with only marginal expression of beta B-mRNA. Corpora lutea in animals treated with GnRH antagonist or prostaglandin had markedly reduced expression of alpha-mRNA while beta B-mRNA was unchanged. In controls, all healthy antral follicles exhibited a high level of expression of beta B-mRNA in the granulosa cells and low expression of alpha-mRNA in theca cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Activins↗