The Roles of GDF-9, BMP-15, BMP-4 and EMMPRIN in Folliculogenesis and In Vitro Fertilization.

Growth differentiation factor 9 (GDF-9) contributes to early ovarian development and oocyte survival. Higher concentrations of GDF-9 in follicular fluid (FF) are associated with oocyte nuclear maturation and optimal embryo development. In in vitro fertilization (IVF), GDF-9 affects the ability of the oocyte to fertilize and subsequent embryonic development. Bone morphogenetic protein 15 (BMP-15) is involved in the regulation of ovarian function and affects oocyte development. During IVF, BMP-15 contributes to the formation of competent blastocysts. BMP-15 may play a role in embryo implantation by affecting endometrial receptivity. Bone morphogenetic protein 4 (BMP-4) is involved in the regulation of follicle growth and development and affects granulosa cell (GC) differentiation. In relation to IVF, BMP-4 is important for embryonic development, influences cell fate and differentiation, and plays a role in facilitating embryo-endometrial interactions during the implantation process. Extracellular matrix metalloproteinase inducer (EMMPRIN) is associated with ovulation and follicle rupture, promotes the release of mature eggs, and affects the modification of the extracellular matrix of the follicular environment. In IVF, EMMPRIN is involved in embryo implantation by modulating the adhesive properties of endometrial cells and promotes trophoblastic invasion, which is essential for pregnancy to occur. The purpose of the current article is to review the studies and recent findings of GDF-9, BMP-15, BMP-4 and EMMPRIN as fundamental factors in normal follicular development and in vitro fertilization.

Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function.

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.

A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human.

Ovarian immature teratomas (OITs) are malignant tumors originating from the ovarian germ cells that mainly occur during the first 30 y of a female's life. Early age of onset strongly suggests the presence of susceptibility gene mutations for the disease yet to be discovered. Whole exon sequencing was used to screen pathogenic mutations from pedigrees with OITs. A rare missense germline mutation (C262T) in the first exon of the BMP15 gene was identified. In silico calculation suggested that the mutation could impair the formation of mature peptides. In vitro experiments on cell lines confirmed that the mutation caused an 84.7% reduction in the secretion of mature BMP15. Clinical samples from OIT patients also showed a similar pattern of decrease in the BMP15 expression. In the transgenic mouse model, the spontaneous parthenogenetic activation significantly increased in oocytes carrying the T allele. Remarkably, a mouse carrying the T allele developed the phenotype of OIT. Oocyte-specific RNA sequencing revealed that abnormal activation of the H-Ras/MAPK pathway might contribute to the development of OIT. BMP15 was identified as a pathogenic gene for OIT which improved our understanding of the etiology of OIT and provided a potential biomarker for genetic screening of this disorder.

Implication of Novel BMP15 and GDF9 Variants in Unexpected Poor Ovarian Response.

Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27 IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744 T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.

A new technology for the treatment of premature ovarian insufficiency: Cell sheet.

Premature ovarian insufficiency (POI) is defined as the development of hypergonadotropic hypogonadism before the age of 40 with definitive treatment being absent. In the current study, we aim to compare the efficacy of the cell sheet method with an intravenous (IV) application of adipose-derived mesenchymal stem cells (AdMSCs) to the POI with an animal model. In the current prospective study, 6-to-8-week-old Sprague Dawley rats were generated four groups: (i) a control group in which only PBS was administered; (ii) an only-POI group generated by cyclophosphamide; (iii) a POI group treated by way of IV AdMSCs; and (iv) a POI group treated by way of the cell sheet method. Twenty-eight days after an oophorectomy was performed, intracardiac blood was taken. Follicle count, immunohistochemical examination for GDF9, BMP15, and TUNEL were conducted, gene expressions of GDF9 and BMP15 were examined, and E2 was measured in the serum samples. With hematoxylin-eosin, in the third group, multi oocytes follicles were the most remarkable finding. In the fourth group, most of the follicles presented normal morphology. GDF9 involvement was similar between the first and fourth groups. BMP-15 immunoreactivity, in contrast to fourth group, was weak in all stages in the second and third groups. The current attempt represents a pioneer study in the literature in which a cell sheet method is used for the first time in a POI model. These results suggest that the cell sheet method may be a feasible and efficient method for the stem cell treatment of models with POI and could be a new treatment approach in POI.

Effects of kisspeptin on the maturation of human ovarian primordial follicles in vitro.

At this time, with advances in medical science, many cancers and chronic diseases are treatable, but one of their side effects is infertility. Some women also want to delay pregnancy for personal reasons. There has been some evidence that kisspeptin activates broad signals by binding to its receptor, suggesting that the role of kisspeptin in direct control of ovarian function includes follicle growth and steroid production. In this study, the effect of kisspeptin on improving the quality and results for human ovarian follicles was investigated. A section of ovary was removed laparoscopically from women between 20 and 35 years of age (n = 12). Pieces were divided randomly into two groups, control and treatment (with 1 µM kisspeptin). Real-time PCR was performed for GDF9, BMP15 and mTOR gene expression assessments. Western blotting was carried out to measure AKT and FOXO3a protein expression. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey's test; means were considered significantly different at a P-value < 0.05. During treatment with the kisspeptin group, maturity genes are expressed. Therefore, kisspeptin is an effective substance to improve the quality of the human ovarian medium as it increases the maturity of follicles.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep

Abstract Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Resumo Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep / Mutações no gene da proteína morfogenética óssea 15 causadoras de defeitos na secreção proteica em ovelhas inférteis Cholistani

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Parsing Genetic and Autoimmune Etiology in Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is a rare cause of primary amenorrhea in adolescents. For young women with uncertain etiology of POI, genetic and autoimmune testing may be recommended to assist in treatment and management decisions. This report presents a case of POI in a 16-year-old adolescent with both poly-autoimmune disease and a heterozygous missense variant in the bone morphogenic factor 15 (BMP15) gene, both potentially involved in the pathogenesis of POI. Accurately distinguishing between autoimmune and genetic causes is crucial for effective treatment and counseling. In addition, given the significant psychological impact and the need for reproductive options counseling, a multidisciplinary approach that includes psychological support is highly recommended.

Impact of GnRH agonist and GnRH antagonist on GDF9 and BMP15 expression in mouse ovaries and oocyte development.

GnRH analogues were widely used for controlld ovary stimulation, but their effects on oocyte quality remain contradictory. This study aimed to explore the influence of GnRH analogues on oocyte quality in mice. A total of 120 mice were randomly assigned to four groups:(i)GnRH-a+PMSG group; (ii) GnRH-ant+PMSG group; (iii) PMSG group; (iv) Control group. Ovaries were collected for quantitative real-time polymerase chain reaction (qRT-PCR) to assess GDF9 and BMP15 mRNA expression, and protein expression were evaluated by western blotting. Moreover, embryo developmental progress in vitro and implantation rate in vivo were recorded. Compared with control group, both GDF9 mRNA and protein expressions were strengthened in PMSG group, but reduced in the presence of GnRH-a or GnRH-ant. The GnRH-a group exhibited decreased BMP15 mRNA expression compared to PMSG group, while the GnRH-ant group did not show the same pattern. BMP15 protein expression were not statisticlly different among the four groups. Notably, there was no statistically difference in the expression of these two factors between GnRH-a and GnRH-ant groups. The percentage of zygotes progressing to the 2-cell stage and percentage of 2-cell advancing to the blastocyst stage were similar in the PMSG group and control group. However, both the GnRH-a and GnRH-ant groups showed decreased embryos development rates compared to other two groups. The embryonic implantation rate in control group (53.3%) was higher than that in the GnRH-a and GnRH-ant groups (33.3% and 30.8%, P<0.05). The difference between the PMSG (45.0%) and GnRHa group was statistically significant (P value of 0.023), but not between the PMSG and GnRH-ant group (P value of 0.486). No statistical difference was confirmed between GnRH-a and GnRH-ant groups. Our findings shed light on the safety of GnRH analogues in ovary stimulation, and highlight the need for further research to establish optimal and effective controlled ovary stimulation protocol.

Effects of obesity on the serum BMP15, GDF9, and kisspeptin concentrations in women of reproductive age.

Background: As BMP15, GDF9, and kisspeptin all play critical roles in folliculogenesis and fertilization, investigating the possible relationship between obesity and these three factors could prove crucial in relation to understanding the role of obesity in infertility. Thus, the present study sought to determine the effects of obesity on the serum BMP15, GDF9, and kisspeptin concentrations in women of reproductive age. Methods: Ninety female participants were equally divided into three groups: class-1 obese (n=30), class-2 obese (n=30), and normal weight (control; n=30). The participants' serum BMP15, GDF9, and AMH concentrations were measured. Moreover, the serum kisspeptin concentrations were evaluated in the class-1 obese and control groups by means of the enzyme-linked immunosorbent assay (ELISA) method while the participants were in their menstrual period. Results: The serum BMP15 and kisspeptin concentrations were found to be much higher in the control group than in both obese groups (p=0.001 and p=0.01, respectively). While the GDF9 concentration exhibited a statistically significant positive correlation with age, the BMP15 concentration exhibited a positive correlation with the kisspeptin and LH concentrations in the control group. In addition, a positive correlation was identified between the BMP15 concentration and both age and the glucose level and a negative correlation with the insulin level in both the obese groups. Conclusions: Obesity appears to reduce the serum BMP15 and kisspeptin concentrations in obese women of reproductive age. This reduction may represent a milestone in reproductive dysfunction and may be used to predict the success of infertility treatment in obese women.

The effect of kisspeptin on the maturation of human ovarian follicles in culture following vitrification-thawing processes.

OBJECTIVE: Ovarian cryopreservation is one of the effective methods to preserve fertility for cancer patients. Still, this approach has some problems, namely ROS, resulting in adverse effects on oocytes and ovarian follicles. Kisspeptin as an antioxidant to control ovarian function, directly or indirectly. In this study, the effect of kisspeptin on follicle maturation was evaluated in culture following ovarian cryopreservation. METHODS: Ovarian tissue samples of women between 20 and 35 years old (n=12) were laparoscopically collected. The samples were randomly divided into four groups: 1) control, 2) vitrification, 3) vitrified+1µM kisspeptin, and 4) vitrified+10µM kisspeptin. After vitrification and thawing processes, the tissues were cultured in DMEM medium for 7 days. H&E staining for histological evaluation, Real-Time PCR for GDF9 and BMP15 gene expression, and immunohistochemical staining for GDF9 and BMP15 protein expression were performed. RESULTS: In the vitrification group, ovarian tissue morphology was incoherent, and more primordial follicles than other follicle types were found. The expression of GDF9 and BMP15 genes and proteins were significantly decreased in this group compared with other groups (p<0.05). In the vitrification groups with kisspeptin (1 and 10 µM), the number of primary and secondary follicles was more than in the vitrification group. Besides, the expression of these genes and proteins was dramatically elevated in the vitrification groups with kisspeptin compared to the vitrification group alone (p<0.05). CONCLUSIONS: It seems that kisspeptin is an effective substance to improve the quality of the human ovarian cryopreservation medium by improving follicle maturation.

Polymorphisms Analysis of BMP15, GDF9 and BMPR1B in Tibetan Cashmere Goat (Capra hircus).

The Tibetan cashmere goat is a prolific goat breed in China. In sheep breeds, natural mutations have demonstrated that the transforming growth factor beta (TGF-ß) super family ligands, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor (BMPR1B), are essential for ovulation and increasing litter size. In this study, 216 female Tibetan cashmere goats were sampled, and candidate genes with fecundity traits were detected via restriction fragment length polymorphism (RFLP) and sequenced. Four polymorphic loci were found in specific amplification fragments of BMP15 and GDF9. Two SNP sites of the BMP15 gene were discovered, namely G732A and C805G. The G732A mutation did not cause the change in amino acids, and the frequencies of each genotype were 0.695 for the GG type, 0.282 for the GA type and 0.023 for the AA type. The C805G mutation caused amino acids to change from glutamine to glutamate. The genotype frequencies were 0.620 for the CC type, 0.320 for the CG type and 0.320 for the CG type. For the GG type 0.060, the G3 and G4 mutations of the GDF9 gene were all homozygous mutations. Two known SNP sites, C719T and G1189A, were detected in the Tibetan cashmere goat GDF9 gene, of which the C719T mutation caused a change of alanine to valine, with a genotype frequency of 0.944 for the CC type and 0.056 for the CT type, whereas no TT type was found. The G1189A mutation caused valine to become isoleucine, and the frequencies of each genotype were 0.579 for the GG type, 0.305 for the GA type and 0.116 for the AA type; G1, B2, B3, B4, FecXH, FecXI, FecXL, G2, G5, G6, G7, G8, FecGE, FecTT and FecB mutations were not found in Tibetan cashmere goats. The results of this study provide a data basis for future studies of BMP15, GDF9 and BMPR1B gene mutations in goats.

Ovarian dysfunction in women with Turner syndrome.

Ovarian dysfunction is one of the most common features of women with Turner syndrome. In these women, oocyte apoptosis is markedly accelerated from the early stage of fetal life. Reduction in the number of germ cells disturbs primordial follicle development and thereby leads to the formation of streak gonads. There are three possible causes of accelerated germ cell loss in 45,X ovaries. First, chromosomal pairing failure due to X chromosomal aneuploidy is believed to induce meiotic arrest. Indeed, it has been suggested that the dosage of the X chromosome is more critical for the survival of the oocytes than for other cells in the ovary. Second, impaired coupling between oocytes and granulosa cells may also contribute to germ cell apoptosis. Previous studies have shown that 45,X ovaries may tend to lose tight junctions which are essential for intercellular interactions. Lastly, ovarian dysfunction in women with Turner syndrome is partly attributable to the reduced dosage of several genes on the X chromosome. Specifically, BMP15, PGRMC1, and some other genes on the X chromosome have been implicated in ovarian function. Further studies on the mechanisms of ovarian dysfunction are necessary to improve the reproductive outcomes of women with Turner syndrome.

Correlation of serum kisspeptin levels, ovarian kisspeptin expression, and ovarian BMP15 expression in rat model of polycystic ovary syndrome.

Background: Kisspeptin is a neuropeptide that has an important role in the female reproductive cycle which is indicated by its role in regulating the hypothalamic-pituitary-gonadal axis. Aims: To analyze the correlation between serum kisspeptin levels, ovarian kisspeptin expression, and ovarian Bone Morphogenic Protein-15 (BMP15) expression in polycystic ovary syndrome (PCOS) model rats. Methods: The research was accurate experimental research with a post-test design-only control group and was carried out from August to October 2022 at the Faculty of Veterinary Medicine Universitas Airlangga. 32 Rattus novergicus rats were divided into a control group and a PCOS model group. Blood serum and ovaries were obtained from all groups. In addition, blood serum was examined for kisspeptin levels by ELISA technique, and kisspeptin expression and BMP15 Ovaries were examined immunohistochemically. Results: Serum kisspeptin levels and ovarian kisspeptin expression of the PCOS model group were not significantly higher than those of the control group (p > 0.05, p > 0.05). The ovarian BMP15 expression of the PCOS model group was not significantly lower (p > 0.05) than that of the control group. Ovarian kisspeptin expression and ovarian BMP15 expression did not significantly correlate with serum kisspeptin levels (p > 0.05). In contrast, there was a significant correlation (p < 0.05) between ovarian kisspeptin expression and ovarian BMP15 expression. Conclusion: Serum kisspeptin levels and ovarian kisspeptin expression of the PCOS model group were not higher than those of the control group, and the ovarian BMP15 expression of the PCOS model group was not lower than that of the control group. There was no correlation between serum kisspeptin levels with ovarian kisspeptin expression and ovarian BMP15 expression. However, a significant correlation was found between ovarian kisspeptin expression and ovarian BMP15 expression.

The Significance of a Multilocus Analysis for Assessing the Biodiversity of the Romanov Sheep Breed in a Comparative Aspect.

The Romanov breed was evaluated using immunological and genetic markers. The seven blood group systems were characterized with a greater accuracy than in previous works on sheep in the Russian Federation, and were compared to eight ruminant species. Unlike other breeds, Romanov sheep shows a higher frequency of HBA than HBB alleles. There are 3-4 genotypes at the transferrin locus whereas in other breeds 6-11 genotypes have been found. At the albumin locus, the majority of the identified genotypes were heterozygotes, unlike in the other breeds studied. In the prealbumin locus, the Romanov breed was the only one where all the genotypes were heterozygous. We speculate that polymorphism at two loci (BMP-15 and BMPR1B) could effect on the high ovulation rates of Romanov sheep. Based on different genetic markers, the prevalence of heterozygotes in the Romanov sheep could determine their higher viability. A cluster analysis showed the close proximity of 12 populations of the Romanov breed, as the breeding stock come from the Yaroslavl region.

Effects of New Mutations in BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 Genes on Litter Size in Sheep.

Prolificacy is a crucial characteristic of livestock, particularly for species such as sheep that have many births. The objectives of this study were as follows: (1) to investigate the genetic diversity of the 13 new and 7 known variants in the BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 genes in Ujimqin (UM), the F1 population of Dorper × Ujimqin crossbred (DPU), the F1 population of Suffolk × Ujimqin crossbred (SFKU), Sonid sheep (SN), Tan sheep (Tan), Hu sheep (Hu), and Small-tailed Han sheep (STH) sheep breeds/populations; (2) to perform an association analysis of the above 20 variants with litter size in 325 UM, 304 DPU, and 66 SFKU sheep populations; (3) to compare the frequencies of the litter-size-related alleles of these 20 variants among 8 sheep breeds/populations (the above seven sheep breeds + Mongolia sheep breed). With the use of the Sequenom MassARRAY®SNP assay technology, these 20 mutations were genotyped. The association analysis results showed that the c.746A>G (FecB) mutation in BMPR1B was significantly associated with the litter size of UM and DPU, the c.994A>G (FecGA) in GDF9 was significantly associated with the litter size of SFKU, and the c.31_33CTTinsdel (B1) in BMP15 was significantly associated with the litter size of UM. Our findings might provide valuable genetic markers for expanding sheep litter sizes.

The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-ß 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-ß 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-ß 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-ß signaling pathway. This study provides a theoretical basis for further complementing the BMP15/TGF-ß signaling pathway to regulate mammalian oocyte maturation.

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

STUDY QUESTION: 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? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: 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-ß 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Melatonin effective to reduce the microscopic symptoms of polycystic ovary syndrome-related infertility: An experimental study.

Polycystic ovary syndrome (PCOS) is an endocrine disorder seen in women of reproductive age and has been gradually increasing over the years. The mechanism of the syndrome has still not been clearly understood. In this study, the possible effects of exogenously administrated melatonin on melatonin (MT1) receptor, Growth Differentiation Factor-9 (GDF9), and Bone Morphogenetic Protein-15 (BMP15) in experimental PCOS were investigated. Thirty-two 6-8-week-old Sprague-Dawley rats were randomly divided into four groups (n = 8 in each) as Sham control (Group 1), Melatonin (Group 2), PCOS (Group 3), and PCOS + Melatonin (Group 4) groups. At the end of the 21st day, the experiment was terminated, the ovary tissues were taken, and Hematoxylin-Eosin staining, MT1, GDF9, BMP15 immunohistochemical labeling, western blot, and quantitative real-time polymerase chain reaction (qPCR) analyses were performed. Serum Luteinizing Hormone (LH)/Follicle Stimulating Hormone (FSH) levels and colpo-cytological examinations were also carried out. The results revealed that melatonin administration increased the expression levels of the MT1 receptor, GDF9, and BMP15 in PCOS at protein and mRNA levels. It was determined that melatonin administration reduced the microscopic symptoms of PCOS. Melatonin was found to be effective via the MT1 receptor in the pathogenesis of PCOS, and it suppressed the transport pathways of GDF9 to granulosa cells in antral follicles.