GDF9 Research Articles

Developmental Transcriptome Profiling of the Tibial Reveals the Underlying Molecular Basis for Why Newly Hatched Quails Can Walk While Newly Hatched Pigeons Cannot.

Birds can be classified into altricial and precocial species. The hatchlings of altricial birds cannot stand, whereas precocial birds can walk and run soon after hatching. It might be owing to the development of the hindlimb bones in the embryo stage, but the molecular regulatory basis underlying the divergence is unclear. To address this issue, we chose the altricial pigeon and the precocial Japanese quail as model animals. The data of tibia weight rate, embryonic skeletal staining, and tibia tissues paraffin section during the embryonic stage showed that the Japanese quail and pigeon have similar skeletal development patterns, but the former had a faster calcification rate. We utilized the comparative transcriptome approach to screen the genes and pathways related to this heterochronism. We separately analyzed the gene expression of tibia tissues of quail and pigeon at two consecutive time points from an inability to stand to be able to stand. There were 2910 differentially expressed genes (DEGs) of quail, and 1635 DEGs of pigeon, respectively. A total of 409 DEGs in common in the quail and pigeon. On the other hand, we compared the gene expression profiles of pigeons and quails at four time points, and screened out eight pairs of expression profiles with similar expression trends but delayed expression in pigeons. By screening the common genes in each pair of expression profiles, we obtained a gene set consisting of 152 genes. A total of 79 genes were shared by the 409 DEGs and the 152 genes. Gene Ontology analysis of these common genes showed that 21 genes including the COL gene family (COL11A1, COL9A3, COL9A1), IHH, MSX2, SFRP1, ATP6V1B1, SRGN, CTHRC1, NOG, and GDF5 involved in the process of endochondral ossification. These genes were the candidate genes for the difference of tibial development between pigeon and quail. This is the first known study on the embryo skeletal staining in pigeon. It provides some new insights for studying skeletal development mechanisms and locomotor ability of altricial and precocial bird species.

АЛЛЕЛЬНЫЕ И ГЕНОТИПИЧЕСКИЕ ВАРИАНТЫ ПОЛИМОРФИЗМА ГЕНОВ GH, GDF9 У ОВЕЦ ПОРОДЫ МАНЫЧСКИЙ МЕРИНОС

The article presents the results of genotyping of sheep of the Manych Merino breed. Using PCR-RFLP analysis, the specificity of the allelic spectrum of the GH and GDF9 genes, which regulate growth processes and affect meat productivity, was established, expressed in different frequencies of alleles and genotypes.

ГЕНЕТИЧЕСКАЯ СТРУКТУРА ОВЕЦ ЗАПАДНО-СИБИРСКОЙ МЯСНОЙ И КУЛУНДИНСКОЙ ТОНКОРУННОЙ ПОРОД ПО ГЕНАМ CAST, GDF9 И KRT1.2

The purpose of the study is to analyze the genotypic structure and population-genetic parameters of the West Siberian meat and Kulunda fine-wool breed of sheep by the CAST, GDF9 and KRT1.2 genes and blood groups. Molecularnogenetic and immunogenetic studies were carried out in the SRIAH (Siberian Research Institute of Animal Husbandry SFSCA RAS) biotechnology laboratory. PCR-RFLP analysis to determine genotypes was performed according to the described and approved methods on a C1000 “BioRad” amplifier; the results were visualized using an E-Box-CX5.TS-20.M gel documentation system. It was found that the frequency of genotypes and alleles of the CAST gene of West Siberian meat and Kulunda fine-wool breeds have no significant differences. In both breeds, CASTMM was predominant, with a frequency of 69.0–75.0%, CASTNN carriers being only 1.6–5.1%. The Kulunda fine-wool breed is characterized by a very high frequency of the GDF9GG genotype 92.7%, which is higher than that of the West Siberian meat sheep by 26.3%. GDF9AA genotype is extremely rare in breeds (0–5%). The GDF9G allele in the compared breeds was found to range from 0.811–0.960 and the GDF9A allele from 0.189–0.040. There are differences in the ratio of genotypes of the KRT1.2 gene in the compared breeds. In the Kulunda fine-wool breed, the vast majority of animals have homozygous genotype KRT1.2MM (95.5%), whereas in the West Siberian meat breed such sheep constitute 32.8%. Consequently, the frequency of the KRT1.2M allele is 0.565–0.978 and the KRT1.2N allele is 0.435–0.022. The genetic balance in the studied genes is not disturbed, χ2 = 0,033–1,025. The index of genetic similarity between breeds, calculated on the basis of the frequencies of genotypes and blood groups is 0.901 ± 0.028 and 0.833 ± 0.024. Population and genetic characteristics are practically identical in the compared breeds, except for gene homozygosity (Ca), which is higher in the Kulunda fine-wool sheep breed by 24.3% for the GDF9 gene and KRT1.2 by 29.1%, compared to the West Siberian meat sheep breed. The number of effective alleles is insignificant (1.04–1.46). The genetic variability (V) by individual genes in the breeds varies from 5.0 to 49.6%. The negative value of the Fis coefficient indicates the absence of inbreeding in sheep the studied breeds.

Effect Of Retinol In The Vitrification Medium On Viability Of Vitrified Ovine Preantral Follicles And Expression Of Key Developmental And Apoptosis Related Genes

BACKGROUND: Vitrification increases the production of reactive oxygen species (ROS) and the antioxidants in the vitrification solution may be beneficial by reducing excessive ROS production. OBJECTIVE: To evaluate the effect of retinol supplementation in vitrification solution on viability, apoptosis and development-related gene expression in vitrified sheep preantral follicles. MATERIALS AND METHODS: Preantral follicles were isolated and randomly assigned into one of five groups: Group1, control fresh preantral follicles; Group 2, vitrification treatment; Group 3, vitrification + 2 μM retinol; Group 4, vitrification + 5 μM retinol ; Group 5, vitrification + 10 μM retinol . Preantral follicles were placed in vitrification solutions and then plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue exclusion method and for gene expression. RESULTS: Vitrification with 5 μM retinol positively affected viability in comparison with vitrification without retinol (P < 0.05). There was no significant difference in viability among the Group 1, Group 2, Group 3 and Group 5. Expression of apoptotic genes BAX and Casp 3 were higher in the vitrified group, and vitrification with 5 μM retinol (Group 4) is comparable to the control fresh. Expressions of other apoptosis-related genes (i. e., BCL2L1, BAD and BAK) showed significant difference between the control fresh group and the vitrification group with 5 μM retinol. Expression of Annexin5 was also significantly different among various groups. The expression of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in the Group vitrified with 5 μM retinol. CONCLUSION: The supplementation of 5 μM retinol in vitrification solution was beneficial for the vitrification of ovine preantral follicles.

​The Analysis of Reproduction-related Genes Provides Insights into the Adaptive Evolution of Fecundity Traits in Yangtze Finless Porpoise

Background: Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis), is the first class protected animal in China. In order to analyze the adaptive evolution of fecundity traits in YFP, the rapidly evolving gene families of YFP were obtained. At the same time, the major genes controlling ovulation, GDF9, BMP15, FSHβ and FSHR were also analyzed. Methods: Orthofinder software was employed to search homologous genes based on protein sequence. CAFÉ software was used to obtain the expansion and contraction gene families of YFP. Then, GO terms and pathway enrichment analyses were performed using TBtools software and Swissprot database. PAML package was used to calculate Ka/Ks (i.e., ω). Evolution rate changes in the positive selected genes were examined using the GU99 process in Diverge (v3.0) program. Result: In YFP, 501 rapid expansion gene families GO enrichment results showed that the reproductive activities related pathways were mainly three significant enrichment process, participation of germline stem cells maintain androgen receptors signaling pathways regulating and male reproductive tract stem cell population to maintain. The most significant GO terms of 220 rapidly contraction gene families associated with reproductive activities mainly consisted of biological processes which were involved in positive regulation of estrogen secretion, mating and estrogen metabolic process. GDF9 and BMP15 genes exhibited purifying selection. However, significant signs of positive selection were detected in FSHR and FSHβ genes, but only FSHβ showed specific changes in the YFP lineage.

Polymorphism of Prolificacy Genes (BMP15, BMPR 1B and GDF9), in the Native Goat (Capra hircus) of Cameroon

The main objective was to contribute to a better understanding of molecular characteristics of the local goat in order to improve its productivity and specifically to: analyse genetic polymorphism of three prolificacy genes (BMP15, BMPR1B, and GDF9) and test the association of genetic polymorphism and prolificacy of local goats. Tissue samples were collected from 446 animals, and 24 representative female goats were selected to analyse the genetic polymorphism of the prolificacy genes. The selected goats were divided into two groups of 12 females for high prolificacy (more than three kids consecutively in four parity) and 12 females for low prolificacy (less than two kids consecutively in four parity). Chi-square was used to test the association between genetic polymorphism and prolificacy of local goat. The main results showed that BMP15 gene is monomorphic, whereas the two other genes (BMPR1B and GDF9) display polymorphism. For BMPR1B gene, the ten mutations found did not change the corresponding amino acid. Allelic and genotypes frequencies of mutations of this gene varied from one mutation to another and between the two groups of females (high and low prolificacy). Chi-square test of the polymorphism of this gene shows that C34T and A120G mutations of exon 3 are significantly associated (p < 0.05) with prolificacy and can be considered as potential genetic markers for improving prolificacy in the native goat. For the GDF9 gene, three mutations were detected in exon 1 with alleles A and G1 of frequency 0.261 and 0.130 for A35G; G2 and C1 of frequency 0.696 and 0.304 for G81C; then G3 and C2 of frequency 0.696 and 0.304 for G255C. The mutations G81C and G255C appeared under BLAST and were missense mutations P27A and A85G respectively while A35G is located in the non-translated 5’ region of the gene. Chi-square test between each genotype for any site and the prolificacy was not significant (P > 0.01) suggesting that these two characters are not associated. Two mutations were detected in exon 2 at C881T and A1160G sites with C and T and A and G alleles respectively. The two mutations changed the corresponding amino acid from Alanine to Valine at the position 273 in the protein and from Valine to Isoleucine at the position 397 in the protein respectively. Allelic and genotypes frequencies of mutations varied from one mutation to another and between the two groups of females (high and low prolificacy). Chi-square test of the polymorphism shows that, although C881T and A1160G mutations were not significantly associated (P > 0.05) with prolificacy, the alleles responsible for the variation of amino acid increased the litter size. Therefore, further studies with increased sample size will help to verify the results.

An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.

Single nucleotide polymorphisms of GDF9 gene/exon 2 region and their associations with milk yield and milk content traits in Karakaş and Norduz sheep breeds

Karakaş and Norduz sheep have been adapted to the Lake Van region of Turkey for many years as being domestic genetic resources and meat, milk, and fleece traits are a reliable economic resource especially preferred by small family businesses around the area. In this study, the data set consisted of milk yield (MY) and milk content components; milk fat (MF), fat-free dry matter (FFDM), dry matter (DM), protein (Pro), lactose (Lac), pH, acidity H (aH), and lactic acid (Lac). Besides, Karakaş (n = 30) and Norduz (n = 26) sheep were chosen to investigate based on the SNP method. Correspondingly, genomic DNA from both breeds exon 2 of the GDF9 gene region was amplified, 815 base pairs (bp) in length, by means of PCR. Therefore, there were three noval SNPs detected in both breeds under investigation. Although SNP1 and 2 with genotypes of GG and AG had statistically significant impacts on both milk production and milk components (p < 0.01), SNP3 with genotypes of TT, TC had no significant effects on the milk characteristics in question.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200–300 μm) were randomly assigned into four groups. Group1 – Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2– FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2– FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (−196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Primordial germ cells can be differentiated by retinoic acid and progesterone induction from embryonic stem cells

This study aimed to examine the expression of the genes associated with different development stages of primordial germ cells (PGCs) in differentiating mouse embryonic stem cells (mESCs). The cells were cultured in three groups of control, 10-8 M of all-trans retinoic acid and the combination of 10-7 M of Progesterone and retinoic acid for 7, 12, 17, and 22 days. Immunofluorescent and Quantitative RT-PCR were used to evaluate the effect of progesterone on the differentiation of mESCs into primordial germ cells. RA-treated cells exhibited increased expression of Fragilis, Stella, Dazl, Stra8, Sycp3, and Gdf9 genes and decreased expression of Oct4, Mvh genes compared to the non-treated controls. Furthermore, RA in combination with progesterone (RA?P) led to increased expression of Oct4, Fragilis, Stella, Dazl, Sycp3, Gdf9 and decreased expression of Mvh, and Stra8 genes compared to the RA-treated scenario. Immunofluorescence detection of Stella and Mvh showed that the expression levels of the cells treated with RA+P are much higher than those of the other groups. Our project showed that under the influence of the induced factors, mESCs can spontaneously differentiate into germ cells. Also, the combination of RA+P can enhance and accelerate the differentiation of mESCs into germ cells.

Monitoring of the allele fund of erythrocyte antigens and DNA polymorphism of genes - markers of beef quality

The article presents the results of the analysis of animals of the Bredy meat type by the allele fund of erythrocyte antigens of blood groups and DNA - polymorphism of the GDF5 and TG5 genes, which are valuable for beef cattle. The studies were carried out on the basis of blood analysis of cows from three micropopulations belonging to well-known breeding enterprises in Russia. Alleles of blood groups were identified for all systems, and the frequency of their occurrence was shown. The work focuses on the ratio of homozygous and heterozygous genotypes, shows the degree of their displacement depending on the micropopulation, determines the indices of genetic similarity (I) and genetic distance (Dn) between micropopulations. The revealed levels of polymorphism of animals of the Bredy meat type for genes GDF5 and TG5 showed an insignificant frequency of occurrence of the desired genotypes, this indicator for the first gene was 8.7, and for the second - 4.3%. Comparison of the frequency of occurrence of the most common alleles of the EAB-system of blood groups of the herd, with the frequency of occurrence of genotypes for genes GDF5 and TG5 in a random sample of cows of the Bredy meat type, did not show any dependence of the polymorphic state of genes on the specific content of alleles.

The decellularized ovary as a potential scaffold for maturation of preantral ovarian follicles of prepubertal mice

ABSTRACT In the current study, a decellularized ovary scaffold was used for the encapsulation and development of preantral follicles. Maturation and the expression of the related genes were evaluated and compared against in vivo conditions. Mature rat ovaries were decellularized and decellularization was confirmed using hematoxylin/eosin and Hoechst stains. Collagen and glycosaminoglycan (GAG) preservation and the extracellular matrix (ECM) architecture were evaluated by Masson’s trichrome staining, Alcian blue staining, and scanning electron microscopy, respectively. Raman confocal microscopy and DNA quantification were performed to show DNA and cell debris depletion and ECM retention. Preantral follicles of mice were cultured for 12 days in the decellularized (3D) and two-dimensional (2D) conditions. Finally, the survival rate, antrum formation rate and diameters of follicles, levels of estradiol (E2) and progesterone (P4), and expression of the ZP2, Gdf9, Bmp6, and Bmp15 genes were evaluated in the 2D and 3D culture systems. On day 13 of culture, oocyte maturation was induced by hCG. Although histochemical tests showed partial retention of ECM, Raman confocal microscopy showed some extent of ECM washing out along with the depletion of DNA and cell debris. However, SEM showed the preservation of the ovarian ECM’s ultrastructure. The survival rate of follicles was similar, but the antrum formation rate, concentrations of E2 and P4, and oocyte maturation rate were significantly higher in 3D compared with 2D. The level of ZP2, Gdf9, Bmp6, and Bmp15 gene expression in follicles matured in 2D, 3D, and in vivo was statistically similar. In all matured follicles, their gene expression levels significantly declined relative to preantral follicles. Ovarian follicles that matured in the decellularized ovaries demonstrated the same follicular growth and maturation gene expression levels as those that grew in the natural ovary. This is consistent with using the decellularized ovary as a scaffold suitable for ovarian reconstruction. Abbreviations GAG: glycosaminoglycan; ECM: extracellular matrix; 2D: two-dimensional; E2: estradiol; P4: progesterone; BMP15: bone morphogenetic protein 15; GDF9: growth differentiation factor 9; ZP2: zona pellucida 2; Gdf9: growth/differentiation factor-9; Bmp6: bone morphogenetic protein 6; Bmp15: bone morphogenetic protein 15.

Association between gene expression levels of GDF9 and BMP15 and clinicopathological factors in the prognosis of female infertility in northeast Indian populations

Female Infertility is a serious health issue, and genetic factors plays crucial role in female infertility. To explore the molecular mechanism involved in female infertility, we had investigated demographic, clinicopathological factors and gene expression profile of BMP15 and GDF9 in primary and secondary female infertility patients. Present study comprised of 469 primary and 210 secondary infertile females, and 419 fertile females (controls). The expression level of GDF9 and BMP15 genes was studied using quantitative real-time PCR and multifactor dimensionality reduction tool (MDR) was used to detect high-risk interaction. Finding revealed that there was down-regulation of the BMP15 and GDF9 gene in both primary and secondary infertile women in comparison to fertile women. A significant difference in the expression level of GDF9 gene among the study subjects for the various levels of TSH and RBS (p < 0.01) indicating that the level of gene expression was influenced by few hormonal factors. The best risk factor model predicted for female infertility was the five-factors model of TSH, LH, LH/FSH ratio, Hb, MCV with 100% CVC and maximum TBA = 0.921 and p < 0.01. Thus, down-regulation of GDF9 and BMP15 gene and interaction of clinicopathological factors significantly persuade female infertility, and these findings might be useful as possible biomarker for the estimation of primary and secondary female infertility.

Alteration of TGFB1, GDF9, and BMPR2 gene expression in preantral follicles of an estradiol valerate-induced polycystic ovary mouse model can lead to anovulation, polycystic morphology, obesity, and absence of hyperandrogenism.

ObjectiveIn humans, polycystic ovary syndrome (PCOS) is an androgen-dependent ovarian disorder. Aberrant gene expression in folliculogenesis can arrest the transition of preantral to antral follicles, leading to PCOS. We explored the possible role of altered gene expression in preantral follicles of estradiol valerate (EV) induced polycystic ovaries (PCO) in a mouse model.MethodsTwenty female balb/c mice (8 weeks, 20.0±1.5 g) were grouped into control and PCO groups. PCO was induced by intramuscular EV injection. After 8 weeks, the animals were killed by cervical dislocation. Blood serum (for hormonal assessments using the enzyme-linked immunosorbent assay technique) was aspirated, and ovaries (the right ovary for histological examinations and the left for quantitative real-time polymerase) were dissected. ResultsCompared to the control group, the PCO group showed significantly lower values for the mean body weight, number of preantral and antral follicles, serum levels of estradiol, luteinizing hormone, testosterone, and follicle-stimulating hormone, and gene expression of TGFB1, GDF9 and BMPR2 (p<0.05). Serum progesterone levels were significantly higher in the PCO animals than in the control group (p<0.05). No significant between-group differences (p>0.05) were found in BMP6 or BMP15 expression. ConclusionIn animals with EV-induced PCO, the preantral follicles did not develop into antral follicles. In this mouse model, the gene expression of TGFB1, GDF9, and BMPR2 was lower in preantral follicles, which is probably related to the pathologic conditions of PCO. Hypoandrogenism was also detected in this EV-induced murine PCO model.

Polymorphism of genes CAST, GH, GDF9 of sheep of the Dagestan mountain breed

Aim. To study the polymorphism of the genes calpastatin (CAST), somatotropin (GH) and differential growth factor (GDF9) of sheep of the Dagestan mountain breed.Material and Methods. Studies on the genetic typing of Dagestan mountain sheep according to CAST, GH and GDF9 genes were carried out in conditions of distant pasture farming in the Republic of Dagestan by the Genetics and Breeding Laboratory of the Federal Agrarian Scientific Centre of the Republic of Dagestan, an accredited laboratory of immunogenetics and DNA technologies of the All-Russian Research Institute of Sheep and Goat Breeding and a branch of the North Caucasus Federal Scientific Agrarian Centre. These investigations were undertaken by the PCR-RFLP method (polymyrase chain reaction - restriction fragment length polymorphism) on a Tersik four-channel programmable thermal cycler from DNA-Technologia (Russia) using specific primers synthesized in the SYNTHOL research and production laboratory (Moscow).Results. Carrying out PCR-RFLP revealed breed-specific alleles with different frequency of occurrence, which was: in the locus of the CAST gene - allele CASTN - 0.03; allele CASTM - 0.97; at the GH gene locus - GHA allele - 100.0, GHB allele - 0; in the locus of the GDF9 gene - the GDF9A allele - 0.25, the GDF9G allele - 0.75, which determined the frequency of occurrence of the homo- and heterozygous genotypes: CASTNN, CASTMM and CASTNM - 93.0; 0 and 7.0%; GHAA, GHBB and GHAB - 100.0 and 0%; GDF9AA, GDF9GG and GDF9АG- 16.0, 66.0 and 19.0%, respectively.Conclusion. The regularity revealed can be considered as an ecological factor that optimizes the adaptive functions of the sheep's organism, on the one hand, an evolutionary selection process that contributes to the creation of a specific way of genetic structures in the population, on the other. The assumption is made that the information obtained can serve as the beginning of a more in-depth study of the unique gene pool of sheep of the Dagestan rock breed for its further improvement. Variants of genetic marker profiles of parental pairs are proposed for accumulation in breeding herds bred in different ecological-geographical zones and genotypes of carriers of selection-significant genetic structures.

Association between miRNA Target Sites and Incidence of Primary Osteoarthritis in Women from Volga-Ural Region of Russia: A Case-Control Study.

Over the past decades, numerous studies on the genetic markers of osteoarthritis (OA) have been conducted. MiRNA targets sites are a promising new area of research. In this study, we analyzed the polymorphic variants in 3′ UTR regions of COL1A1, COL11A1, ADAMTS5, MMP1, MMP13, SOX9, GDF5, FGF2, FGFR1, and FGFRL1 genes to examine the association between miRNA target site alteration and the incidence of OA in women from the Volga-Ural region of Russia using competitive allele-specific PCR. The T allele of the rs9659030 was associated with generalized OA (OR = 2.0), whereas the C allele of the rs229069 was associated with total OA (OR = 1.43). The T allele of the rs13317 was associated with the total OA (OR = 1.67). After Benjamini-Hochberg correction, only rs13317 remained statistically significant. According to ethnic heterogeneity, associations between the T allele (rs1061237) with OA in women of Russian descent (OR = 1.77), the G allele (rs6854081) in women of Tatar descent (OR = 4.78), the C allele (rs229069) and the T allele (rs73611720) in women of mixed descent and other ethnic groups (OR = 2.25 and OR = 3.02, respectively) were identified. All associations remained statistically significant after Benjamini-Hochberg correction. Together, this study identified miRNA target sites as a genetic marker for the development of OA in various ethnic groups.

Генетическая структура стада по генам GDF9, GH у овец Волгоградской и Эдильбаевской пород

Aim. The study of the genetic structure of sheep of the Volgograd and Edilbaevsky breeds according to the GDF9, GH genes. Materials and Methods. The research was carried out in herds of sheep from leading breeding plants, on the basis of Don State Agrarian University and Volga Region Research Institute of Manufacture and Processing of Meat-and-Milk Production. Biosamples of skin and cartilage tissue from the auricles were taken from sheep by plucking an area of 1 cm² for molecular genetic studies. Evaluation was performed by PCR-RFLP method. Endonucleases BstHH HaeIII were used to restrict the amplified regions of the GDF9 and GH genes. The presence and frequency of alleles and genotypes for the GDF9, GH genes were determined based on the results of a molecular genetic study. Results. The article is devoted to the study of the genetic structure of sheep of the Volgograd and Edilbaevsky breeds. One of the important resources for creating highly productive animals is the use of marker selection as an additional criterion for the selection and selection of breeding animals. The use of DNA markers for the selection of animals containing desirable alleles of genes of economically valuable traits is one of the new scientific approaches to solve this problem. The development of sheep breeding in our country will allow using the available natural and human resources in rural areas, as well as obtaining high-quality lamb through the use of resource-saving technologies. The aim of the work was to study the genetic structure of sheep of the Volgograd and Edilbaevsky breeds according to the GDF9, GH genes. To perform molecular genetic studies, samples of ear plucks (tissue from the auricle) with an area of 1 cm2 were taken from sheep. The assessment was carried out by PCR-PDRF. In all the studied groups of sheep, A and B allelic variants of the GDF9 gene, alleles A and B of the GH gene were established. The analysis of the data revealed a breed aspect in the distribution of the frequency of alleles and genotypes of the GDF9, GH genes in the populations under consideration. Conclusion. The results of the study of the genetic structure of the populations of sheep of the Volgograd and Edilbaevsky breeds showed that the frequency distribution of alleles and genotypes of the growth hormone (GH) and differential growth factor (GDF9) genes have some features associated with the breed affiliation of the studied sheep.

Characterization of a novel mutation V136L in bone morphogenetic protein 15 identified in a woman affected by POI

BackgroundPremature ovarian insufficiency (POI) is an ovarian defect characterized by primary or secondary amenorrhea, hypergonadotropism and hypoestrogenism which occurs before the age of 40 years with a major genetic component. In this study we performed clinical evaluation and genetic analysis of a group of 18 patients with POI.The study involved 18 consecutive women with POI. Karyotiping and genetic analysis for research of mutations in GDF9 (Growth Differentation Factor 9) and BMP15 (Bone morphogentic protein 15) genes and FMR1 (Fragile X Mental Retardation 1) premutation were carried out. In vitro functional study of the novel BMP15 mutation was performed using COV434 (Human ovarian granulosa tumour cells 434) cells of ovarian granulosa, which consistently express BMP responsive element, and luciferase reporter assay.ResultsThree patients (17%) had a family history of POI. Ten patients (56%) had a family history of autoimmune diseases and nine patients (50%) showed a personal history of one or more autoimmune diseases. Of patients for whom morphological assessment was available, almost half (44%) had poor follicle assets or small ovaries’s size at pelvic US. Two patients (13%) showed reduced bone density at DEXA (Dual Energy X-ray Absorptiometry). All the women had normal female kariotype and no mutations in the GDF-9 gene or FMR1 premutations were found. A novel heterozygous mutation c.406G > C (V136L) of BMP15 gene was identified in one patient. After transfection in COV434 cells, BMP15 variant showed a significantly reduced luciferase activity compared to wild type.ConclusionsPOI is a multifactorial disease with several health implications. Autoimmunity and genetics represent the most common aetiology. We identified and characterized a novel BMP15 mutation, providing an additional elucidation of molecular basis of this complex disorder.

A study on mutation points of GDF9 gene and their association with prolificacy in Egyptian small ruminants

BackgroundGenetic variants of the GDF9 gene were considered to be the potent gene markers for improving fecundity traits in Egyptian sheep and goats. Also, these favorable gene variants could be applied in the breeding program by gene-assisted selection (GAS), aiming towards the potential amelioration of reproduction and production in such small ruminants. The present investigation was designed to evaluate the genetic variants of the GDF9 gene on fecundity traits including the mean number of lambing “MNL” and mean number of twin production “MNTP” of Egyptian sheep and goats. ResultsThis experiment involved 113 mothers, 83 of sheep and 30 of goats, at first, second, third, and fourth parity, and also 26 young females, 12 of sheep and 14 of goats at age of sexual maturation. T-ARMS-PCR analysis was performed on five mutation points (G1, G4, G6, G7, and G8). In sheep, the heterozygous mothers of G4 had significant elevation (P ≤ 0.05) of MNL and MNTP than wild-type homozygous ewes. However, the heterozygous mothers of G1 and G6 gave a reduction of MNL and MNTP as compared to mothers with wild-type genotypes. The ewes of G7 had heterozygous genotype (AG), and the ewes of G8 had wild type (CC). In goat, G4 and G7 were polymorphic, and G1, G6, and G8 were monomorphic type. Based on these findings, it must be selected the young sheep females of heterozygous in G4, and the young goat females of heterozygous in G4 and G7 for participating in a successful breeding program, because they will have potential high fecundity traits. ConclusionThe present results confirmed that the genetic variants of the GDF9 gene were considered to be the major gene markers for enhancement of the prolificacy in Egyptian sheep and goats and could be applied in a successful breeding program by gene-assisted selection (GAS) in small ruminants.

Abnormal early folliculogenesis due to impeded pyruvate metabolism in mouse oocytes†.

Fetal ovarian germ cells show characteristic energy metabolism status, such as enhanced mitochondrial metabolism as well as glycolysis, but their roles in early folliculogenesis are unclear. We show here that inhibition of pyruvate uptake to mitochondria by UK5099 in organ cultures of fetal mouse ovaries resulted in repressed early folliculogenesis without affecting energy production, survival of oocytes, or meiosis. In addition, the abnormal folliculogenesis by UK5099 was partially rescued by α-ketoglutarate and succinate, intermediate metabolites in the TCA cycle, suggesting the importance of those metabolites. The expression of TGFβ-related genes Gdf9 and Bmp15 in ovarian germ cells, which are crucial for folliculogenesis, was downregulated by UK5099, and the addition of recombinant GDF9 partially rescued the abnormal folliculogenesis induced by UK5099. We also found that early folliculogenesis was similarly repressed, as in the culture, in the ovaries of a germ cell-specific knockout of Mpc2, which encodes a mitochondria pyruvate carrier that is targeted by UK5099. These results suggest that insufficient Gdf9 expression induced by abnormal pyruvate metabolism in oocytes results in early follicular dysgenesis, which is a possible cause of defective folliculogenesis in humans.