Search

Examines whether meiosis-activating sterols (MAS) play a physiological role in the resumption of meiosis in the rat when using ketoconazole as an inhibitor of sterol synthesis. Inhibition of ovulation; Ketoconazole-treated ovaries; Maturation and degeneration of oocytes; Luteinizing hormone-stimulated oocyte maturation; Follicular progesterone production.

The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation., Competing Interests: The authors have declared that no competing interests exist.

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. Here we show that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. Our results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. We now show that Stat92E also promotes GSC differentiation. Our results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation., (© 2014. Published by The Company of Biologists Ltd.)

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.

Steroids mediate the gonadotropic stimulus of oocyte maturation in fish and amphibians. Such a role of steroids in mammals has not been confirmed until recently. A series of studies presented data suggesting that steroids might be involved in meiosis of mouse oocytes. Here we examined this suggestion using in vitro cultures of rat and mouse follicle-enclosed oocytes (FEOs) and cumulus-enclosed oocytes (CEOs). In FEOs that mature only in response to gonadotropins or other stimuli, we tested the ability of steroids to trigger meiosis and whether addition of steroid receptor antagonists blocks LH/human chorionic gonadotropin stimulation of meiosis. In CEOs that mature spontaneously, we tested whether steroid antagonists block maturation and whether steroids overcome the inhibition of maturation by hypoxanthine (Hx), a mild inhibitor of meiotic resumption. The progesterone antagonists mifepristone (RU 486) and Organon 31710 as well as the estrogen antagonist faslodex did not prevent LH-triggered maturation of rat or mouse FEOs or the spontaneous maturation of CEOs. In accordance, the progesterone agonist promegestone (R5020) and estradiol did not stimulate the resumption of meiosis in rat and mouse FEOs, and both did not overcome the Hx inhibition of meiosis in rat and mouse CEOs. Flutamide, an androgen antagonist, did block meiosis in rat FEOs, but this action could not be affected by adding dihydrotestosterone, suggesting that it was not androgen receptor mediated. Flutamide did not affect spontaneous maturation of rat CEOs, and dihydrotestosterone could not stimulate meiosis inhibited by Hx. Thus, in contrast to lower vertebrates, in mammals, steroids do not seem to serve as an obligatory signal by which the somatic cells of the follicle transfer the gonadotropic stimulation of meiosis to the oocyte.

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG., ((c) 2006 Wiley-Liss, Inc.)

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

In-vitro studies of mouse oocytes have provided evidence that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on resumption of meiosis. These sterols are synthesized by cytochrome P450 (CYP) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. Our studies in the rat with specific inhibitors and molecular approaches did not support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. (i) Specific inhibitors of MAS synthesizing enzymes did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. At higher doses, they caused degeneration of oocytes. (ii) The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. (iii) The preferential localization of LDM protein in the oocytes suggests MAS production in oocytes, rather than its transport from the somatic compartment as expected by the suggested role of MAS in the regulation of meiosis as a putative cumulus-oocyte signal molecule. (iv) AY-9944, which supposedly increases MAS levels by inhibiting its metabolism, induced the maturation of follicle-enclosed oocytes that was much delayed as compared with gonadotropic stimulation. Thus, the resumption of meiosis induced by added MAS [Biol. Reprod. 61 (1999) 1362, Biol. Reprod. 64 (2001) 418] or presumed endogenous MAS accumulation by AY-9944, resulted in oocyte maturation with remarkably slower kinetics than observed with LH stimulation. This delay in meiosis after MAS stimulation, the studies with LDM inhibitors and its spatial and temporal expression, cast serious doubts whether MAS is indeed mediating the meiosis inducing action of the gonadotropins, as suggested.

In vitro studies on mouse oocytes have shown that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on the resumption of meiosis. These sterols are synthesized by cytochrome P(450) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. We have used specific inhibitors of LDM, azalanstat (RS-21607) and RS-21745, to test whether MAS is an obligatory mediator in the resumption of meiosis in the rat. Addition of azalanstat and RS-21745 (1-200 microM) to culture medium of rat isolated cumulus-enclosed oocyte and preovulatory follicle-enclosed oocyte stimulated by LH/hCG did not allow separation between their inhibition of the resumption of meiosis and the degeneration of oocytes. In both models, doses of the drug that inhibited oocyte maturation also increased oocyte degeneration. The inhibitors only partially suppressed follicular progesterone production. We have examined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunocytochemistry the ovarian expression of LDM mRNA and protein during the preovulatory period. We did not find evidence for the stimulation of this enzyme by LH/hCG. The strongest staining by LDM antiserum was obtained in primordial and primary oocytes, and the staining was reduced with oocyte growth. In addition, strong LDM staining could be observed in some of the granulosa cells, especially of the corona radiata localized in close proximity to the oocyte. In conclusion, our results with specific inhibitors and molecular approaches do not reveal evidence to support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. Specific inhibitors of MAS synthesis did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. Much higher concentrations of the inhibitors, which affected meiosis, were detrimental to oocytes, leading to their degeneration. The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. Finally, the preferential localization of LDM protein to the oocytes suggests MAS production in oocytes rather than its transport from the somatic compartment as implied by the proposed role of MAS as a cumulus-oocyte signal molecule.

We have analyzed mouse fetuses and neonates for the presence of epidermal growth factor (EGF)-specific mRNA. No detectable EGF-specific mRNA was found in fetuses, fetal membranes, or placentae from Day 9 of gestation through birth or in the early neonatal period. While the kidneys begin to produce EGF specific transcripts by two weeks postpartum, the salivary glands begin to produce detectable levels of EGF mRNA only after weaning and even then at levels far below the adult amount. Reports of EGF and EGF-related material in rodent fetuses failed to determine whether this material was of maternal or fetal origin. We now conclude that authentic EGF in these embryos is probably of maternal origin. We have performed experiments designed to determine whether EGF can be transported into the fetus. A small percentage of 125I-EGF administered to pregnant females either systemically or directly into the uterine arteries reached the fetus itself. The uterus and the placenta attained a high level of labeling, whereas the amniotic fluid and yolk sac were virtually devoid of the tracer. In the neonatal period, milk may be the physiologically relevant source of EGF. We have found that 125I-EGF ingested by neonates was absorbed into the circulation, reached many internal organs, and was eventually excreted in the urine. Previously demonstrated EGF receptors in mouse embryonic cell types may be activated by either alpha type transforming growth factor or maternal EGF transported via the placenta.