C-Type Natriuretic Peptide Pre-Treatment Improves Maturation Rate of Goat Oocytes by Maintaining Transzonal Projections, Spindle Morphology, and Mitochondrial Function.

C-type natriuretic peptide (CNP) is a peptide molecule naturally found in follicles and can be used to extend meiotic resumption and enhance the potential for oocytes to develop. However, the mechanism by which CNP improves goat oocyte quality remains unclear. In this study, cumulus-oocyte complexes (COCs) from goats were pre-treated with CNP prior to IVM, and the results showed that pre-treatment with CNP enhanced goat oocyte maturation. First, we discovered that CNP maintained communication between cumulus cells and oocytes by regulating the transzonal projections (TZPs). We then found that CNP treatment reduced abnormal spindle formation and increased the expression of genes associated with spindle assembly and the spindle assembly checkpoint. Moreover, further analysis showed that oocytes exhibited better antioxidant ability in the CNP treatment group, which mainly manifested in higher glutathione (GSH) and lower reactive oxygen species (ROS) concentrations. Enhanced mitochondrial activity was signified via the augmented expression of mitochondrial oxidative metabolism and fusion and fission-related genes, thus diminishing the apoptosis of the oocytes. Overall, these results provide novel insights into the potential mechanism by which CNP treatment before IVM can improve oocyte quality.

The formins inhibitor SMIFH2 inhibits the cytoskeleton dynamics and mitochondrial function during goat oocyte maturation.

The cytoskeleton plays a crucial role in facilitating the successful completion of the meiotic maturation of oocytes. Its influence extends to the process of oocyte nuclear maturation and the proper functioning of various organelles during cytoplasmic maturation. The formin family of proteins plays a crucial role in the molecular regulation of cytoskeletal assembly and organization; however, its role in goat oocytes are not fully understood. Our study examined the inhibition of formins activity, which revealed its crucial role in the maturation of goat oocytes. We observed that the inhibition of formins resulted in meiotic defects in goat oocytes, as evidenced by the hindered extrusion of polar bodies and the expansion of cumulus cells. Additionally, the oocytes exhibited altered actin dynamics and compromised spindle/chromosome structure upon formins inhibition. The results of the transcriptomic analysis highlighted a noteworthy alteration in the mRNA levels of genes implicated in mitochondrial functions and oxidative phosphorylation in formins inhibited oocytes. Validation experiments provided evidence that the meiotic defects observed in these oocytes were due to the excessive early apoptosis induced by reactive oxygen species (ROS). Our findings demonstrate that the involvement of formins in sustaining the cytoskeletal dynamics and mitochondrial function is crucial for the successful meiotic maturation of goat oocytes.

The Impact of Arp2/3 Complex Inhibition on Cytoskeleton Dynamics and Mitochondrial Function during Goat Oocyte Meiosis.

F-actin is of critical importance in oocyte meiotic maturation. Actin assembly and its dynamics are mainly regulated by actin nucleation factors. The actin-related protein complex 2/3 (Arp2/3) is responsible for the organization of F-actin filaments. However, the role of Arp2/3 complex in goat oocytes has not been fully elucidated. Our findings demonstrate that Arp2/3 complex activity is necessary for the maturation of goat oocytes. The Arp2/3 complex-specific inhibitor CK666 impairs the maturation of goat oocytes and alters the genes associated with cumulus expansion, both of which suggest that normal meiosis is affected. Arp2, one of the subunits of the Arp2/3 complex, was found to be mainly accumulated at the oocyte cortex and to co-localize with F-actin during goat oocyte maturation in our results. Thus, we further investigated the cytoskeleton dynamics and found that Arp2/3 complex inhibition disrupts the F-actin assembly and spindle organization. Further analysis revealed that, in addition to direct effects on the cytoskeleton, Arp2/3 complex could also induce ROS accumulation and oxidative stress by disrupting mitochondrial distribution and function, ultimately increasing the rate of early apoptosis in goat oocytes. Our study provides evidence that the Arp2/3 complex is a key regulator of goat oocyte maturation through its regulation of the cytoskeleton dynamics and mitochondrial function.

The presence of synthetic polymers in the maturation medium affects the cryotolerance and developmental capacity after parthenogenic activation of vitrified goat oocytes.

The purpose of this present study is to assess if addition of the synthetic polymers in maturation medium can influence cryotolerance and subsequently embryonic development of mammalian oocytes. We examined the roles of two polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on in vitro maturation (IVM), embryonic developmental capacity, and cryotolerance of goat oocytes. The present study includes two parts. At first, goat cumulus-oocyte complexes (COCs) were matured in a medium supplemented with 10% fetal bovine serum (FBS), 3 mg/ml PVP, or 1 mg/ml PVA, respectively. Data of oocyte with first polar body, cleavage, and blastocyst following parthenogenetic activation (PA) were recorded. Secondly, after maturation in the above medium, oocytes were vitrified using the Cryotop technique and then the morphology, cleavage and blastocyst formation of vitrified oocytes have been checked. The results demonstrated that the adding of PVP or PVA in maturation medium can't affect IVM of goat oocytes in comparison with FBS, as concern cumulus cell expansion, first polar body formation, and embryonic development. Additionally, without plunging into liquid nitrogen, only exposure to the vitrification and warming solutions cannot also influence the quality of oocytes, in terms of morphology, cleavage, and blastocyst formation. However, after IVM with synthetic polymers and vitrification, the ratio of oocytes with standard morphology in PVP or PVA group was only 59.47% ± 3.56% or 54.86% ± 5.19%, respectively, and was significantly less than that in the FBS group (89.37% ± 4.52%, P < 0.05). Furthermore, the cleavage ratio of oocytes in PVP or PVA group was 37.41% ± 4.17% or 27.71% ± 3.91% and was considerably less than that in the FBS group (64.97% ± 4.69%, P < 0.05). In addition, the cleavage ratio in PVP group was statistically higher than that in PVA group (P < 0.05). In terms of blastocyst development, a significant difference was observed between the synthetic polymer group and the FBS group (24.96% ± 3.62%, P < 0.05). However, the blastocyst ratio in the PVA group (7.51% ± 1.68%) was statistically less than the PVP groups (13.20% ± 4.59%, P < 0.05) and the FBS group (P < 0.05). In conclusion, two potential serum replacements, either PVP or PVA, can support IVM and embryonic development of goat oocytes at the concentration used in this study. But IVM with synthetic polymers supplemented to maturation medium may reduce the cryotolerance of oocytes. Additionally, the supportive function of PVP on embryonic development of vitrified oocytes might be better than that of PVA.

Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 µM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 µM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 µM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 µM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 µM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 µM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

Ooplast transfer of triploid pronucleus zygote improve reconstructed human-goat embryonic development.

Poor development of the interspecies somatic cell nuclear transfer (iSCNT) embryos was due to nuclear-mitochondrial incompatibility. In humans, it has been known that ooplast transfer (OT) could support normal fertilization, the development of embryos and prevents the transmission of mtDNA disease. To investigate whether OT could support development of the iSCNT embryos, the ooplast of Triploid Pronucleus (3PN) zygote which would be discarded in IVF lab was transferred into the enucleated goat oocytes to construct humanized iSCNT embryos in our study. The results showed the 3PN-OT could significantly improve the early development of humanized iSCNT embryos. The percentage of blastocyst development of OT group was also higher than that of the control group. Interestingly, the morphology of some OT-iSCNT blastocysts was similar to normal human blastocysts in vitro fertilization, while the morphology of iSCNT blastocysts from control group was similar to goat blastocysts. Importantly, the pluripotent marker Oct4 of the OT-iSCNT blastocyst was expressed stronger than that of the control group. These results suggested that 3PN-OT could improve the developmental potency of human iSCNT embryos and would facilitate establishing ESCs from iSCNT blastocysts.

All-trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation.

All-trans retinoic acid (t-RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t-RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus-oocyte complexes (COCs) were matured in vitro in the absence or presence of t-RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real-time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t-RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t-RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t-RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t-RA inhibits the apoptosis of cumulus cells (P < 0.01). t-RA treatment up-regulated the expression of B-cell lymphoma 2 (BCL-2), catalase (CAT) (P < 0.05) and down-regulated the expression of Caspase-8 (P < 0.05). In conclusion, t-RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.