Maternal Kdm2a-mediated PI3K/Akt signaling and E-cadherin stimulate the morula-to-blastocyst transition revealing crucial roles in early embryonic development.

Histone methylation plays an essential role in oocyte growth and preimplantation embryonic development. The modification relies on histone methyl-transferases and demethylases, and one of these, lysine-specific demethylase 2a (Kdm2a), is responsible for modulating histone methylation during oocyte and early embryonic development. The mechanism of how Kdm2a deficiency disrupts early embryonic development and fertility remains elusive. To determine if maternally deposited Kdm2a is required for preimplantation embryonic development, the expression profile of Kdm2a during early embryos was detected via immunofluorescence staining and RT-qPCR. The Kdm2a gene in oocytes was specifically deleted with the Zp3-Cre/LoxP system and the effects of maternal Kdm2a loss were studied through a comprehensive range of female reproductive parameters including fertilization, embryo development, and the number of births. RNA transcriptome sequencing was performed to determine differential mRNA expression, and the interaction between Kdm2a and the PI3K/Akt pathway was studied with a specific inhibitor and activator. Our results revealed that Kdm2a was continuously expressed in preimplantation embryos and loss of maternal Kdm2a suppressed the morula-to-blastocyst transition, which may have been responsible for female subfertility. After the deletion of Kdm2a, the global H3K36me2 methylation in mutant embryos was markedly increased, but the expression of E-cadherin decreased significantly in morula embryos compared to controls. Mechanistically, RNA-seq analysis revealed that deficiency of maternal Kdm2a altered the mRNA expression profile, especially in the PI3K/Akt signaling pathway. Interestingly, the addition of a PI3K/Akt inhibitor (LY294002) to the culture medium blocked embryo development at the stage of morula; however, the developmental block caused by maternal Kdm2a loss was partially rescued with a PI3K/Akt activator (SC79). In summary, our results indicate that loss of Kdm2a influences the transcriptome profile and disrupts the PI3K/Akt signaling pathway during the development of preimplantation embryo. This can result in embryo block at the morula stage and female subfertility, which suggests that maternal Kdm2a is a potential partial redundancy with other genes encoding enzymes in the dynamics of early embryonic development. Our results provide further insight into the role of histone modification, especially on Kdm2a, in preimplantation embryonic development in mice.

The Biological Characteristics and Differential Expression Patterns of TSSK1B Gene in Yak and Its Infertile Hybrid Offspring.

This study aimed to investigate the spatially and temporally expressed patterns and biological characteristics of TSSK1B in male yaks and explore the potential correlation between TSSK1B and male sterility of the yak hybrid offspring (termed cattle-yak). First, the coding sequence (CDS) of TSSK1B was cloned by RT-PCR, and bioinformatics analysis was conducted with relevant software. Quantitative real-time PCR (RT-qPCR) was employed to detect the expression profile of TSSK1B in various tissues of male adult yaks, the spatiotemporal expression of TSSK1B in different stages of yak testes, and the differential expression of TSSK1B between yak and cattle-yak testes. The cellular localization of TSSK1B was determined by immunohistochemistry (IHC). Furthermore, the methylation status of the TSSK1B promoter region was analyzed by bisulfite-sequencing PCR (BSP). The results showed that TSSK1B was 1235 bp long, including 1104 bp of the CDS region, which encoded 367 amino acids. It was a conserved gene sharing the highest homology with Bos mutus (99.67%). In addition, the bioinformatics analysis revealed that TSSK1B was an unstable hydrophilic protein mainly containing the alpha helix of 34.06% and a random coil of 44.41%, with a transmembrane structure of 29 amino acids long. The RT-qPCR results demonstrated that TSSK1B was specifically expressed in yak testes compared with that in other tissues and especially highly expressed in adult yak testes. On the contrary, TSSK1B was hardly expressed in the testis of adult cattle-yak. IHC confirmed that TSSK1B protein was more strongly expressed in the testes of adult yaks than in their fetal and juvenile counterparts. Interestingly, nearly no expression was observed in the testes of cattle-yak compared with the corresponding testes of yak. Bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the TSSK1B promoter region of cattle-yak was significantly higher than that in the yak. Taken together, this study revealed that TSSK1B was specifically expressed in yak testes and highly expressed upon sexual maturity. Moreover, the rare expression in cattle-yak may be related to the hypermethylation of the promoter region, thereby providing a basis for further studies on the regulatory mechanism of TSSK1B in male cattle-yak sterility.

Analysis of PPP1R11 expression in granulosa cells during developmental follicles of yak and its effects on cell function.

The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non-pregnant female yaks (4-year-old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (<3.0 mm), medium (3.0-5.9 mm) and large (6.0-9.0 mm) follicles was detected by RT-qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si-PPP1R11 and si-NC (negative control) were transfected into GCs. RT-qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation- and apoptosis-related genes were detected by RT-qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si-PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway-related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation-related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis-related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development.

Oocyte-Specific Knockout of Histone Lysine Demethylase KDM2a Compromises Fertility by Blocking the Development of Follicles and Oocytes.

The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17ß-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.

MicroRNA-342-3p regulates yak oocyte meiotic maturation by targeting DNA methyltransferase 1.

MicroRNAs (miRNAs) play vital roles in the development of oocytes and ovarian follicles. We have previously shown differential expression of miR-342-3p during yak oocyte maturation. In this study, we investigated the role of miR-342-3p in meiotic maturation of yak oocytes and the underlying mechanism. The profile of ovarian DNA methyltransferase 1 (DNMT1) expression was investigated in yak by RT-qPCR and western blot analyses. The pattern of Dnmt1 expression in various meiotic stages (GV stage, MI stage and MII stage) of yak oocyte maturation was then measured by immunofluorescence staining. The interaction between Dnmt1 and miR-342-3p was verified by dual-luciferase reporter assay. Finally, miR-342-3p inhibitors were microinjected into yak cumulus-oocyte complex to evaluate the effects on oocyte maturation. MiR-342-3p expression was upregulated in oocytes during meiotic maturation, with significantly higher levels in the MII stage compared with the GV- and MI stages (p < .05), whereas the opposite pattern of Dnmt1 expression was detected. In the period to sexual maturity (3-year-old), DNMT1 showed an age-related pattern of ovarian expression at both the gene and protein levels. Immunohistochemistry analysis also indicated maturation-stage-related differences in DNMT1 expression in the ovarian follicles and corpus luteum, with expression predominantly detected in cumulus cells and oocytes. MiR-342-3p inhibitors effectively upregulated Dnmt1 expression and significantly inhibited oocyte meiotic maturation. Taken together, our results indicate that miR-342-3p plays a vital role in the meiotic maturation of yak oocytes by targeting the 3'-untranslated regions (UTR) of Dnmt1 and provide a new perspective on the mechanism of this process.

Polymorphisms of SORBS1 Gene and Their Correlation with Milk Fat Traits of Cattleyak.

This study aimed to find the SNPs in the SORBS1 gene of cattleyak, analyze the relationship between its polymorphisms and the milk fat traits, and find potential molecular markers for the milk fat traits of cattleyak. The polymorphism of the SORBS1 gene in 350 cattleyak from Hongyuan County (Sichuan, China) were detected by PCR and DNA sequencing, and the correlation between these SNPs and the milk production traits of cattleyak was analyzed. The results showed that there were nine SNPs in the CDS and their adjacent non-coding regions of the SORBS1 gene, and all SNPs have three genotypes. The correlation analysis found that the genotypes with superior milk fat traits in the other eight alleles were homozygous genotypes with a high genotype frequency except the g.96284 G > A (c.3090 G > A) (p < 0.05). However, at locus g.96284 G > A, the milk fat percentage, monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) of the GA genotype were significantly higher than that of GG and AA genotypes (p < 0.05). Among these SNPs, three SNPs (g.6256 C > T (c.298 C > T), g.24791 A > G (c.706 A > G) and g.29121 A > G (c.979 A > G)) caused the amino acids change. The genotypes of the three SNPs consist of three haplotypes and four diplotypes. The amino acid mutation degree of diplotype H1-H1 (CCAAAA) was the highest, and its milk fat percentage, MUFAs, PUFAs and SFAs were also the highest (p < 0.05). Taken together, we found nine SNPs in the SORBS1 gene that are closely related to the milk fat traits of cattleyak. Moreover, the mutation of amino acids caused by SNPs had positive effects on the milk fat traits of cattleyak. H1-H1 is the dominant diplotype which significantly related to the milk fat traits of cattleyak. This study provides a new molecular marker and theoretical basis for screening the milk fat traits of cattleyak.