BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and ...predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 µM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1β was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in α-minimum essential medium (α-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1β mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1β expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.
BACKGROUND: Follicular fluid meiosis‐activating sterol (FF‐MAS) overcomes hypoxanthine (HX)‐mediated meiotic arrest in mammalian oocytes. METHODS: In order to determine whether chromosome segregation ...was normal in oocytes matured in FF‐MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX‐arrested mouse oocytes cultured in the absence or presence of FF‐MAS. RESULTS: FF‐MAS‐induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis (∼40 and ∼90% polar body formation respectively). The majority of oocytes stimulated by FF‐MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX‐arrested oocytes cultured with or without FF‐MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX‐arrested oocytes cultured in the presence of FF‐MAS but not lanosterol. CONCLUSIONS: FF‐MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF‐MAS‐stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF‐MAS appears to protect mammalian oocytes from precocious chromatid segregation.
BACKGROUND: In the context of mammalian oocyte maturation, it has been suggested that intermediates of cholesterol biosynthesis may represent the physiological signal that instructs the oocyte to ...reinitiate meiosis. METHODS: Endogenous levels of follicular fluid meiosis‐activating sterol (FF‐MAS) were monitored in rabbit ovarian tissue, and the influence of exogenous gonadotrophins on sterol formation was assessed. The involvement of cAMP in FF‐MAS‐induced versus spontaneous oocyte maturation in vitro in mice was also investigated, as was the direct microinjection of FF‐MAS into mouse oocytes. RESULTS: Levels of FF‐MAS in rabbit ovaries were significantly elevated 1 h after hCG/LH induction and remained so for 4 and 12 h after induction. In naked oocytes undergoing spontaneous maturation, a significant decrease in cAMP was detected after 30 min of culture. However, FF‐MAS‐mediated induction of oocyte maturation in hypoxanthine‐arrested naked oocytes was not associated with any detectable decrease in intracellular cAMP levels. Microinjected FF‐MAS failed to induce any noticeable meiosis. CONCLUSIONS: A rapid increase in FF‐MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice.
Other isotype-selective estrogen receptor (ER) agonists, the selective ERα agonist 3,17-dihydroxy-19-nor-17α-pregna-1,3,5 (10)-triene-21, 16α-lactone and the selective ERβ agonist 8-vinylestra-1,3,5 ...(10)-triene-3,17β-diol, were used in hypophysectomized rats, gonadotropin-releasing hormone antagonist-treated mice, as well as intact rats to elucidate the effects of isotype-selective estrogens on the physiology of folliculogenesis and ovulation. In hypophysectomized rats and gonadotropin-releasing hormone antagonist-treated mice, the ERβ agonist caused stimulation of early folliculogenesis, a decrease in follicular atresia, induction of ovarian gene expression, and stimulation of late follicular growth, accompanied by an increase in the number of ovulated oocytes similar to 17β-estradiol (E2). In contrast, the ERα agonist had little or no effect on these parameters, implying that direct estrogen effects on ovarian follicular development are mediated by ERβ. In intact rats, E2 and the ERα agonist dose-dependently inhibited ovulation, in contrast to the ERβ agonist. On the other hand, the ERβ agonist did not stimulate uterine weight in intact rats, in contrast to E2 and the ERα agonist. This finding is in line with the assumption that estrogen mediated ovulation inhibition and stimulation of uterine growth are mediated by ERα but not by ERβ.
Meiosis-activating sterols (MAS) have been found to induce meiotic maturation in mouse oocytes in vitro. In the present study we have extended these observations by investigating the effects of ...follicular fluid MAS (FF-MAS) on rat oocyte maturation in vitro and ex vivo. Rat oocytes freed from their follicles were cultured with FF-MAS (0 μM, 1 μM, 3 μM, 10 μM, 30 μM) for 22 h in a medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 250 μM). A dose-dependent significant increase in germinal vesicle breakdown (GVB) was observed after adding FF-MAS to the culture medium in both cumulus-enclosed (CEO) and denuded (DO) oocytes. A time course study (0, 3, 8, 14, and 22 h) showed a significant increase in GVB after 14 h when DO and CEO were cultured in the presence of 10 μM FF-MAS + 250 μM IBMX. Furthermore immature rats were primed with eCG (20 IU) and 48 h later perfused ex vivo for 12 h in a recirculating system with either FF-MAS (0 μM, 10 μM, 30 μM, 60 μM), cholesterol (60 μM), or LH (0.2 μg/ml) in the presence of 200 μM IBMX, respectively. In addition, ovarian perfusion was carried out with FF-MAS (30 μM, 60 μM) or 0.2 μg/ml LH in the absence of IBMX. After 12 h, oocytes were freed from the ovaries and checked for GVB. By using the ex vivo perfused rat ovary, we found that FF-MAS, starting at 30 μM, was dose-dependently able to overcome IBMX-induced meiotic arrest leading to a comparable increase in GVB as was observed for LH. Furthermore, it was found that FF-MAS in the absence of IBMX was also able to induce meiotic maturation. Our data are consistent with the notion that the maturation-inducing effects of FF-MAS are mediated by different mechanisms compared to spontaneous maturation.
The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS follicular-fluid meiosis-activating sterol) from
human follicular fluid has recently been identified as a compound that induces ...the resumption of meiosis. FF-MAS and various
oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological
activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate
whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent
transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with
FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes
were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100
mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of
germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in
both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed
similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both
the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol,
25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation
compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported
here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in vitro in both
NkO and CEO irrespective of the inhibitory substance used. In contrast, the oxysterols and cholesterol had no significant
biological activity on this oocyte function, and consequently we found no correlation between LXRalpha activation and meiosis
stimulation.
ABSTRACT—Both known estrogen receptors, ERα and ERβ, are expressed in blood vessels. To gain further insight into the role of ERα in a functional setting, we investigated the effect of the novel ...highly selective ERα agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17β-estradiol (E2, 2 μg/kg body wt per day), the selective ERα agonist Cpd1471 (30 μg/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (Rmax, 53%±3% versus sham, 79%±2%; P <0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (Rmax, 70%±2%; resp, 73%±2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53±2% versus 77%±5%; P <0.01) and was normalized by both E2 (70%±2%) and Cpd1471 (70%±3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ERα agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ERα activation mediates positive vascular effects.
Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic
maturation of mouse oocytes in vitro. We therefore compared ...FF-MAS-induced maturation of naked mouse oocytes arrested in prophase
I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame
the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively.
We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes
by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a
barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small
cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that
in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations
suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a
delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly
and by delaying CG release, which is known to contribute to reduced fertilization.
Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone ...concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.
PDF
