Expression Of GDF9 Research Articles

Cilostamide and forskolin maintain gap junction function of incubated dog follicles

Disruption of the communication between the oocyte and granulosa cells is one of the major causes of poor development of in vitro grown ovarian follicles and oocytes. The present study investigated the effect of two cAMP modulators, cilostamide and forskolin, on in vitro growth of isolated dog secondary follicles and enclosed oocytes, communication between the gamete and surrounding granulosa cells, expression of GJA1 and GDF9, as well as cAMP level. Secondary follicles were incubated with cilostamide or forskolin alone or a combination of 20 μM cilostamide +1 μM forskolin, and the diameter of the incubated follicles and enclosed oocytes assessed every 72 h. Gap junction activity, GJA1 and GDF9 expression and cAMP level were assessed on Days 6 and 12 and transzonal projection (TZP) density was evaluated on Day 12. Neither cilostamide nor forskolin alone enhanced in vitro growth of dog follicles and the enclosed oocytes (P > 0.05). However, these two cAMP modulators dose dependently sustained gap junction activity and stimulated cAMP production compared with the non-supplemented control. Cilostamide at the high dosage (20 μM) also upregulated GJA1 expression. The combination of cilostamide and forskolin supported oocyte growth during the first 9 days and upregulated GJA1 and GDF9 expression at Day 12 of in vitro culture. This combination treatment also sustained gap junction activity, cAMP production, and increased TZP function (calcein intensity: TZP density ratio). The findings indicated that a combination of cilostamide and forskolin supported growth and survival of oocytes enclosed within cultured follicles by sustaining cAMP production and gap junction activity.

Quantitative expression of mRNA encoding BMP/SMAD signalling genes in the ovaries of Booroola carrier and non-carrier GMM sheep.

The GMM sheep is a carrier of Booroola fecundity (FecB) gene, which produces the twins and triplets in one lambing. The homozygous carrier GMM (FecBBB ), non-carrier GMM and Malpura (FecB++ ) ewes were synchronized by progesterone sponges, and the plasma progesterone concentration was measured by RIA. The results showed that the progesterone concentration did not differ significantly (p>.05) in homozygous carrier GMM (5.74±1.2ng/ml), non-carrier GMM (5.42±1.4ng/ml) and non-carrier Malpura ewes (5.67±1.5ng/ml). Further, quantitative expression of BMP factors/receptors and SMAD signalling genes were analysed in the ovaries of sheep by qRT-PCR. The study showed that the expression of BMP2 was slightly higher (p>.05) in carrier GMM than that of non-carrier GMM, but it was almost similar to Malpura ewes. Expression of BMP4 and BMP7 was significantly higher (p<.001; p<.05) in carrier GMM than that of non-carrier GMM and Malpura ewes. Although BMP6 expression was higher (p>.05) in carrier GMM than that of non-carrier GMM, but lower (p>.05) than the Malpura ewes. Expression of BMP15 (p<.05), GDF5 (p<.01) and GDF9 (p<.05) was significantly higher in carrier GMM than non-carrier GMM ewes. Surprisingly, BMPR1B expression was significantly higher (p<.001) in non-carrier GMM and Malpura than the carrier GMM ewes, while TGFβRI did not differ significantly (p>.05) among both GMM genotypes. On the other hand, expression of BMPR1A (p>.05) and BMPRII (p<.05) was higher in carrier GMM than the non-carrier GMM, but significantly lower (p<.001) than the Malpura ewes. It was interesting to note that the expression of SMAD1 (p>.05), SMAD2 (p<.001), SMAD3 (p<.05), SMAD4 (p<.001), SMAD5 (p<.001) and SMAD8 (p<.001) was lower in the carrier GMM than that of non-carrier GMM ewes. It is concluded that the FecB mutation alters the expression of BMPR1B and SMAD signalling genes in the ovaries of homozygous carrier GMM ewes.

Luteinizing Hormone effect on the GDF-9 and BMPR-1a Expression of Bovine Granulosa Cells culture

Growth Differentiation Factor-9 (GDF-9) and Bone Morphogenetic Protein Receptor 1a (BMPR-1a) was a member of the transforming growth factor-β (TGF-β) superfamily known to regulated ovarian functions in mammals. In addition, Luteinizing Hormone (LH) also has an important role in ovarian function. The objectives of this research are to observe effect of LH on the GDF-9 and BMPR-1a expression in granulosa cells (GCs) culture. Bovine ovaries were obtained from a slaughterhouse and carried to the laboratory within 30 min in a container kept at 37 °C in saline containing gentamicin and amphotericin. The ovaries were washed with pre-warmed PBS supplemented with gentamicin and amphotericin. Granulosa cells were collected from follicles by aspiration using a syringe with needle (20G). The cells were centrifuged for 1 min at 800g at room temperature and washed twice in DMEM/F12 medium and filtered through a stainless steel filter (100 um) to get the GCs then seeded in 6 well culture plates. The cells were cultured at 37 °C in a 5% CO2 atmosphere for 24 h and then the wells were washed with PBS to remove unattached cells. The culture medium was replaced with serum-free medium supplemented with LH at 100 ng/mL and cultured for 24 h. Total RNA was extracted from GCs using TRIsure. The RNA quality and quantity were estimated by using spectrophotometer at 260/280 nm. Aliquots (1 ug) of total RNA from each pool of GCs were independently reverse-transcribed to cDNA. The mRNA expression of GDF-9 and BMPR-1a was estimated by the quantitative real-time PCR method. The results showed that LH added to the culture medium can increase the expression of GDF9 and BMPR-1a of granulosa cells.

Intergenerational response of steroidogenesis-related genes to maternal malnutrition.

We sought to examine whether rat maternal food restriction (MFR) affects the expression of steroidogenesis-related genes Cyp19, Cyp17a1, Insl3 and Gdf-9 in the ovaries of offspring from the first (FRG1) and second (FRG2) generations at pre-pubertal age (week 4) and during adulthood (week 8). At week 4, MFR significantly increased the expression of RNAs for all analyzed genes in both FRG1 and FRG2 females, which may indicate that MFR affects the onset of the reproductive lifespan, by inducing early pubertal onset. At week 8, the Cyp19 gene was still upregulated in MRF-subjected animals (Cyp19: P=0.0049 and P=0.0508 in FRG1 and FRG2, respectively), but MFR induced a significant decrease in Cyp17 and Gdf-9 gene expression in the offspring of both FRG1 and FRG2 females when compared with the controls (Cyp17: P=0.0018 and P=0.0016, respectively; Gdf-9: P=0.0047 and P=0.0023, respectively). This suggests that females at week 8, which should normally be in their optimal reproductive capacity, experience premature ovarian aging. At week 4, the activation of Cyp19 and Cyp17 was higher in the FRG1 ovaries than in the FRG2 ovaries, whereas the extent of Insl3 and Gdf-9 activation was lower in the FRG1 ovaries. This may indicate that FRG2 females were more vulnerable to MFR than their mothers (FRG1) and grandmothers, which is consistent with the 'predictive adaptive response' hypothesis. Our findings reveal that MFR may induce intergenerational ovarian changes as an adaptive response to ensure reproductive success before death.

Effect of myostatin inhibitor (myostatin pro-peptide) microinjection on in vitro maturation and subsequent early developmental stages of buffalo embryo

Expression of myostatin (MSTN, also known as growth differentiation factor 8, GDF8) was recently detected in cumulus-oocytes complexes (COCs), however little is known about its role in in vitro maturation (IVM) and fertilization (IVF) in large animals. Therefore, this study was designed to investigate the effect of MSTN inhibition on IVM of buffalo oocytes through investigation of IVM efficiency and expression of some specific genes in COCs from IVM till subsequent developmental stages following IVF. To reach this goal, we prepared a construct of adeno-associated virus (AAV) carrying MSTN pro-peptides (AAV-MSTNP) to inhibit MSTN. Over-expression of MSTNP was verified by upregulated expression of MSTNP and downregulated expression of the TGFβ receptor ActRIIb, the TGFβ signal transducer SMAD2 in COCs using qPCR. Microinjection of AAV-MSTNP to oocytes before IVM yielded a significant decrease in maturation rate as revealed by less cumulus cells expansion, fewer oocytes reaching metaphase II, and downregulation of cumulus expansion-related genes pentraxin 3 (Ptx3) and prostaglandin-endoperoxide synthase 2 (Ptgs2) as compared to the control and vehicle groups. These changes were also accompanied by elevated intracellular reactive oxygen species (ROS), upregulated expression of the apoptotic Bax gene, reduced antioxidant enzymes (SOD, CAT, GPX) activities, and downregulated expression of the antioxidant gene nuclear factor erythroid 2 like 2 (Nrf2), and the anti-apoptotic gene Bcl2 in COCs after IVM. Overexpression of MSTN inhibitor, MSTNP, also inhibited GDF9 and BMP15 genes expression in COCs. Additionally, both the fertilization efficiency and cleavage and blastocyst rates were significantly lower in MSTNP group than in the control and vehicle groups. The obtained data suggest an important role for MSTN during IVM and the subsequent developmental stages probably through, at least in part, inhibition of ROS production and apoptosis and modulation of IVM-related gene expression in COCs.

Neonatal exposure to a glyphosate-based herbicide alters the histofunctional differentiation of the ovaries and uterus in lambs

The aim of the present study was to compare the effect of oral and subcutaneous exposure to a glyphosate-based herbicide (GBH) on the female reproductive system, specifically in the ovaries and uterus of prepubertal lambs. To this end, ewe lambs were exposed to a s.c. (n: 5) or an oral (n: 5) environmentally relevant dose of GBH (2 mg/kg/day) or to vehicle (controls, n: 12), from postnatal day (PND) 1 to PND14. Serum glyphosate and aminomethylphosphonic acid (AMPA) concentrations were measured on PND15 and PND45. The ovaries and uterus were obtained and weighed on PND45. Ovarian follicular dynamics and uterine morphological features were determined by picrosirius-hematoxylin staining. The proliferation marker Ki67 was evaluated by immunohistochemistry in ovarian and uterine samples. Glyphosate but not AMPA was detected in serum of exposed lambs on PND15, whereas neither glyphosate nor AMPA were detected on PND45. Controls were negative for glyphosate and AMPA on PND15 and PND45. GBH exposure did not affect ovarian or uterine weight. However, on PND45, the ovary of GBH-exposed lambs showed altered follicular dynamics, increased proliferation of granulosa and theca cells, and decreased mRNA expression of FSHR and GDF9, whereas their uterus showed decreased cell proliferation but no alterations in the histomorphology or gene expression. In conclusion, GBH exposure altered the ovarian follicular dynamics and gene expression, and the proliferative activity of the ovaries and uterus of lambs. It is noteworthy that all the adverse effects found in the ovaries and uterus of both GBH-exposed groups were similar, independently of the administration route.

The combination of retinoic acid and estrogen can increase germ cells genes expression in mouse embryonic stem cells derived primordial germ cells

Generation of germ cells from embryonic stem cells in vitro could have great application for treating infertility. The temporal expression profile of several genes was expressed at different stages of germ cell development and examined in differentiation the mouse embryonic stem cells. Cells were treated in three groups of control, with 10−8 M of all-trans retinoic acid and the combination of 10−9 M of 17β-Estradiol and retinoic acid for 7, 12, 17 or 22 days. Quantitative RT-PCR and Immunofluorescent were used to investigate the possible inductive effects of estrogen on mouse embryonic stem cell-derived primordial germ cells. mRNA expression of Oct4 and Dazl were downregulated in embryonic stem cells by the retinoic acid group, whereas Mvh transcription was reduced by retinoic acid and estrogen group in these cells compared to the control group. But, retinoic acid with estrogen group-treated cells exhibited increased mRNA expression of Stra8, Fragilis, Sycp3, GDF9, and Stella compared to untreated controls. The expression of Stella and Mvh proteins were remarkably increased in cell colonies. This study shows that estrogen affects the expression of specific markers of primordial germ cells. Also, estrogen and retinoic acid speed up and increase the level of expression of specific markers.

Expression Analysis of the Prolific Candidate Genes, BMPR1B, BMP15, and GDF9 in Small Tail Han Ewes with Three Fecundity (FecB Gene) Genotypes.

Simple SummaryAs important prolific candidate genes, BMPR1B, BMP15, and GDF9 may affect the lambing performance of sheep. Therefore, regarding the three FecB genotypes of Small Tail Han (STH) sheep (FecB BB, FecB B+, and FecB ++), this study explored the gene expression characteristics of different tissues using reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR). The results showed that BMPR1B, BMP15, and GDF9 expression differed between the selected tissues, with all being highly expressed in the ovaries. Further analysis indicated that there was no significant difference in BMPR1B expression among the three FecB genotypes, but both GDF9 and BMP15 had the highest expression in FecB B+. As for other non-ovarian tissues, expression also varied. This study is relevant to understanding the high prolificacy of the STH breed.The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic–pituitary–gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction.

Expresión génica del factor de crecimiento BMP15, GDF9, FGF2 y sus receptores en células foliculares bovinas

Introducción. El crecimiento y la maduración folicular implican una serie de transformaciones de diversos componentes del folículo, como el ovocito, las células de la granulosa y de la teca. Varios factores de crecimiento, incluyendo factor de crecimiento de diferenciación 9 (GDF9), proteína morfogénica ósea 15 (BMP15) y factor de crecimiento de fibroblastos básico (FGF2) son importantes para el desarrollo folicular y maduración de ovocitos, por su capacidad de aumentar la proliferación de las células de la granulosa, teca y el estroma ovárico. Objetivo. Evaluar la expresión de los factores de crecimiento GDF9, BMP15, FGF2 y sus principales receptores: el receptor 1 del factor de crecimiento transformante beta (TGFβ-R1), el receptor de la proteína morfogénica del hueso tipo IB (BMPR-IB) y el receptor del factor de crecimiento fibroblástico 2 (FGFR2) en células foliculares bovinas. Materiales y métodos. Se realizó la extracción de ARN total de pooles de ovocitos (OOs) y células del cumulus (CCs) de complejos cumulus-ovocito (COCs) y del pellet de células foliculares (PCs) provenientes de 70 ovarios obtenidos de 96 vaquillonas para carne, colectados en un frigorífico local. Los patrones de expresión de los factores de crecimiento y sus receptores en las células foliculares bovinas fueron evaluados por retro-transcripción seguida de la reacción en cadena de la polimerasa (RT-PCR). Resultados. La presencia de los transcriptos de ARNm de los genes GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB y FGFR2 fue detectada por RT-PCR en todas las células estudiadas. Es la primera vez que se reporta en ovocitos bovinos la expresión de los receptores TGFβ-R1 y BMPR-IB. Conclusiones. La presencia de transcriptos de factores de crecimiento y sus receptores en las células estudiadas, indica que estos factores podrían actuar como reguladores paracrinos y autocrinos de la foliculogénesis.

Impairment of ovaries by 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat

2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero had been shown to affect ovarian development and functions. However, its mechanism remained unknown. In this study, to investigate the effect of maternal exposure to TCDD on ovaries, the pregnant Sprague Dawley (SD) rats were treated with TCDD (100 ng/kg or 500 ng/kg) or only vehicle and corn oil on the day 8–14 of gestation through the gavage with a stainless-steel feeding needle (once a day). The vaginal opening and estrous cycle of female offspring rats (F1) were monitored twice a day. The ovarian histology, follicle counts, real-time PCR, western blotting and DNA methylation analysis about Gdf9 and Bmp15 were carried out in F1 rats. The results showed that exposure to TCDD (especially the dose of 500 ng/kg) in utero on GD8-14 might change the ovary weight, the concentration of E2 and FSH, the estrous cycles and the numbers of primordial and secondary follicles of the offspring rats. In addition, the mRNA and protein expression of GDF9 and BMP15 was down-regulated, while the methylation patterns of Gdf9 and Bmp15 were not affected. In conclusion, maternal exposure to TCDD could affect the ovary development and functions which were possibly associated with down-regulation of mRNA and protein expression of GDF9 and BMP15. However, the down-regulation was not related to the pattern of methylation of Gdf9 and Bmp15.

Xenotransplantation of pre-pubertal ovarian cortex and prevention of follicle depletion with anti-Müllerian hormone (AMH).

To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7days and then harvested after 14days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.

Is there a correlation between follicle size and gene expression in cumulus cells and is gene expression an indicator of embryo development?

BackgroundIn an article published in 2017, we discussed the results of the first part of our study into the morphokinetic development of embryos in relation to follicle diameter and homogeneity of follicular development. Our findings showed that embryos coming from small follicles in heterogeneous cycles had significantly higher rates of arrest or failure to reach blastocyst than embryos coming from large follicles in homogenous cycles. The aim of this further study was to investigate the relationship between follicular size and gene expression of cumulus cells (CCs) and evaluate whether gene expression could be an indicator of embryo development.MethodsThis study was based on 2495 COCs from 184 patients. CC expressions of five genes (TNFAIP6, PTGS2, HAS2, PTX3 and GDF9) were studied by generalized linear mixed models (GLMMs) regarding follicular size. CC expressions were then separately analysed regarding patient-specific variables (age, BMI, AMH and follicular size) in relation to embryos reaching blastocyst (eRB) or top or good quality blastocysts (TQ + GQ) using GLMMs with logit link.ResultsFollicular size significantly correlated with the potential of an oocyte to develop into a blastocyst: oocytes developing from large follicles were more than twice as likely to develop into an eRB than oocytes from small follicles (p < 0.001). Gene expression of HAS2 and GDF9 correlated with blastocyst quality when separately evaluated with follicular size and the patient specific variables of age, BMI and AMH. However, no such correlation was found in other gene expressions studied.ConclusionsOur findings suggest that differences in the expression of genes studied could be related to follicular size rather than to embryo quality. Although gene expression of HAS2 and GDF9 correlated with blastocyst quality, the only variable correlating with eRB and TQ and GQ blastocysts for each of these five models was follicular size.Trial registrationThis prospective cohort study was registered at clinicaltrials.gov (NCT02230449).

Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos

Changes in abundance of mRNAs during oocyte growth and maturation and during pre-implantation embryo development have been documented using quantitative real-time RT-PCR (qPCR), microarray analyses, and whole genome sequencing. However, these techniques require amplification of mRNAs, normalization using housekeeping genes, can be biased for abundant transcripts, and/or require large numbers of oocytes and embryos which can be difficult to acquire from mammalian species. We optimized a single molecule RNA fluorescence in situ hybridization (RNA-FISH) protocol, which amplifies fluorescence signal to detect candidate transcripts, for use with individual oocytes and embryos. Quantification using the software Localize showed patterns of Gdf9 and Pou5f1 mRNA expression in oocytes and embryos that were consistent with previously published data. Interestingly, low levels of Nanog mRNA were also accurately and reproducibly measured in oocytes and one- and two-cell embryos suggesting that RNA-FISH could be used to detect and quantify low abundance transcripts. Unlike other techniques, RNA-FISH is also able to detect changes in the localization patterns of mRNAs which may be used to monitor post-transcriptional regulation of a transcript. Thus, RNA-FISH represents an important technique to investigate potential mechanisms associated with the synthesis and stability of candidate mRNAs in mammalian oocytes and embryos.

The protective effects of mangiferin on metabolic and organs functions in the adolescent rat model of alcohol abuse

This study was carried out to investigate the beneficial effects and potential mechanism of mangiferin (MG) against the disorders of metabolic and organs functionsinduced by alcoholabuse (40% alcohol, 10 mL/kg day−1 BW, ig) in adolescent rats. MG administration (100 mg/kg·day−1 BW, ig) can effectively prevent the above disorders. The mechanisms of potential effect of MG were related to scavenged free radicals, increased the levels of serum calcium, estradiol, testosterone and bone density, and up-regulated brain GFAP, testes IRS-2 and ovaries GDF-9 expression levels. In addition, lipid accumulation and blood glucose level were effectively regulated by MG, which are also important for adolescent metabolic and organs functions. These systems and balances in vivo were not isolated from each other, but interrelated and influenced each other. These results suggested that MG can be a novel phytochemicals for preventing the disorders of metabolic and organs functions induced by alcohol abuse.

Therapeutic effectiveness of rat bone marrow stem cells in Poly Cystic Ovary Syndrome Mice Model on folliculogenesis, TGF-β, GDF-9 expression, and estrogen, TNF- and androgen Levels

Objectives: to identify therapeutic effectiveness of Rat Bone Marrow stem cell in PCOS rats model on folliculogenesis, TGF-beta and GDF-9 expression and on estrogen, TNF-a and androgen levels.Material and Methods: this study is a laboratory experimental research with using animal testing. PCOS was induced by the administration of testosterone propionate hormone into 30 mice. The subjects of this study are divided into 2 groups: stem cell group and control group. The mice were injected with testosterone then vaginal swab was performed to determine the mice cycle. After determining mice in anestrous cycle, stem cell was injected. TNF-a was measured with immunohistochemistry and androgen was examined using ELISA. The data was measured by student t-test.Result: The average number of TNF-a expression in control group was lower than stem cell group (5.35 vs 2.34; p= 0.0026). The average androgen level for stem cell group was lower than mean for control group (2.31 vs 0.40; p= 0.0026).Conclusion: In this study of polycystic model mice, stem cell decreased the expression of TNF-a and androgen level

Effects of 2,3-Bis(4-hydroxyphenyl)-propionitrile on Induction of Polyovular Follicles in the Mouse Ovary.

Neonatal diethylstilbestrol (DES) treatment induces polyovular follicles (PFs), which contain more than two oocytes in a follicle, through estrogen receptor (ER) β, not ERα. 2,3-Bis(4-hydroxyphenyl)-propionitrile (DPN) is a specific ERβ agonist; the effects of neonatal DPN exposure on PF induction and gene expression in the mouse ovary were examined. Histological analysis and real-time reverse transcription-polymerase chain reaction were performed. The PF incidence was significantly high in the ovary of neonatally DPN-exposed mice compared to that in oil-exposed mice. The gene expression of growth differentiation factor 9 (Gdf9), Mullerian-inhibiting substance, steroidogenic factor 1 (Sf1) and steroidogenic acute regulatory protein (Star) in the ovary was significantly increased in the mice neonatally exposed to 40 μg DPN compared to oil-treated mice. Since SF1 is an important transcription factor of several genes involved in ovarian function, up-regulation of Sf1 expression by neonatal exposure to DPN, through ERβ, might affect expression of Gdf9, Mis and Star, resulting in increased PFs in mouse ovary.

Effect of FH535 on invitro maturation of porcine oocytes by inhibiting WNT signaling pathway.

Wingless-int (WNT) signaling pathway is vital to modulate life processes, including cell fate determination, cell differentiation, cell proliferation, cell apoptosis and embryogenesis. To demonstrate the uncertain effect of the canonical WNT signaling pathway on oocyte maturation, immature porcine oocytes were collected and cultured invitro with the WNT/β-catenin inhibitor FH535. The concentrations of FH535 were selected as 0.00, 0.01, 0.10, 1.00 and 10.00μmol/L. The results showed that the optimum concentration of FH535 on oocyte maturation was 1.00μmol/L. In this concentration, the proportion of MII oocytes increased (P<0.05). The rate of cleavage was the same with the control (P>0.05), while the rate of blastocysts in the 1.00μmol/L FH535 treated group was higher than that of control (P<0.01). Additionally, the average number of nuclei in blastocysts raised significantly (P<0.05). The inhibition of WNT could regulate expression of maturation-related genes, including Cdc-2, Bmp-15, Gdf-9 and Mos. In the 1.00μmol/L FH535 treated group, the messenger RNA level of β-catenin showed no significant change compared to the control (P>0.05), but the protein abundance was decreased (P<0.05). This study revealed that the inhibition of FH535 on the WNT signaling pathway could promote the maturation of porcine oocytes and altered gene expressions invitro.

Robust gdf9 and bmp15 expression in the oocytes of ovotestes through the Figla-independent pathway in the hermaphroditic black porgy, Acanthopagrus schlegelii.

More than 1,500 fish species are hermaphroditic, but no hermaphroditic lineage appears to be evolutionarily ancient in fishes. Thus, whether more than one sex at a time was present during the evolutionary shift from gonochorism to hermaphroditism in fishes is an intriguing question. Ectopic oocytes were created in the ovotestes of protandrous black porgy via the withdrawal of estradiol (E2) administration. These ectopic oocytes reprogrammed the surrounding cells, which changed from Sertoli cells to follicle-like cells. We observed that gdf9 and bmp15 expression was localized in the primary oocytes and gradually decreased after oocytes entered a secondary oocyte stage. Robust expression of gdf9 and bmp15 in ectopic oocytes was associated with the surrounding Sertoli cells. However, blocking Cyp19a1a activity and increasing androgen levels did not stimulate the expression of gdf9 and bmp15. Thus, the robust gdf9 and bmp15 expression was not related to the inappropriate male microenvironment. Furthermore, in vitro data demonstrated that gdf9 and bmp15 were not downstream genes of Figla signaling. Therefore, our results suggest that there are two independent mechanisms, a Figla-dependent pathway and a Figla-independent pathway, by which oocyte-surrounding cells are altered from a male somatic fate to a female somatic fate. This functional switch might clarify how oocytes created an appropriate microenvironment during the transition from the ancient gonochorism to the present hermaphroditism.

Comparative analysis of developmental and molecular correlates of developmental competence of buffalo oocytes derived from small and large follicles

The present study was conducted to correlate the developmental competence of large follicle (LF, &gt;6 mm) and small follicle (SF, &lt;6 mm) derived buffalo oocytes in terms of morphological assessment and molecular characteristics. A pool of culture grade cumulus oocytes complexes (COCs) from both the groups separately were subjected to brilliant cresyl blue (BCB) staining. LF derived oocytes had significantly higher percentage of BCB+ve oocytes and were larger in size and maturation status was also visibly superior. Cleavage and blastocysts rate was also significantly higher in LF group than SF group. Relative mRNA expression analysis revealed that expression of GDF9 was significantly higher in LF group than SF group while expression of BMP15 increased insignificantly. Expression of GREM1, EGFR and PTGS2 was significantly higher in LF group than SF group while expression of HAS2 was insignificantly higher. Similarly, expression of GLUT1, OCT4 and IFN- in in vitro produced blastocyts was significantly higher in LF group than SF group. Thus, it can be concluded that LF derived oocytes are larger in size and express higher level of competence markers and resulted in better quality embryos. This information may be further utilized for the enhancement of developmental competence of small follicle derived oocytes.

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.