Expression Of Bone Morphogenetic Protein 15 Research Articles

The Potential Regulatory Role of miR-378 in the Expression of Bone Morphogenetic Protein-15 in Infertile Women with Hyperprolactinemia (HPL)

The aim of this study is to investigate the relationship between microRNA 378 and BMP15 gene expression levels in blood samples collected from 50 healthy fertile females as controls and 50 hyperprolactinemia infertile females by quantitative real-time polymerase chain reaction (RT-qPCR). Specific primers were designed for this purpose based on the sequences of microRNA 378 and BMP15 retrieved from NCBI and designed by primer 3 software. The result assessing the expression level of BMP15 in hyperprolactinemia (HPL) was 0.220, while the control group's fold change value was 1.000. The HPL group showed downregulation in the expression of the BMP15 gene. While the fold expression values of the miRNA378 gene in the hyperprolactinemia (HPL) group were 2.227 and the control group's mean value was 1.000, the HPL group showed up-regulation in the expression of the miRNA378 gene. The results of the study revealed that miR-378 has directly regulated BMP15 gene expression; based on this, BMP15 gene expression was obviously decreased. Therefore, the expression level of BMP15 was negatively correlated with miR-378 expression.

Determination of the Expression of Bone Morphogen Protein 15 and its Receptors in Laying Hens’ Ovary

Abstract The objective of the current research was to determine expression, function and regulation of bone morphogenetic protein 15 (BMP15) during follicular development in laying hens. A trial was conducted with 40 layers from Lohman Klassik Brown breed (40 weeks old). At the end of the study fifteen layers were humanely killed and their ovaries were then dissected. Ribonucleic acid (RNA) expression of BMP15 was analyzed in the ooplasm and in granulosa cells. It was significantly higher in the ooplasm (p<0.01). BPM15 expression was not found in the granulosa cells from 6-8 mm and >9 mm follicles. The expression for bone morphogenetic protein 15 receptors (BMPR1B and BMPR2) in the granulosa cells was in significant positive correlation with the follicle size (p<0.05). The results obtained in this study demonstrate the possible role of BMP15 in developing oocytes. BMP15 expression is important for the growth regulation and signaling in the follicular cells in the preovulatory phase.

The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-β signaling pathway

Bone morphogenetic protein 15 (BMP15) is specifically expressed in oocytes in pigs at all stages from early stages to ovulation and has an important role in oocyte maturation. However, there are few reports on the molecular mechanisms by which BMP15 affects oocyte maturation. In this study, we identified the core promoter region of BMP15 using a dual luciferase activity assay and successfully predicted the DNA binding motif of the transcription factor RUNX1. The effect of BMP15 and RUNX1 on oocyte maturation was examined using the first polar body extrusion rate, a reactive oxygen species (ROS) assay and total glutathione (GSH) content at three time points of 12, 24 and 48 h of in vitro culture of porcine isolated oocytes. Subsequently, the effect of the transcription factor RUNX1 on the TGF-β signaling pathway (BMPR1B and ALK5) was further verified using RT–qPCR and Western blotting. We found that the overexpression of BMP15 significantly increased the first polar body extrusion rate (P < 0.01) and total glutathione content of oocytes cultured in vitro for 24 h and decreased reactive oxygen levels (P < 0.01), whereas interference with BMP15 decreased the first polar body extrusion rate (P < 0.01), increased reactive oxygen levels in oocytes cultured in vitro for 24 h (P < 0.01), and decreased glutathione content (P < 0.01). The dual luciferase activity assay and online software prediction showed that RUNX1 is a potential transcription factor binding to the core promoter region (−1203/−1423 bp) of BMP15. Overexpression of RUNX1 significantly increased the expression of BMP15 and oocyte maturation rate, while inhibition of RUNX1 decreased the expression of BMP15 and the oocyte maturation rate. Moreover, the expression of BMPR1B and ALK5 in the TGF-β signaling pathway increased significantly after overexpression of RUNX1, whereas their expression decreased after inhibition of RUNX1. Overall, our results suggest that the transcription factor RUNX1 positively regulates the expression of BMP15 and influences oocyte maturation through the TGF-β signaling pathway. This study provides a theoretical basis for further complementing the BMP15/TGF-β signaling pathway to regulate mammalian oocyte maturation.

Decreased GDF9 and BMP15 in follicle fluid and granulosa cells and outcomes of IVF-ET among young patients with low prognosis.

To analyze the level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in follicle fluid (FF) and granulosa cells (GCs) derived from young patients with low prognosis for in vitro fertilization and embryo transfer (IVF-ET) treatment. A prospective cohort study was carried out by enrolling 52 young patients with low prognosis according to the POSEIDON classification group 3 (low prognosis group) and 51 young patients with normal ovarian reserve (control group). The concentration of the GDF9 and BMP15 proteins in FF was determined by enzyme-linked immunosorbent assay. The mRNA level of the GDF9 and BMP15 in the GCs was measured by quantitative real-time PCR. The concentration of GDF9 (1026.72 ± 159.12 pg/mL vs. 1298.06 ± 185.41 pg/mL) and BMP15 (685.23 ± 143.91 pg/mL vs. 794.37 ± 81.79 pg/mL) in FF and the mRNA level of GDF9 and BMP15 in the GCs and the live birth rate per treatment cycle started (30.77% vs. 50.98%) and oocytes retrieved (4.25 ± 1.91 vs.12.04 ± 4.24) were significantly lower, whereas the canceled cycle rate was significantly higher (9.62% vs. 0) in the low prognosis group compared with the control group (P < 0.05). The expression of GDF9 and BMP15 in the ovary was positively correlated with live birth (P < 0.05). The expression of GDF9 and BMP15 in the ovary was decreased in young patients with low prognosis accompanied by a poorer outcome of IVF-ET treatment. ChiCTR1800016107 (Chinese Clinical Trial Registry), May 11, 2018. ( http://www.chictr.org.cn/edit.aspx?pid=27216&htm=4 ).

Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of Guizhou black goat

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

Altered Expression of GDF9 and BMP15 Genes in Granulosa Cells of Diminished Ovarian Reserve Patients: A Case-Control Study.

Objective Diminished ovarian reserve (DOR) is a challenging issue encountered during assisted reproductive technology. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) belong to the transforming growth factor-beta (TGF-β) superfamily which are essential for folliculogenesis. We aimed to the evaluation of the GDF9 and BMP15 expression in the granulosa cells (GCs) of DOR patients. Materials and Methods This case-control study included 14 women with DOR and 12 controls, who were between 28- 40 years of age undergoing controlled ovarian stimulation with a gonadotropin releasing hormone (GnRH) antagonist protocol. DOR patients were selected by the Bologna criteria. The GCs were extracted from the aspirated follicular fluids and RNA isolated from this. The fold change of gene expressions was assessed by real-time polymerase chain reaction (PCR). Results GDF9 expression in patients was 0.23 times lower than the control group, which was significant (P<0.0001). BMP15 expression in patients was 0.32 times lower than the control group, which was significant (P<0.0001). The number of archived oocytes, MII, and two pronuclei (PN) embryos was higher in the control group and these differences were statistically significant (P<0.05). ConclusionGiven that GDF9 and BMP15 are specifically involved during follicular recruitmen., we expect expression of these two genes in DOR patients which is greatly reduced by reducing follicular reserve.

High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

O-034 Xenotransplantation of ovarian cortex tissue from young girls with Turner Syndrome in a mice model: is normal follicular development possible?

Abstract Study question Does aneuploidy of granulosa and stromal cells affect folliculogenesis of small follicles in girls with mosaic Turner Syndrome (TS)? Summary answer Our results showed that despite high level aneuploidy in granulosa cells of small follicles and stromal cells folliculogenesis was not affected in mosaic TS patients. What is known already In 2018 the TurnerFertility study was initiated to explore if ovarian tissue cryopreservation (OTC) is a viable option for TS patients to preserve their fertility at an early age. In this study, karyotyping of ovarian cells in 7 mosaic TS patients showed that the majority of oocytes had a normal X chromosomal content, while granulosa and stromal cells were aneuploid. Until now, it remains unclear if follicular development is affected, and consequently the success rate of autotransplantation after OTC in girls with TS. Study design, size, duration A collaboration project was initiated between Radboudumc, The Netherlands and UCLouvain, Belgium to assess folliculogenesis of small follicles in TS patients by using a xenograft mice model. Ovarian cortex tissue was obtained from 18 mosaic TS patients and 13 age-matched controls. Participants/materials, setting, methods After unilateral ovariectomy, one fragment of the ovarian tissue was used for research. One part of this fragment was used for fluorescence in situ hybridization (FISH) to determine the X chromosomal content of ovarian cells in non-grafted tissue. The other part was xenografted into severe combined immunodeficient mice. After 5 months, grafts were retrieved and analysed for aneuploidy by FISH. The expression of 6 proteins essential for folliculogenesis was analysed by immunohistochemistry and immunofluorescence. Main results and the role of chance The mean follicle density in ovarian tissue of TS patients before grafting was significantly lower compared to controls. FISH analysis showed that 97% of the oocytes in non-grafted tissue had a normal X chromosomal content. Follicles of TS patients contained mainly or exclusively 45,X granulosa cells, but different levels of X chromosome mosaicism between TS patients and between follicles of the same patient were observed. In total, 12/18 grafts contained follicles after 5 months xenografting. Follicle density of both TS patients and controls decreased significantly after xenotransplantation. Despite the presence of high level aneuploidy in granulosa and stromal cells in the tissue before grafting, secondary and even antral follicles were observed after xenotransplantation. Remarkably, a shift from high to low percentage of 45,X granulosa cells was observed during folliculogenesis. Immunohistochemistry showed that proliferating cell nuclear antigen (PCNA) positive follicles from TS patients increased during grafting to almost 100%. Secretion of anti-Müllerian hormone by granulosa cells was impaired before grafting in peri/postpubertal TS girls, but recovered after grafting. Expression of c-kit receptor and bone morphogenetic protein 15 (BMP15) in peri/postpubertal TS patients remained abnormal after xenotransplantation, while secretion of growth differentiation factor 9 (GDF9) and kit ligand was similar to controls. Limitations, reasons for caution In this study, only ovarian tissue of mosaic TS patients was examined, because the chances of finding ovarian follicles in this subgroup is considerably higher than in other TS patients. Subtle effects of reduced expression of c-kit receptor and BMP15 on folliculogenesis might have gone unnoticed. Wider implications of the findings Small follicles of mosaic TS patients are able to grow to secondary and antral follicles, despite the presence of aneuploid granulosa and stromal cells before grafting. Therefore, OTC could be a realistic option for young mosaic TS patients to preserve their fertility provided that sufficient numbers of follicles are present. Trial registration number NCT03381300

The effect of gold nanoparticles synthesized with Achillea biebersteinii on gene expression in Cultured preantral Follicles derived from NMRI mice ovary

Infertility is one of the global public health problems facing society today. Studies on the causes of infertility have greatly expanded with the advancement of new technologies. The increasing use of nanomaterials has raised concerns about nanoparticles' potential dangers. The use of plant extracts is an easy and cost-effective approach to synthesize metal nanoparticles. In this comparative study, the effects of green and chemically synthesized Gold nanoparticles (GNPs), as well as Achillea biebersteinii (AM) (medicinal plant) extract, were investigated on the maturity of preantral Follicles (PF) of mice in vitro. The expression of GDF-9, BMP-15, GPX, and SOD-1 genes was analyzed by real-time polymerase chain reaction (Real Time-PCR). Twenty PF in control and experimental groups were treated with 50 μg/ml of AM extract, green GNPs, and GNPs, and were then used to determine intracellular ROS (Reactive oxygen species) in the first and fifth days after culture. AM extract also increased the expression of genes that stimulate preantral follicle growth (PF), including Growth differentiation factor-9 (GDF-9)and Bone morphogenetic protein 15 (BMP-15). Expression of the glutathione peroxidase (GPX) gene was observed in the group of NPs synthesized by a green method. Due to its antioxidant properties, this gene expression was also higher in the plant extract group than commercially synthesized GNPs. Overall, this study's findings showed that AM extract, due to its antioxidant effects, improves PF development, and AM-GNPs did not significantly impact PF growth, but GNPs inhibited PF growth. Findings suggest that the AM extract that covered the surface of AM-GNPs may be responsible for reducing the toxic effects of GNPs.

Effects of Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15 overexpression on the steroidogenic metabolism in bovine granulosa cells in vitro.

Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P4 ) and estradiol (E2 )) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P4 secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non-described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism.

Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

BackgroundGrowth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment.MethodsA total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.ResultsCompared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P < 0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P < 0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P < 0.05).ConclusionsFor poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant.Trial registrationChinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

The Effect of Isotretinoin on Oocyte Maturation in Adolescent Female Rats

Objective: Isotretinoin is used in acne vulgaris treatment for more than 20 years. Isotretinoin has serious side effects on many organs, but there are no comprehensive studies investigating its possible toxic effects on reproductive organs. Thus, we aimed to investigate the possible toxic effects of isotretinoin administration on oocyte maturation in female rat gonads in this study. Methods: Thirty-two adolescent female rats (Wistar Albino, 220 ± 35 g) were randomly divided into 4 groups with 8 subjects in each group: group 1, group 2, group 3, and group 4. Different doses of isotretinoin which was dissolved in sesame oil were given to rats by gavage: 7.5 mg/kg/day in group 3 and 15 mg/kg/day in group 4. The rats in group 2 received sesame oil by gavage. To create gavage stress, only gavage was administered to the rats in group 1. The gavages for each group continued once a day and at a certain time for 30 days. To determine the effect of isotretinoin on oocyte maturation, the periodic acid-Schiff reaction was performed for histochemical and histomorphometric evaluation of the zona pellucida, and staining of growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) was performed for immunohistochemical analysis. Results: When the thickness of the zona pellucida was evaluated, a statistically significant difference was found between group 1 and experimental groups (group 3 and group 4). In the experimental groups, it was determined that the thickness of the zona pellucida was decreased depending on the increase in dose. GDF-9 and BMP-15 expressions in oocytes of primordial and primary follicles decreased significantly in the experimental groups compared to group 1 and group 2. However, the expression of GDF-9 and BMP-15 in oocytes of secondary follicles was not significantly different between group 1 and group 2 and the experimental groups. Conclusions: In our study, we showed toxic effect of isotretinoin on oocyte maturation in female rats.

Cyclooxygenase 2 messenger RNA levels in canine follicular cells: interrelationship with GDF-9, BMP-15, and progesterone

Cyclooxygenase 2 (COX-2) encoded by the Cox-2 gene within the periovulatory follicles is a critical mediator of oocyte development. Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) participate in the modulation of certain target genes in the ovary, possibly influencing the Cox-2 gene expression. However, this relationship has not been characterized in canines. This study aimed to examine the possible relationships among BMP-15, GDF-9, progesterone, and Cox-2 gene expression in granulosa-cumulus cells in dogs. Granulosa cells from antral follicles and their corresponding cumulus-oocyte complexes and follicular fluid (FF) were separately obtained from 56 ovaries collected from adult bitches at estrus (n = 15) and proestrus (n = 13) after ovariohysterectomy. Total RNA extraction was performed in follicular cells, and Cox-2 gene expression was assessed by quantitative PCR analysis. Progesterone, BMP-15, and GDF-9 were determined in the FF samples using ELISA assays. Cumulus-oocyte complexes were subjected to in vitro maturation (IVM) with or without (control) recombinant GDF-9 and BMP-15. After 72 h of culture, Cox-2 transcript analyses were performed in cumulus cells via quantitative PCR. Data were evaluated by ANOVA. An increase (P < 0.05) in Cox-2 messenger RNA levels was observed in follicular cells from follicles at estrus with respect to those at proestrus. However, the levels of BMP-15 and GDF-9 in FF decreased (P < 0.05), whereas progesterone increased (P < 0.05) from the proestrus phase to the estrus phase. The expression of Cox-2 gene in cumulus cells was 4-fold greater (P < 0.01) than that in the control when both growth factors were added to the IVM culture. In conclusion, although BMP-15 together with GDF-9 appears to upregulate the levels of Cox-2 transcripts during IVM, the inverse relationship of these paracrine factors with Cox-2 gene expression and the positive correlation of progesterone with Cox-2 transcripts suggest that the high progesterone levels could be more relevant in the local mechanisms regulating the Cox-2 gene expression.

Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process.

Simple SummaryThe knowledge of physiological events associated with canine reproduction involving oocyte developmental potential is essential to increase the success of reproductive biotechnologies in this species. In mammals, the oocytes are closely surrounded by a group of cells known as the cumulus cells. Although it is not well-known how these cells interact with the oocyte to promote maturation, they may provide important answers concerning oocyte development. The competence to undergo expansion is a unique characteristic of cumulus cells which is critical for normal oocyte maturation, however, the complete expansion of these cells takes longer in canines, which has been associated with the lengthy maturation process of the oocyte. Growth Differentiation Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are described as relevant players in the oocyte–cumulus cells’ regulatory mechanisms. Cumulus cells express many important genes from a very early stage, therefore, we proposed to study the gene expression of GDF-9 and BMP-15 in canine cumulus cells in relation to cumulus expansion and the maturation process. We demonstrate, for the first time, that these genes are differentially expressed in canine cumulus cells throughout the estrous cycle and that this expression is related to cumulus expansion and maturity status, suggesting specific regulation.The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.

Rapamycin preserves the primordial follicle pool during cisplatin treatment in vitro and in vivo

Rapamycin has been proven to effectively inhibit the activation of primordial follicles while cisplatin-induced the loss of primordial follicles due to the over-activation of the primordial follicle stockpile. Whether rapamycin could inhibit the loss of primordial follicles induced by cisplatin is still unknown. The ovaries of neonatal Sprague Dawley rats were cultured in vitro in different doses of rapamycin (0.08, 0.16, and 0.32 μg/ml) and cisplatin (0.1, 0.4, and 0.8 μg/ml). The immature BALB/c mice were administered cisplatin with or without rapamycin by intraperitoneal injection. Ovaries were collected to analyze the histomorphology, the messenger RNA (mRNA) expression ofanti-Mullerian hormone (AMH), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) and the expression of key proteins of mammalian target of rapamycin (mTOR) pathway. Growing follicle counts of ovaries cultured in vitro in the R0.16 and R0.32 groups were decreased and the ratio of growing to primordial follicles was also decreased in a dose-dependent manner. In the C0.8 group, growing follicles were decreased compared with the other groups while the ratio was substantially increased in the C0.4 and C0.8 group. Co-treatment attenuated primordial follicle loss and reduced the upregulated ratio induced by cisplatin. Ovarian follicle dynamics in vivo was consistent with the in vitro results. Primordial follicles counts were statistically increased and the ratio was reduced in the rapamycin group compared with the control group. Primordial follicle counts were dramatically reduced in the cisplatin group whereas co-treatment with rapamycin slightly recovered its counts. There was no obvious difference in the number of growing follicles between the cisplatin group and other groups. The ratio was significantly increased in cisplatin-treated mice whereas decreased in the co-treatment group. The apoptosis rate of antral follicles in cisplatin-treated mice was higher than the other groups while the apoptosis rate was decreased in the co-treatment group in vivo. Compared with the control and rapamycin group, the mRNA expression of AMH, GDF9, and BMP15 were downregulated in the cisplatin group. The co-treatment group recovered the mRNA expression of BMP15. In addition, the expression of key protein of mTOR pathway rpS6 and its phosphorylated forms were increased in the cisplatin-treated group while co-treatment decreased their expression. Rapamycin attenuated the loss of primordial follicles induced by cisplatin through the inhibitory effect of rapamycin on the mTOR pathway. These results suggest that rapamycin may be an effective drug for the protection of ovarian function during chemotherapy.

The antioxidant curcumin postpones ovarian aging in young and middle-aged mice.

Reproductive senescence is accompanied by a reduced number and quality of ovarian follicles in response to the accumulation of free radicals and the process of apoptosis. Having selected mice as models, we examined the hypothesis that curcumin as an antioxidant and anti-inflammatory agent might prevent or retard ovarian aging. Female NMRI 21-day-old mice were divided into control, vehicle and curcumin groups. In the treatment group the mice received curcumin at 100mgkg-1day-1 intraperitoneally. After 6, 12 and 33 weeks several parameters were examined including ovarian reserve, oocyte quality, oxidative status, invitro fertilisation and expression of ovulation-related (growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15)) and anti-aging-related (sirtuin 1 (SIRT-1) and SIRT-3) genes. Curcumin treatment up to 12 and 33 weeks resulted in increased ovarian volume and number of follicles and was associated with elevated anti-Müllerian hormone and oestrogen and diminished FSH serum levels. Furthermore, enhanced oocyte maturation, fertilisation and embryo development plus reduced oxidative stress were seen in the curcumin group. Also, the expression of GDF-9, BMP-15, SIRT-1 and SIRT-3 genes was increased in the curcumin group. Concerning gestational age, the findings of the study suggested that administration of curcumin could delay the process of oocyte aging in a mouse model.

Neonatal exposure to agonists and antagonists of sex steroid receptors induces changes in the expression of oocyte-derived growth factors and their receptors in ovarian follicles in gilts

The objective of the present study was to examine the effects of neonatal exposure to either agonists or antagonists of androgen and estrogen receptors on the expression of growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) and their cognate receptors (TGFBR1, BMPR1B, and BMPR2) in ovarian follicles of adult pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen, at 20 mg/kg bw), flutamide (FLU, an antiandrogen, at 50 mg/kg bw), 4-tert-octylphenol (OP, an estrogenic compound, 100 mg/kg bw), ICI 182,780 (ICI, an antiestrogen, 400 μg/kg bw), or corn oil (control) between postnatal Days 1 and 10 (n = 5/group). Ovarian follicles were excised from adult pigs on Days 8–11 of the estrous cycle. The expression of GDF9, BMP15, TGFBR1, BMPR1B and BMPR2 were examined in the population of preantral and small antral ovarian follicles using real-time PCR, Western blot and immunohistochemistry. In preantral follicles, the upregulation of GDF9 mRNA and protein expression was found in pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMP15. TGFBR1 and BMPR2 mRNA and protein expression were upregulated in preantral follicles of adult pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMPR1B. In small antral follicles, the mRNA and protein for TGFBR1 and BMPR2 were upregulated, while BMPR1B was downregulated in response to neonatal OP treatment. In addition, treatment with FLU upregulated BMPR1B and BMPR2 mRNA and protein expression, while downregulated the expression of TGFBR1. Moreover, GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles obtained from both control and treated ovaries. TGFBR1, BMPR1B and BMPR2 receptors were observed in the oocytes and granulosa cells of preantral follicles as well as in granulosa and theca cells of small antral follicles. In conclusion, the present study demonstrated neonatal exposure to either agonists or antagonists of androgen and estrogen receptors affected GDF9 and BMP15 signalling in ovaries of adult pigs. It seems that neonatal androgen excess or deficiency may lead to the acceleration of initial follicle recruitment, while neonatal exposure to compounds with antiandrogenic and estrogenic activity may disturb small antral follicles fate. Therefore, it confirms that neonatal window is critical for programming of ovarian function in pigs.

Экспрессия факторов паракринной регуляции в яичниках крыс при коррекции экспериментальных функциональных кист яичников

To study the expression of vascular endothelial growth factor (VEGF), bone morphogenetic protein – 15 (BMP-15) and connexin 37 (Cx-37) in the rat’s ovaries with the model of functional ovarian cysts and with their correction, an experiment was conducted on mature white rats of Wistar line. Group I (main, n = 25) — modelling of functional ovarian cysts (introduction of follicle-stimulating hormone) followed by treatment with bevacizumab. Group II (comparison, n = 35) — modelling of functional ovarian cysts without further treatment. The control group (n = 20) is intact animals. Rats were removed from the experiment on the 7 th , 15 th , 30 th day. An immunohistochemical study of the expression of VEGF, BMP-15, Cx-37 has been conducted. Modelling of functional ovarian cysts in white rats was accompanied by an increase in the specific volume of blood vessels in the ovaries, increased expression of VEGF in the cyst wall, excessive stimulation of folliculogenesis, accompanied by increased expression of BMP-15 and Cx-37 at the early stages of the experiment. Introduction of an inhibitor of vascular endothelial growth factor caused a decrease in the number and specific volume of cysts, a decrease in VEGF expression and the approximation of specific vascular volume and VEGF expression to the values of the control group on the 15 th day. By day 30, expression of BMP-5 and Cx-37 did not differ from that of the intact control group. Thus, the introduction of bevacizumab accelerates the regression of functional ovarian cysts by reducing vascularization in the cyst wall, accompanied by a decrease in the expression of VEGF. The revealed decrease in the number of growing follicles, the enhancement of atresia processes in the follicles and the change in the expression of factors of oocyte-somatic interaction (BMP-15, Cx-37) are of short duration.

BMP15 mRNA profile in granulosa cells from endometriosis patients undergoing in vitro fertilization

Endometriosis is a female reproductive disorder that can cause infertility by lowering oocyte quality due to a low quality of oocytes. Granulosa cell assessment is a minimally invasive procedure for determining oocyte quality. Bone morphogenetic protein 15 (BMP15) also acts as a growth factor for oocyte derivatives that regulate proliferation and granulosa cell differentiation. In endometriosis, higher BMP15 expression is assumed to induce oocyte growth. This study aimed to determine the mRNA levels of BMP15 in granulosa cells from endometriosis patients undergoing in vitro fertilization. We analyzed samples from 12 patients with endometriosis and seven normal women using real-time PCR at Yasmin Clinic, Kencana, Cipto Mangunkusumo Hospital. Granulosa cells collected during oocyte retrieval were preserved using RNAlater® QIAGEN and were evaluated by absolute quantitative real-time PCR with pre-manufactured oligonucleotides as the standard. Results showed that although BMP15 did not significantly differ between the endometriosis patients and the controls (p > 0.05), the endometriosis patients had higher BMP15 mRNA levels than the controls. Therefore, further study is warranted to gain more comprehend data.