Follicular Fluid Meiosis-activating Sterol Research Articles

Follicular fluid meiosis-activating sterol prevents porcine ovarian granulosa cells from hypoxia-induced apoptosis via inhibiting STAT4 expression.

Follicular fluid meiosis-activating sterol (FF-MAS) is a small molecule compound found in FF, named for its ability to induce oocyte resumption of meiosis. Granulosa cells (GCs) within the follicle are typically located in a hypoxic environment under physiologic conditions due to limited vascular distribution. Previous research suggests that hypoxia-induced cell cycle arrest and apoptosis in GCs may be crucial triggering factors in porcine follicular atresia. However, the impact of FF-MAS on GCs within follicles has not been explored so far. In this study, we uncovered a novel role of FF-MAS in facilitating GC survival under hypoxic conditions by inhibiting STAT4 expression. We found that STAT4 expression was upregulated in porcine GCs exposed to 1% O2. Both gain and loss of function assays confirmed that STAT4 was required for cell apoptosis under hypoxia conditions, and that the GC apoptosis caused by hypoxia was markedly attenuated following FF-MAS treatment through inhibition of STAT4 expression. Correlation analysis in vivo revealed that GC apoptosis was associated with increased STAT4 expression, while the FF-MAS content in follicular fluid was negatively correlated with STAT4 mRNA levels and cell apoptosis. These findings elucidate a novel role of FF-MAS-mediated protection of GCs by inhibiting STAT4 expression under hypoxia, which might contribute to the mechanistic understanding of follicular development.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-β family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. None. Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.

Follicular Fluid Meiosis-Activating Sterol in Assisted Reproductive Techniques: A Systematic Review and Meta-analysis of Randomized Controlled Trials

Background: Follicular fluid meiosis-activating sterol (FFMAS) is one of several molecular compounds that has been added into in vitro maturation (IVM) technique with contradictory results. Our study aimed to investigate the effects of FFMAS in assisted reproductive technology (ART). Methods: We searched systematically in PubMed, Web of Science, Scopus, Cochrane Registry of clinical trials, WHO registry of clinical trials, clinicaltrials.gov, Google Scholar until July 2017. Meta-analysis was used to investigate the efficacy and safety outcomes of FFMAS. Following the retrieval of potential articles, two independent reviewers screened and extracted included papers rigorously. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were computed for the individual outcome when data was available. Quality of included studies was assessed using Cochrane collaboration tool. Results: A total of seven randomized controlled trials involving 1198 participants with 3105 oocytes were explored in this study. Most of the studies were at low risk of bias. Our random effects model meta-analyses in maturation and abnormal embryo rate between FFMAS-exposed oocytes compared with controls revealed no significant differences (summary OR 1.00, 95% CI 0.46–2.16, [Formula: see text] value = 0.99 and summary OR was 1.31, 95% CI 0.84–2.04, [Formula: see text] value = 0.23, respectively). Beside, our study showed a significant increase in uniformly abnormal embryo rate in FFMAS group compared with vehicle control group (summary OR 1.98, 95% CI 1.09–3.61, [Formula: see text] value = 0.03). No significances were noted on meta-analyses of normal embryo rate, uniformly abnormal embryo rate, uniformly normal embryo rate, mosaic embryo rate, abnormal blastomere rate, normal blastomere rate, aneuploidy mosaic embryo rate, and chaotic mosaic embryo rate. Conclusions: FFMAS showed no efficacy in maturation process in human oocytes and there was some evidence for detrimental effects in comparison to vehicle controls. We discouraged any further trials due to safety concern.

Follicular fluid meiosis-activating sterol (FF-MAS) promotes meiotic resumption via the MAPK pathway in porcine oocytes

Follicular fluid meiosis-activating sterol (FF-MAS) exerts beneficial effects on the meiotic resumption of mammalian oocytes and their subsequent early embryonic development, but the signaling pathway underlying these effects has not been elucidated. Therefore, the objective of the present study was to investigate whether the mitogen-activated protein kinase (MAPK) pathway is involved in the FF-MAS-induced in vitro resumption of meiosis in porcine oocytes. Porcine cumulus oocyte complexes (COCs) were allocated in several groups cultured in TCM-199 medium with different concentration of AY 9944-A-7 (20, 30, 40 μmol/L) or ketoconazole (20 μmol/L) to increase or decrease endogenous accumulation of FF-MAS. Each experimental condition was repeated at least six times. After maturation for 44 h, the resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) and the first polar body (PBI) extrusion, The relative expressions of related genes in MAPK pathway [c-mos, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK1/2)] at both transcriptional and translational levels were detected to investigate the kinetic trend of expression throughout oocyte maturation in vitro in response to the addition of AY 9944-A-7 or ketoconazole to the maturation medium. Results indicated that AY 9944-A-7 promoted, while ketoconazole inhibited, the in vitro maturation (IVM) of porcine oocytes. Relative expression of meiosis related genes was upregulated by AY 9944-A-7 and downregulated by ketoconazole. With extended culturing time, c-mos mRNA expression levels reached their peak at 12 h of maturation and decreased gradually thereafter, while MEK, ERK1 and ERK2 expression increased after an initial decrease peaking at 44 h of culture in the AY 9944-A-7-group. And the trend of the protein expression of c-mos, MEK, ERK1/2 was basically consistent with the mRNA expression of these genes. These results imply that the endogenous accumulation of FF-MAS is beneficial to resumption of meiosis in porcine oocytes and that MAPK signaling is involved in FF-MAS-induced meiotic resumption.

Selected cholesterol biosynthesis inhibitors produce accumulation of the intermediate FF-MAS that targets nucleus and activates LXRα in HepG2 cells

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners.

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

Effects of AY9944 A-7 on gonadotropin-induced meiotic resumption of oocytes and development of parthenogenetic embryos in sheep

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus–oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7–induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes.

AY9944 A-7 promotes meiotic resumption and preimplantation development of prepubertal sheep oocytes maturing in vitro

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 μM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 μM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 μM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 μM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro.

Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro

Meiosis activating sterol, produced directly by lanosterol 14-alpha-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins' induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs' meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.

Stage-specific Expression of Lanosterol 14-Demethylase in Mouse Oocytes in Relation to Fertilization and Embryo Development Competence

Follicular fluid meiosis-activating sterol (FF-MAS) has been suggested as a positive factor which could improve the oocyte quality and subsequent embryo development after in vitro fertilization. However, FF-MAS is a highly lipophilic substance and is hard to detect in studying the relationship between MAS and quality of oocyte maturation. The present study focused on the expression of lanosterol 14α-demethylase (LDM), a key enzyme that converts lanosterol to FF-MAS, on mouse oocyte maturation and its potency on development. LDM expression was strong in gonadotropin-primed germinal vesicle stage oocytes, weak after germinal vesicle breakdown (GVBD), and then strong in MII stage oocytes. The LDM-specific inhibitor azalanstat significantly inhibited oocyte fertilization (from 79.4% to 68.3%, p<0.05). Also, azalanstat (5 to 50 μM) decreased the percentage of blastocyst development dose-dependently (from 78.7% to 23.4%, p<0.05). The specific inhibition of sterol Δ14-reductase and A7-reductase by AY9944 accumulates FF-MAS and could increase blastocyst development rates. Additionally, in the AY9944 group, the rate of inner cell mass (ICM)/ total cells was similar to that of in vivo development, but the rate was significantly decreased in azalanstat treatment. In conclusion, LDM, the key enzyme of FF-MAS production, may play an important role in fertilization and early development of the mouse embryo, especially in vitro.

Effect of rescuing donated immature human oocytes derived after FSH/hCG stimulation following in vitro culture with or without Follicular Fluid Meiosis Activating Sterol (FF-MAS)—an embryo chromosomal and morphological analysis

Studies in mice and humans have shown that Follicular Fluid - Meiosis Activating Sterol (FF-MAS) induces meiotic maturation of immature oocytes in vitro. A multicenter, prospectively randomised study evaluated chromosomal status of embryos from FSH/hCG primed human immature oocytes, cultured with or without FF-MAS. Denuded immature oocytes (n=365) were randomly allocated into inert control, FF-MAS 5 microM or 20 microM. Seventy +/-2 hours after ICSI on matured oocytes, all cleaved embryos were fixed for fluorescence in situ hybridisation analysis. Only 15% of oocytes resulted in cleaved embryos. GV oocytes matured at significantly lower rates (14% and 7%) in the two FF-MAS groups compared to the inert control group (47%). High rates of chromosomal abnormalities were found in all groups. Immature oocytes showed poor development with high rates of embryo chromosomal abnormalities. Exposure to FF-MAS in the concentrations, duration and/or formulation used in this study did not improve the results.

Principal findings from a multicenter trial investigating the safety of follicular-fluid meiosis-activating sterol for in vitro maturation of human cumulus-enclosed oocytes

To evaluate the safety of applying follicular-fluid meiosis-activating sterol (FF-MAS) in vitro to immature human oocytes. Phase I bicenter, randomized, parallel-group, controlled, partially blinded trial. Third-level referral academic centers, including reproductive biology and genetics laboratories. Endocrinologically normal women with a medical indication for IVF or intracytoplasmic sperm injection, or healthy volunteers. Subjects were randomized at a ratio 1 to 6 into either conventional GnRH-agonist and recombinant FSH stimulation (IVO) for oocyte retrieval, or minimally stimulated in vitro maturation (IVM) with the use of recombinant FSH. Retrieved immature oocyte cumulus complexes were cultured for 30 or 36 hours in one of six IVM culture conditions containing FF-MAS (range, 0.1-20 microM). Polar body-extruded oocytes from the IVO and IVM groups were processed for chromosomal analysis. The primary endpoint was the incidence of metaphase II stage oocytes with numeric chromosomal abnormalities, using full (spectral karyotyping) or partial (fluorescent in situ hybridization with seven probes) karyotyping or Giemsa count. A secondary objective was to document the frequency of metaphase II oocytes after IVM with FF-MAS supplements. Oocyte cumulus complexes obtained from the IVO (mean, 8.9) and IVM (mean, 6.2) groups had equal maturation rates. Compared to IVO, exposure of germinal-vesicle oocytes for a maturation period of 30 hours did not increase aneuploidy. An exposure period of 36 hours doubled the aneuploidy rate, but this was significant only for the 20-muM dose of FF-MAS. Inclusion of 1-10 microM FF-MAS in a 30-hour IVM protocol is safe.

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes

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. 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 microM 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 SMC1beta was compared between oocytes matured with or without FF-MAS. 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 alpha-minimum essential medium (alpha-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 Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. 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 Smc1beta 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.

Follicular sterol composition in gonadotrophin stimulated women with polycystic ovarian syndrome

This is the first study evaluating whether oocyte development and fertilization competence are related to intrafollicular concentration of cholesterol, meiosis-activating sterols and progesterone, after human chorionic gonadotrophin (HCG) administration of women with polycystic ovarian syndrome (PCOS). The concentration of follicular fluid meiosis-activating sterol (FF-MAS) significantly increased in the periovulatory period from 10–14 to 34–38h after HCG administration, while the concentration of testis meiosis-activating sterol (T-MAS) decreased, suggesting a HCG-dependent inhibition of sterol Δ14-reductase. There was no correlation between follicular lanosterol, FF-MAS, T-MAS, and progesterone concentrations and the presence or absence of MII oocytes. Interestingly, free cholesterol level was significantly lower and FF-MAS/cholesterol and progesterone/cholesterol ratios significantly higher in follicles containing MII oocytes compared to follicles from which oocytes were not retrieved. Yet, fertilization and embryo quality did not correlate with follicular sterols. This knowledge should be beneficial for the implementation of protocols for in vitro maturation process, usually used in PCOS patients.

Effect of Garcinia cambogia Administration on Female Reproductive Organs in Rats

We investigated the effect of administering Garcinia cambogia (GA), which contains (−)-hydroxycitric acid ((−)-HCA), to female rats, focusing especially on changes in sexual hormones and its safety in terms of histopathological changes. A diet containing a high level of (−)-HCA (154 mmol/kg diet) was given to seven-week-old female rats with matched estrous cycles for two or four weeks. There were no significant differences in any of the serum hormones tested (follicle-stimulating hormone, luteinizing hormone, estradiol and progesterone) between the control group and the GA group during the observation period. Also, there were no abnormal morphological findings in the follicle and corpus luteum, nor were there any significant differences in the ovarian 4,4-dimethyl-5α-cholesta 8,14,24-triene-3β-ol (follicular fluid meiosis-activating sterol; FF-MAS) and 4,4-dimethyl-5α-cholesta-8,24-diene-3β-ol (testis meiosis-activating sterol; T-MAS) concentrations between the control and the GA groups. On the other hand, the final body weight of the GA group was statistically lower than that of the control group. The weight of abdominal fat in the GA group was markedly lower than that in the control group even after two weeks of feeding. Therefore, although we had confirmed in our previous study that GA had a marked effect on sexual maturation in male rats, no such deleterious influence was observed in the female rats.

Impact of follicular-fluid meiosis-activating sterol in an albumin-based formulation on the incidence of human pre-embryos with chromosome abnormalities

To evaluate the effect of adding follicular-fluid meiosis-activating sterol (FF-MAS) in a novel 0.2% recombinant human albumin-based formulation to cumulus-enclosed oocytes on chromosomal status and development of pre-embryos. Multicenter, prospective, randomized, open (double-blind for vehicle and FF-MAS groups), four parallel groups, controlled trial. Four public IVF clinics in Denmark. Two hundred eighteen women undergoing IVF donated 483 oocytes. Follicle-stimulating hormone/hCG-primed cumulus-enclosed oocytes randomized to 4 hours of exposure to medium with 1 or 10 micromol/L of FF-MAS dissolved in 0.2% recombinant human albumin, medium with 0.2% recombinant human albumin (vehicle control), or medium alone (control) before insemination. Primary endpoint: incidence of human pre-embryos with chromosomal abnormalities. Secondary endpoint: fertilization rate, cleavage rate, and pre-embryo quality assessed after 68 hours of culture. At pre-embryo level, the overall abnormality rates in the control, vehicle control, and 1- and 10-micromol/L FF-MAS groups were 53%, 39%, 42%, 53%, respectively, and at blastomere level 49%, 44%, 44%, and 48%, respectively. After 20 and 26 hours, the fertilization rates were between 67% and 71% in all groups. No differences in the cleavage rates were observed. The concentrations of FF-MAS in a novel 0.2% recombinant human albumin-based formulation of FF-MAS did not increase the risk of chromosomal abnormalities in pre-embryos or blastomeres. No statistically significant differences in fertilization rate, cleavage rate, or number of good quality pre-embryos were found among the four groups.

Mouse oocyte meiotic resumption and polar body extrusion in vitro are differentially influenced by FSH, epidermal growth factor and meiosis-activating sterol.

In this study, we compared the relative ability of FSH (100 mIU/ml), epidermal growth factor (EGF) (10 ng/ml), and follicular-fluid meiosis-activating sterol (FF-MAS, 10 micromol/l) to induce meiotic resumption and polar body I (PBI) extrusion in mouse oocytes. Cumulus-enclosed oocytes (CEO) were co-incubated with meiosis-arresting agents, including 4 mmol/l hypoxanthine (Hx), 0.3 mmol/l dibutyryl cAMP (dbcAMP), and 8.5 micromol/l cilostamide, a selective inhibitor of the oocyte-specific phosphodiesterase 3 (PDE 3). In Hx-treated oocytes, FSH, EGF and FF-MAS induced meiosis resumption at very high rates, but only FSH and EGF also promoted PBI extrusion with high frequency. In experiments conducted in the presence of dbcAMP, FF-MAS was unable to promote an increase in germinal vesicle breakdown (GVBD) rate, whereas FSH and EGF generated a response similar to the Hx groups. Neither FSH, EGF nor FF-MAS caused any change in the meiotic status of CEO when meiotic arrest at the germinal vesicle (GV) stage was maintained by cilostamide. In the presence of Hx, naked oocytes (NkO) co-cultured with their cumulus cells were able to respond to the GVBD-inducing effect of FSH and EGF by resuming meiosis at high rate. Collectively, these results indicate that: (i) a signal triggered in cumulus cells by either FSH or EGF, but not necessarily coincident with FF-MAS, may contribute to meiotic maturation, supporting GVBD and extrusion of PBI; (ii) the transmission of this signal can occur in a paracrine fashion, at least with reference to the breakdown of the GV. It also appears that concomitant regulation of intra-oocyte cAMP degradation is a prerequisite for meiosis resumption.

A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro.

Follicular fluid-meiosis-activating sterol (FF-MAS) is a factor present in the pre-ovulatory follicle during the time of oocyte maturation. In mouse oocytes maturing in vitro, FF-MAS promotes the completion of meiotic maturation to metaphase II (MII) and improves competence to complete the 2-cell stage to blastocyst transition. We produced analogues of FF-MAS and selected three on the basis of potency to promote the resumption of meiosis by mouse oocytes maintained in meiotic arrest by hypoxanthine. The objective of this study was to determine whether these FF-MAS analogues also affect the quality of oocytes maturing in vitro with respect to the completion of meiotic maturation and augmenting the frequency of development to the blastocyst stage after fertilization in vitro. Cumulus cell-enclosed oocytes were isolated from the small antral follicles of 18 or 20 day post-natal mice. These oocytes normally have a reduced competence to complete meiotic maturation and preimplantation embryo development. Oocytes were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0.1% ethanol, 1 micromol/l FF-MAS, or 0.1-10 micromol/l FF-MAS analogues ZK255884 (884), ZK255933 (933) and ZK255991 (991). Oocytes that progressed to MII were fertilized in vitro and the percentage developing to the 2-cell and blastocyst stages was determined. At 1 micromol/l, 991 and 933 increased the portion of oocytes progressing to MII, whereas the lowest dose of 991 and 884 was ineffective. Treatment of maturing oocytes with either 0.1 or 1 micromol/l 933 dramatically increased oocyte competence to complete preimplantation development. The synthetic analogue of FF-MAS, ZK255933, is a potent agonist that improves the quality of mouse oocytes matured in vitro. This compound may therefore have therapeutic value for treatment of oocytes from women undergoing therapy for infertility owing to poor oocyte quality.

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes.

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus–oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 μM) or in combination with Butyrolactone-I (12.5 μM Rosco + 6.25 μM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4–9% blastocyst rate versus 14–17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 μM) of follicular fluid–meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73–86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.