Yak Oocytes Research Articles

Hypoxia-Inducible Factor 1α Affects Yak Oocyte Maturation and Early Embryonic Development by Regulating Autophagy.

In animal assisted reproductive technology, the production of high-quality oocytes is crucial. The yak, having lived in the Qinghai-Tibet Plateau for an extended period, has reproductive cells that are regulated by hypoxia-inducible factor 1α (HIF-1α). This study aimed to investigate the impact of HIF-1α on yak oocyte maturation and early embryonic development in vitro through the regulation of autophagy. The in vitro maturation process of yak oocytes involved the addition of the HIF-1α inducer DFOM and the inhibitor LW6 to examine their effects on yak oocyte maturation, early embryonic development, cell autophagy, cytochrome P450s (CYP450s) enzyme expression, and cumulus diffusion factors. The findings revealed that DFOM significantly upregulated the expression of HIF-1α, resulting in increased the cumulus diffusion area, elevated first polar body expulsion rate of oocytes, enhanced mitochondrial and actin levels, decreased ROS production, and reduced early apoptosis levels of oocytes. Moreover, DFOM promoted the expression of autophagy-related proteins, CYP450s enzymes, and cumulus diffusion factors, thereby enhancing oocyte maturation and early embryonic development. Conversely, LW6 exhibited opposite effects. The inhibition of autophagy levels with 3-MA during DFOM treatment yielded similar outcomes. Furthermore, reducing autophagy led to increased apoptosis levels at all stages of early embryonic development, as well as a significant decrease in total cell number and ICM/TE ratio of blastocysts. Studies have shown that during the in vitro maturation of yak oocytes, HIF-1α can affect the cumulus expansion area of oocytes by regulating autophagy, the first polar body excretion rate, mitochondrial level, actin level, ROS and early apoptosis level, the CYP450s enzyme, and the expression of cumulus expansion factors, thereby improving the in vitro maturation and early embryonic development of yak oocytes. These findings offer valuable insights into the reproductive regulation mechanism of yaks in hypoxic environments and suggest potential strategies for the advancement of yak assisted reproductive technology.

C-type natriuretic peptide treatment before in vitro maturation enhances maturation of yak oocytes and developmental competence of their crossbred embryos

ABSTRACT The effects of C-type natriuretic peptide (CNP) pre-treatment on the in vitro maturation (IVM) and developmental competence of yak (Bos grunniens) oocytes were evaluated. The results showed that 100 nM CNP could effectively maintain meiotic arrest of yak oocytes in vitro within 6 h. Yak oocytes matured in two-step IVM (pre-IVM with 100 nM CNP for 6 h followed by conventional IVM for 28 h) yielded a significantly (P < .05) increased cleavage rate, blastocyst rate after in vitro fertilization with cattle spermatozoa compared to the conventional one-step IVM method. The density of transzonal projections (TZPs) decreased after 6 h standard IVM (P < .05), whereas, COCs pre-IVM with 100 nM CNP for 6 h maintained the same density of TZPs compared to the fresh COCs. This pre-IVM resulted in lesser reactive oxygen species level (P < .05). Two-step IVM resulted in the increased glutathione levels, mRNA expression levels of BCL2, BMP15, and GDF9 (P < .05) in oocytes. In conclusion, these results indicate that CNP treatment (100 nM, 6 h) before IVM could enhance the maturation of yak oocytes and developmental competence of their crossbred embryos. This may have important applications in in vitro production of yak crossbred embryos.

Factors Influencing the Maturation and Developmental Competence of Yak (Bos grunniens) Oocytes In Vitro.

The yak (Bos grunniens) is a unique breed living on the Qinghai-Tibet Plateau and its surrounding areas, providing locals with a variety of vital means of living and production. However, the yak has poor sexual maturity and low fertility. High-quality mature oocytes are the basis of animal breeding technology. Recently, in vitro culturing of oocytes and embryo engineering technology have been applied to yak breeding. However, compared to those observed in vivo, the maturation rate and developmental capacity of in vitro oocytes are still low, which severely limits the application of in vitro fertilization and embryo production in yaks. This review summarizes the endogenous and exogenous factors affecting the in vitro maturation (IVM) and developmental ability of yak oocytes reported in recent years and provides a theoretical basis for obtaining high-quality oocytes for in vitro fertilization and embryo production in yaks.

Dynamic Changes in Proteome during Yak Oocyte Maturation Analyzed Using iTRAQ Technology

The aim of this study was to investigate protein regulation at different time points during the in vitro maturation of yak oocytes. Yak oocytes at GV, MI, and MII stages were collected during in vitro maturation, and differential proteomics sequencing was performed using iTRAQ technology. GO functional classification indicated that the differential proteins were closely associated with biological processes such as “metabolic processes”, and molecular events such as “binding” molecular-function-related categories were active. KOG analysis showed that energy-metabolism-related activities were vigorous during oocyte development from the GV phase to MI phase, and genetic material preparation activities were more active when oocytes developed from the MI stage to MII stage. KEGG pathway analysis showed that the PPAR metabolic pathway, Hippo signaling pathway, and ECM–receptor interaction and metabolic pathway were enriched from the GV to the MI stages. The PI3K-Akt, TGF-β, and phagosome pathways were enriched from the MI stage to the MII stage. These results indicate that transient dynamic changes occurred in the proteome during the maturation of yak oocytes, and the physiological functions mediated by these were also different. The accurate identification of the differential proteins in the three stages of GV, MI, and MII was helpful in further analyzing the molecular regulatory mechanism of yak oocyte maturation.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3β-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

Leukemia inhibitory factor enhances the development and subsequent blastocysts quality of yak oocytes in vitro.

Leukemia inhibitory factor (LIF) is a multipotent cytokine of the IL-6 family which plays a critical role in the maturation and development of oocytes. This study evaluated the influence of LIF on the maturation and development ability of yak oocytes, and the quality of subsequent blastocysts under in vitro culture settings. Different concentrations of LIF (0, 25, 50, and 100 ng/mL) were added during the in vitro culture of oocytes to detect the maturation rate of oocytes, levels of mitochondria, reactive oxygen species (ROS), actin, and apoptosis in oocytes, mRNA transcription levels of apoptosis and antioxidant-related genes in oocytes, and total cell number and apoptosis levels in subsequent blastocysts. The findings revealed that 50 ng/mL LIF could significantly increase the maturation rate (p < 0.01), levels of mitochondria (p < 0.01) and actin (p < 0.01), and mRNA transcription levels of anti-apoptotic and antioxidant-related genes in yak oocytes. Also, 50 ng/mL LIF could significantly lower the generation of ROS (p < 0.01) and apoptosis levels of oocytes (p < 0.01). In addition, blastocysts formed from 50 ng/mL LIF-treated oocytes showed significantly larger total cell numbers (p < 0.01) and lower apoptosis rates (p < 0.01) than the control group. In conclusion, the addition of LIF during the in vitro maturation of yak oocytes improved the quality and the competence of maturation and development in oocytes, as well as the quality of subsequent blastocysts. The result of this study provided some insights into the role and function of LIF in vitro yak oocytes maturation, as well as provided fundamental knowledge for assisted reproductive technologies in the yak.

Effect of rapamycin treatment on oocyte in vitro maturation and embryonic development after parthenogenesis in yaks

Autophagy plays an important role in mammalian oocyte maturation and early embryonic development and rapamycin is well known for inducing autophagy. Although previous studies have reported the effects of rapamycin on oocytes in vitro maturation (IVM) in different species, few studies have been reported on the role of rapamycin in yak oocytes IVM and embryonic development. Therefore, the objective of this study was to examine the effect of rapamycin treatment on yak oocytes IVM and early embryonic development. Specifically, immature yak oocytes during IVM or parthenogenetic (PA) embryos were treated with different rapamycin concentrations to select an optimal dose. Then evaluated its effect on maturation rates, cleavage, and blastocyst formation rates, mitochondrial membrane potential, ROS levels. Related genes and proteins expression in matured oocytes and blastocysts were also evaluated. The results show that 10 nM rapamycin treatment during IVM significantly improved oocyte maturation rates of oocytes and blastocyst formation rates. Treatment with 10 nM rapamycin reduced ROS level but increased mitochondrial membrane potential. Correspondingly, mRNA and protein expressions of LC3, Beclin-1, and Bcl-2 up-regulated while Bax down-regulated in matured yak COCs. When parthenogenetic embryos were treated with different rapamycin concentrations, 10 nM rapamycin treatment showed higher 8-cell and blastocyst formation rates. Also, CDX2, POU5F1, SOX2, and Nanog levels in blastocysts were upregulated. In summary, our findings demonstrate that rapamycin treatment improves oocytes maturation probably by increasing mitochondrial membrane potential, reducing ROS levels, and regulating the apoptosis in mature yak oocytes. Rapamycin treatment also improves embryonic developmental competence in the yak.

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.

Low oxygen concentration improves yak oocyte maturation and inhibits apoptosis through HIF-1 and VEGF.

The gas-phase environment of in vitro culture system plays an important role in the development of oocytes, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effect of different oxygen concentrations (20%, 10%, 5% or 1% O2 ) in yak oocyte maturation and to detect the expression of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF) and cell apoptosis in yak COCs. First, the maturation rate of oocytes, cleavage rate and blastocysts rate following parthenogenetic activation in the group with 5% oxygen concentration were significantly higher (p<.05) than the other groups. Then, TUNEL analysis showed that the 5% oxygen concentration group significantly inhibited apoptosis of cumulus-oocyte complexes (COCs) compared to the other group, and the transcription and protein levels of pro-apoptotic factor Bax, HIF-1α and VEGF in yak COCs significantly reduced, while anti-apoptotic factor Bcl-2 significantly increased. Furthermore, immunohistochemical staining results indicated that HIF-1α protein was mainly located in theca follicle interna, mural follicular stratum granulosum, corona radiata and ovarian stroma in the follicular ovarian tissue, while VEGF protein was mainly located in the granulosa and theca cell layers. In summary, our findings demonstrate that 5% oxygen concentration may promote maturation and inhibit apoptosis of oocytes through HIF-1α-mediated VEGF expression.

GPR50 participates in and promotes yak oocyte maturation: A new potential oocyte regulatory molecule

Numerous molecular regulatory mechanisms are involved in the formation of mammalian oocytes, but many of their key proteins and molecules remain unknown. GPR50, the lone G protein-coupled receptor located in the X chromosome, is one of the superfamily members of the G protein-coupled receptor. GPR50 has been recently proved to play an important role in various physiological activities. However, the role of GPR50 in reproduction is currently unclear. In our previous research, we proved that the GPR50 receptor is present in yak oocytes. In the present study, the expression level and subcellular localization of GPR50 in the in vitro maturation process of yak oocytes were investigated to explore further its role in the maturation of yak oocytes. In the germinal vesicle (GV) stage, GPR50 was expressed and positioned in the cell membrane. By comparison, in the metaphase II (MII) stage, GPR50 had the highest expression level and was highly diffused in the cytoplasm. On the basis of these observations, the knockdown and overexpression of GPR50 yak oocyte models were constructed. Results showed that the expression level of GPR50 knockdown significantly reduced the excretion rate and maturity level of the yak oocyte PB1 (P < 0.01). However, the expression level of GPR50 overexpression exerted no significant influence on the excretion rate and maturity level of the yak oocytes (P > 0.05). This study verified that GPR50 plays an important role in the in vitro maturation process of yak oocytes.

Ascorbic acid protects the toxic effects of aflatoxin B1 on yak oocyte maturation.

High-quality oocytes are a prerequisite for successful fertilization. Mammals feeding on aflatoxin-contaminated feed can cause reproductive toxicity, including follicular atresia, poor oocyte development and maturation, and aberrant epigenetic modifications of oocytes. In addition, the important role of ascorbic acid (AA) in reproductive biology has been confirmed, and AA is widely used as an antioxidant in cell culture. However, the toxic effects of aflatoxin B1 (AFB1 ) on yak oocytes and whether AA has protective effects remain unknown. In this study, we found that exposure to AFB1 impedes meiotic maturation of oocytes, promotes apoptosis by triggering high levels of reactive oxygen species (ROS), and disrupts mitochondrial distribution and actin integrity, resulting in a decrease in the fertilization ability and parthenogenetic development ability of oocytes. In addition, these injuries changed the DNA methylation transferase transcription level of mature oocytes. After adding 50 μg/ml AA, the indices recovered to levels close to those of the control group. The results showed that AA could protect yak oocytes from the toxic effects of AFB1 and improve the quality of oocytes.

Estrogen improves the development of yak (Bos grunniens) oocytes by targeting cumulus expansion and levels of oocyte-secreted factors during in vitro maturation.

The estrogen-signalling pathway is critical for normal follicular development; however, little is known about its importance during in vitro maturation (IVM) in large animals, particularly yaks (Bos grunniens). Through the present study, we aimed to determine the mechanisms underlying estrogen involvement in cumulus expansion and the subsequent development of cumulus-oocyte complexes (COCs). COCs were cultured in the maturation medium supplemented with different concentrations (10−6–10−3 mM) of 17β-estradiol (E2) or its receptor antagonist, fulvestrant, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine the expression of cumulus-expansion related factors and oocyte-secreted factors (OSFs). The cumulus expansion of COCs was observed using an inverted microscope, and COCs developmental ability were judged by the evaluation of cleavage and blastulation rates per inseminated oocytes by IVF, and the number of cells in the blastocyst. Cumulus expansion increased with 10−6–10−3 mM E2, but decreased with fulvestrant. HAS2, PTGS2, PTX3 and OSFs expression increased in the 10−6–10−3 mM E2 groups. Significantly higher cleavage and blastocyst rates were observed in the 10−4 mM E2 group than in the fulvestrant and 0 mM E2 groups. Moreover, in the 10−4 mM group, blastocysts at 7 days had higher cell counts than the other groups. In conclusion, the increase in cumulus expansion and subsequent oocyte development after the addition of E2 to IVM medium may have resulted from increased cumulus-expansion-related factor expression and OSF levels.

Effects of oxygen concentrations on developmental competence and transcriptomic profile of yak oocytes.

Oxygen concentration influences oocyte quality and subsequent embryo development, but it remains unclear whether oxygen concentrations affect the developmental competence and transcriptomic profile of yak oocytes. In this study, we investigated the effects of different oxygen concentrations (5% versus 20%) on the developmental competence, reactive oxygen species (ROS) levels, glutathione (GSH) content, and transcriptomic profile of yak oocytes. The results showed that a low oxygen concentration significantly increased the maturation rate of yak oocytes (81.2 ± 2.2% vs 75.9 ± 1.3%) and the blastocyst quality of yak in vitro fertilized embryos. Analysis of ROS and GSH showed that a low oxygen concentration reduced ROS levels and increased the content of GSH (75.05 ± 7.1 ng/oocyte vs 50.63 ± 5.6 ng/oocyte). Furthermore, transcriptomic analysis identified 120 differentially expressed genes (DEGs) between the two groups of oocytes. Gene enrichment analysis of the DEGs indicated multiple cellular processes, including oxidative phosphorylation, transcription regulation, mitochondrial regulation, oestrogen signalling pathway, HIF-1 signalling pathway, TNF signalling pathway, were involved in the response to oxygen concentration alterations. Taken together, these results indicated that a low oxygen concentration improved the developmental competence of yak oocytes.

Low oxygen concentrations improve yak oocyte maturation and enhance the developmental competence of preimplantation embryos

Mammalian oocyte maturation and early embryo development are highly sensitive to the in vitro culture environment, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effects of different oxygen concentrations (20%, 10%, 5% or 1% O2) on yak oocyte maturation, in vitro fertilization (IVF), and embryo development competence, as well as its effects on the oxidative response, metabolism, and apoptosis in cumulus-oocyte complexes (COCs) and the embryo. The results revealed that the maturation rate of oocytes, blastocysts rate and hatched blastocysts rate in the group with 5% oxygen concentration were significantly higher (P < 0.05) than other groups, but the cleavage rate with 5% oxygen concentration was significantly lower (P < 0.05) than the 20% and 10% oxygen concentrations. The maturation rate of oocytes, the cleavage rate, blastocysts rate and hatched blastocysts rate with the 1% oxygen concentration were the lowest. The blastocyst cultured with 5% oxygen concentration had significantly greater (P < 0.05) numbers of total cells, inner cell mass (ICM) cells and trophectoderm (TE) cells compared to the other groups. Analysis of the apoptosis index of oocytes and blastocyst cells by transferase dUTP nick end labeling (TUNEL) showed that the number of apoptotic cells significantly reduced (P < 0.05) with 5% oxygen concentration, but increased significantly (P < 0.05) in the 1% oxygen concentration group. Also, the qRT-PCR and western immunoblotting analysis confirmed that the transcription levels of the metabolism genes, antioxidant response genes, apoptosis genes, oocyte competence genes and embryonic developmental markers showed significant differences (P < 0.05) in the COCs or blastocysts matured in 5% oxygen concentration group compared to the other groups. In summary, our findings demonstrate that 5% oxygen concentration improves oocyte maturation and blastocyst development in the yak, increases blastocyst cell numbers, reduces apoptosis rate in the oocyte and blastocyst as well as reduces embryo cleavage rate.

Identification of differential abundances of mRNA transcript in cumulus cells and CCND1 associated with yak oocyte developmental competence

The development of an accurate and noninvasive preselection process for competent oocytes is essential to achieve a highly efficient in vitro production (IVP) of embryos. Cumulus cells (CCs) have important functions in oocyte growth, development, maturation, and fertilization. It, therefore, is important to know if the quality of oocytes can be ascertained by assessment of gene expression of the surrounding CCs or not. The aim of this study was to identify differentially expressed genes in yak CCs from oocytes with varying developmental competences as possible biomarkers for distinguishing oocyte competence. The isolated CCs were pooled into immature and mature groups in accordance with the maturation outcome of oocytes. A total of 9516 genes were differentially expressed in the two CC categories (P < 0.05). With a minimum change of 2.5-fold, 45 up-regulated and 79 down-regulated genes were observed in CCs belonging to the mature group compared with those in the immature group (P < 0.01). These genes were primarily enriched for the cell cycle, meiosis, cell signaling, metabolism, and apoptosis. The selected candidate genes (CCND1, BMP15, GDF9, H19, KLF4, GPC1, SYCP3, and CTSB) were validated using quantitative real-time polymerase chain reaction (RT-qPCR) and there were expression patterns similar to those detected with transcriptome analysis. The CCs from fertilized oocytes arrested at the 2-cell (2-cell group), or 8-cell (8-cell group) stages or that developed into blastocysts (the blastocyst group) had a 1.5-, 1.8-, and 2.3-fold increase, respectively, in mRNA relative abundance of CCND1 compared with CCs from unfertilized oocytes (P < 0.05). The results with the RT-qPCR analysis confirmed that the relative abundance of CCND1 mRNA in CCs was associated with oocyte developmental competence. In conclusion, RNA-Seq is useful in extracting transcriptomes and selecting markers associated with oocyte developmental competence. Furthermore, the expression of the CCND1 gene in yak CCs can be used to preselect oocytes for IVP efficiency.

FGF10 enhances yak oocyte fertilization competence and subsequent blastocyst quality and regulates the levels of CD9, CD81, DNMT1, and DNMT3B.

The fusion of sperm and oocytes determines the fertilization competence and subsequent development of embryos, which, in turn, can be affected by various proteins and DNA methylation. However, several factors in this whole regulation process remain unknown, especially in yaks. Here, we report that fibroblast growth factor 10 (FGF10) is an important growth factor that can enhance the maturation rate of yak oocytes and the motility of frozen spermatozoa. Subsequent blastocyst quality was also improved by increasing the total cell number and level of pregnancy-associated protein in blastocysts. These effects were significantly high in the group that received the 5 ng/ml FGF10 treatment, during both in vitro maturation (IVM) and capacitation. Our data show that the effects of FGF10 were dose-dependent at vital steps of embryogenesis in vitro. Furthermore, quantitative polymerase chain reaction, western blot analysis, and immunofluorescence demonstrated that the levels of CD9, CD81, DNMT1, and DNMT3B in both mature cumulus-oocyte complexes and capacitated sperms were regulated by FGF10, which was also highly expressed in the group treated with 5 ng/ml FGF10 during both IVM and capacitation. From our present study, we concluded that FGF10 promotes yak oocyte fertilization competence and subsequent blastocyst quality, and could also regulate CD9, CD81, DNMT1, and DNMT3B to optimize sperm-oocyte interactions and DNA methylation during fertilization.

Transcriptional profiles of crossbred embryos derived from yak oocytes in vitro fertilized with cattle sperm

During mammalian pre-implantation embryonic development, dramatic and orchestrated changes occur in gene transcription. Pregnancy rates were low when yak females were crossbred with cattle breeds, but few studies exist to describe the unique molecular network regulation behind the pre-implantation development of these embryos. We determined the transcriptomes of crossbred embryos derived from yak oocytes in vitro fertilized with Jersey sperm using Illumina RNA-seq for the first time in this study. Embryos were sampled at the 2-, 4-, and 8-cell, morula and blastocyst stages. The results showed that in total, 291.9 million short reads were generated from the five libraries of 2-, 4-, and 8-cell, morula and blastocyst stages, with 276.2 million high-quality reads selected for further analysis. Eighty to 91% of the clean reads were aligned against the yak reference genome. A total of 19,072 transcripts were identified in five libraries, of which 7,785 transcripts were co-expressed in each stage and 2,013 transcripts were stage-specific. When a |log2 ratio| ≥1 and q-value ≤ 0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 3,690 to 10,298 DEGs between any two consecutive stages. Based on the results of GO and KEGG enrichment, some of these DEGs potentially play an important role in regulating pre-implantation development, but they are most likely stage-specific. There were 2,960, 7,287, 6,420, 7,724 and 10,417 DEGs in 2-, 4-, 8-cell, morula and blastocyst stages between the crossbred embryos and purebred embryos of the yak, respectively, leading to a large difference in GO terms and pathways. In conclusion, we sequenced transcriptomes of in vitro-produced crossbred embryos of yak and cattle during pre-implantation and provided comprehensive examinations of gene activities. These will be helpful for development of assisted reproductive technology and better understanding the early maternal-fetal or maternal-embryonic dialog in inter-species crossbreeding.

Effects of Zinc Supplementation During In Vitro Maturation on Meiotic Maturation of Oocytes and Developmental Capacity in Yak.

Zinc (Zn) is an essential trace element that is required during mammalian developmental processes. The objective of this study was to investigate the effects of Zn supplementation during in vitro maturation (IVM) on the developmental capacity of yak (Bos grunniens) oocytes. Cumulus expansion, nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, subsequent embryonic development, and the expression of Zn transporters (ZnTs) and Zrt and Irt-like proteins (ZiPs) were evaluated. The Zn concentrations in yak plasma and follicular fluid were 0.740 ± 0.012 and 0.382 ± 0.009μg/mL, respectively. The cumulus expansion did not show significant differences in COCs after matured with or without Zn supplementation (P > 0.05). The intracellular GSH was higher in oocytes matured with 1 or 2mg/L Zn than in control group (0mg/L) (P < 0.05). However, ROS levels of oocytes matured with 1 or 2mg/L Zn were reduced significantly compared with the control and 0.5mg/L groups (P < 0.05). The SOD activity was increased significantly after Zn supplementation. The cleavage rate was not significantly different after Zn supplementation (P > 0.05). Percentages of matured oocytes that developed into the blastocyst stage after IVF were 47.9, 50.5, 60.4, and 58.9% for 0, 0.5, 1, and 2mg/L Zn groups, respectively. Gene expression analysis revealed that the expression patterns associated with Zn were changed after Zn supplementation. In conclusion, Zn supplementation to IVM improved yak oocyte maturation and subsequent development by increasing GSH and SOD activity, decreasing ROS in oocytes.

Selenium supplementation during invitro maturation enhances meiosis and developmental capacity of yak oocytes.

The objectives of the present study were to investigate the effects of selenium (Se) supplementation during invitro maturation (IVM) on the developmental capacity of yak (Bos grunniens) oocytes. Nuclear maturation, DNA integrity, glutathione peroxidase (GSH-Px) activity, subsequent embryonic development, and gene expression after invitro fertilization (IVF) were evaluated. The Se concentrations in yak plasma and follicular fluid were 0.142 and 0.069μg/mL, respectively. The DNA damage in cumulus cells decreased significantly with 2 and 4μg/mL supplementation of sodium selenite to IVM medium (P<0.05). Total GSH-Px activity in oocytes increased in all Se supplementation groups, and the 2 and 4μg/mL groups were significantly higher than the control group (0μg/mL). However, the cleavage rate was not significantly different after Se supplementation (P>0.05). The IVF blastocyst formation rates of 0, 1 and 4μg/mL sodium selenite groups were 47.7%, 51.2% and 58.9%, respectively. The 2μg/mL sodium selenite group had the highest blastocyst formation rate (60.5%). Gene expression analysis revealed that the quantity of transcripts associated with selenoprotein and protein synthesis were high in the 2 and 4μg/mL groups. In conclusion, both GSH-Px activity of oocytes and DNA integrity of cumulus cells significantly increased with supplemental Se during oocyte IVM. Considering that embryonic development is responsive to Se supplementation, we inferred that appropriate Se concentrations during IVM were beneficial for yak oocyte maturation and subsequent development.

Cytoskeleton Genes Expression and Survival Rate Comparison Between Immature and Mature Yak Oocyte After OPS Vitrification

ABSTRACTThis study was conducted to investigate the effect of vitrification on survival rate and cytoskeleton gene expression during yak oocyte maturation. The yak oocytes were incubated for 0 h [germinal vesicle (GV) stage] and in vitro matured for 24 h [metaphase II (MII) stage] to obtain immature and mature oocytes. Survival rate after vitrification were compared between immature and mature yak oocytes and cytoskeleton-related genes [cytokeratin 8 (CK8), β-actin (ACTB), and gap junction protein, alpha 1 (GJA1)] were tested by real-time PCR. Our results showed that MII stage survival rate after open pulled straw vitrification (35.60%) is significantly higher than GV stage (25.90%) oocytes. Furthermore, expression of CK8, ACTB, and GJA1 in MII stage oocytes are also significantly higher than GV stage oocytes. In conclusion, our study demonstrated that higher expression of GJA1, CK8, and ACTB in vitrify-warmed MII stage oocytes when compared with GV stage oocytes and such discrepancy might result in higher survival rate in vitrify-warmed MII stage oocytes.