Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes.
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The functions of estrogen receptors (ERs) in mouse ovary and genital tracts were investigated by generating null mutants for ERalpha (ERalphaKO), ERbeta (ERbetaKO) and both ERs (ERalphabetaKO). All ERalphaKO females are sterile, whereas ERbetaKO females are either infertile or exhibit variable degrees of subfertility. Mast cells present in adult ERalphaKO and ERalphabetaKO ovaries could participate in the generation of hemorrhagic cysts. Folliculogenesis proceeds normally up to the large antral stage in both ERalphaKO and ERbetaKO adults, whereas large antral follicles of ERalpha+/-ERbetaKO and ERalphabetaKO adults are markedly deficient in granulosa cells. Similarly, prematurely developed follicles found in prepubertal ERalphaKO ovaries appear normal, but their ERalphabetaKO counterparts display only few granulosa cell layers. Upon superovulation treatment, all prepubertal ERalphaKO females form numerous preovulatory follicles of which the vast majority do not ovulate. The same treatment fails to elicit the formation of preovulatory follicles in half of the ERbetaKO mice and in all ERalpha+/-/ERbetaKO mice. These and other results reveal a functional redundancy between ERalpha and ERbeta for ovarian folliculogenesis, and strongly suggest that (1) ERbeta plays an important role in mediating the stimulatory effects of estrogens on granulosa cell proliferation, (2) ERalpha is not required for follicle growth under wild type conditions, while it is indispensable for ovulation, and (3) ERalpha is also necessary for interstitial glandular cell development. Our data also indicate that ERbeta exerts some function in ERalphaKO uterus and vagina. ERalphabetaKO granulosa cells localized within degenerating follicles transform into cells displaying junctions that are unique to testicular Sertoli cells. From the distribution pattern of anti-Mullerian hormone (AMH) in ERalphabetaKO ovaries, it is unlikely that an elevated AMH level is the cause of Sertoli cell differentiation. Our results also show that cell proliferation in the prostate and urinary bladder of old ERbetaKO and ERalphabetaKO males is apparently normal. (+info)
Human Mullerian-inhibiting substance promoter contains a functional TFII-I-binding initiator.
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Mullerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I. (+info)
Hormones in male sexual development.
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Classical embryology has provided a clear view of the timing and hormonal cues that govern sexual differentiation. Molecular biology has added important details to this picture. The cloning of SRY, MIS, and INSL3 provide insight into the molecular signals that provide important cues at the cellular level. Continued understanding of these pathways may provide the necessary information to one day reverse defects of sexual differentiation. (+info)
Mullerian inhibitory substance induces growth of rat preantral ovarian follicles.
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Mullerian inhibitory substance (MIS), also known as anti-Mullerian hormone, is best known as the hormone that regulates the regression of the Mullerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Mullerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis. (+info)
Differences in follicular function of 3-month-old calves and mature cows.
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After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Mullerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Mullerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Mullerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Mullerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation. (+info)
Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo.
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Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer. (+info)
Mullerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents.
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Mullerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect. (+info)
Nuclear receptor Dax-1 represses the transcriptional cooperation between GATA-4 and SF-1 in Sertoli cells.
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A crucial step in mammalian sex differentiation is the regression of the Mullerian ducts in males. This is achieved through the action of Mullerian inhibiting substance (MIS), a key hormone produced by fetal Sertoli cells. Proper spatiotemporal expression of the MIS gene requires the concerted action of several transcription factors that include Sox9, SF-1, WT-1, GATA-4, and Dax-1. Indeed, SF-1 contributes to MIS gene expression by transcriptionally cooperating with other factors such as GATA-4 and WT-1. Dax-1 is coexpressed with SF-1 in many tissues, including the gonads, where it acts as a negative modulator of SF-1-dependent transcription. We now report that Dax-1 can repress MIS transcription in Sertoli cells by disrupting transcriptional synergism between GATA-4 and SF-1. Dax-1-mediated repression of GATA-4/SF-1 synergism did not involve direct repression of GATA-dependent transactivation, but rather, it occurred through a direct protein-protein interaction with DNA-bound SF-1. It is interesting that SF-1, Dax-1, and GATA factors are coexpressed in several tissues such as the pituitary, the adrenals, and the gonads. Because we have shown that other GATA family members also have the ability to synergize with SF-1, Dax-1 repression of GATA/SF-1 synergism may represent an important mechanism for fine-tuning the regulation of SF-1-dependent genes in multiple target tissues. (+info)