Testosterone Supplementation Promotes Estrogen Synthesis of Buffalo Cumulus Cells Surrounding In Vitro-Matured Oocytes.

Cumulus cells (CCs) synthesize estrogens that are essential for follicular development. However, the effects of androgen on estrogen production in buffalo CCs remain unknown. In the present study, the impacts of testosterone on estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes were investigated. The results showed that testosterone supplementation improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 17ß-HSD) and the secretion levels of estradiol in buffalo CCs surrounding in vitro-matured oocytes. Furthermore, testosterone treatment enhanced the sensitivity of buffalo CCs surrounding in vitro-matured oocytes to follicle-stimulating hormone (FSH). This study indicated that testosterone supplementation promoted the estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes mainly through strengthening the responsiveness of CCs to FSH. The present study serves as a foundation of acquiring high-quality recipient oocytes for buffalo somatic cell nuclear transfer.

Effects of MEK1/2 inhibitor U0126 on FGF10-enhanced buffalo oocyte maturation in vitro.

Fibroblast growth factor 10 (FGF10) plays critical roles in oocyte maturation and embryonic development; however, the specific pathway by which FGF10 promotes in vitro maturation of buffalo oocytes remains elusive. The present study was aimed at investigating the mechanism underlying effects of the FGF10-mediated extracellular regulated protein kinases (ERK) pathway on oocyte maturation and embryonic development in vitro. MEK1/2 (mitogen-activated protein kinase kinase) inhibitor U0126, alone or in combination with FGF10, was added to the maturation culture medium during maturation of the cumulus oocyte complex. Morphological observations, orcein staining, apoptosis detection, and quantitative real-time PCR were performed to evaluate oocyte maturation, embryonic development, and gene expression. U0126 affected oocyte maturation and embryonic development in vitro by substantially reducing the nuclear maturation of oocytes and expansion of the cumulus while increasing the apoptosis of cumulus cells. However, it did not have a considerable effect on glucose metabolism. These findings suggest that blocking the MEK/ERK pathway is detrimental to the maturation and embryonic development potential of buffalo oocytes. Overall, FGF10 may regulate the nuclear maturation of oocytes and cumulus cell expansion and apoptosis but not glucose metabolism through the MEK/ERK pathway. Our findings indicate that FGF10 regulates resumption of meiosis and expansion and survival of cumulus cells via MEK/ERK signaling during in vitro maturation of buffalo cumulus oocyte complexes. Elucidation of the mechanism of action of FGF10 and insights into oocyte maturation should advance buffalo breeding. Further studies should examine whether enhancement of MEK/ERK signaling improves embryonic development in buffalo.

Zinc improves the developmental ability of bovine in vitro fertilization embryos through its antioxidative action.

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 µg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 µg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 µg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation.

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.

Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Follistatin (FST), a member of the transforming growth factor-ß (TGF-ß) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-ß/SMAD pathway-related genes TGF-ß, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-ß/SMAD pathway-related genes.

Testosterone supplementation improves estrogen synthesis of buffalo (Bubalus bubalis) granulosa cells.

Granulosa cells (GCs) synthesize estrogens needed for follicular growth. However, the effects of androgen on estrogen production in buffalo GCs remain unclear. In this study, the impacts of testosterone on estrogen synthesis in buffalo GCs were examined. The results showed that testosterone that was added to cell medium at a concentration of 10-7 mol/L and applied to GCs for 48 or 72 h enhanced the estrogen synthesis of buffalo GCs. This study provides a theoretical basis for further exploration of ovarian endocrine mechanism for steroidogenesis.

Hypoxia enhances steroidogenic competence of buffalo (Bubalus bubalis) granulosa cells.

Granulosa cells (GCs) synthesize estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo GCs remain unclear. In this study, the impacts of hypoxic conditions (5% oxygen) on estrogen synthesis in buffalo GCs were examined. The results showed that hypoxia improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3ß-HSD) and the secretion levels of estradiol in buffalo GCs. Hypoxic conditions promoted the sensitivity of buffalo GCs to FSH. Furthermore, inhibition of cAMP/PKA signaling pathway (H89, a cAMP/PKA signaling pathway inhibitor) reduced both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3ß-HSD) and the secretion levels of estradiol in hypoxia-cultured buffalo GCs. Besides, inhibition of cAMP/PKA signaling pathway lowered the responsiveness of buffalo GCs to FSH under hypoxic conditions. The present study indicated that hypoxia enhanced the steroidogenic competence of buffalo GCs principal by affecting cAMP/PKA signaling pathway and subsequent sensitivity of GCs to FSH.

Beneficial Effects of Catalpol Supplementation during In Vitro Maturation of Porcine Cumulus-Oocyte Complexes.

Oxidative stress degrades oocytes during in vitro maturation (IVM). Catalpol, a well-known iridoid glycoside, exhibits antioxidant, anti-inflammatory, and antihyperglycemic effects. In this study, catalpol supplementation was tested on porcine oocyte IVM and its mechanisms. Corticalgranule (GC) distribution, mitochondrial function, antioxidant capacity, DNA damage degree, and real-time quantitative polymerase chain reaction were used to confirm the effects of 10 µmol/L catalpol in the maturation medium during IVM. Catalpol treatment significantly increased the first-pole rate and cytoplasmic maturation in mature oocytes. It also increased oocyte glutathione (GSH), mitochondrial membrane potential and blastocyst cell number. However, DNA damage as well as reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Mitochondrial membrane potential and blastocyst cell number were also increased. Thus, the supplementation of 10 µmol/L catalpol in the IVM medium improves porcine oocyte maturation and embryonic development.

Epicatechin promotes oocyte quality in mice during repeated superovulation.

The negative impacts of repeated superovulation on mitochondrial function and oocyte quality remain unresolved. Epicatechin (EC), a polyphenolic compound found in the human diet with strong antioxidant activity, was investigated for its effects and underlying mechanism on embryonic development after repeated superovulation. The results showed that as the number of superovulation cycles increased, the number of 2-cell embryos decreased, the development of embryos in subsequent in vitro culture was delayed, the apoptosis rate of blastocyst cells increased and the number of blastocyst cells decreased. However, intraperitoneal injection of EC (10 mg/kg body-weight) for two consecutive days during repeated superovulation increased mitochondrial DNA copies in 2-cell embryos of mice. It also promoted the expression of antioxidant enzyme genes in ovaries, increased the content of glutathione (GSH) content and improved the antioxidant capacity of ovaries. Altogether, these results revealed that intraperitoneal injection of EC could increase the embryonic mitochondrial DNA copy number (mtDNA-CN) and enhance the ovary's antioxidant capacity and GSH content, ultimately promoting the quality of mouse embryos in the process of repeated superovulation.

Regulation of hypoxia-inducible factor 1α by optimal oxygen concentration enhances oocyte maturation and early embryonic development in buffalo.

Despite significant progress in vitro maturation (IVM) and in vitro culture (IVC) of oocytes and embryos, their developmental competence remains low. To address this issue, we used buffalo oocytes as a model system to investigate the effects and mechanisms of oxygen concentration on IVM and IVC. Our findings demonstrated that culturing buffalo oocytes with 5% oxygen significantly enhanced the efficiency of IVM and developmental competence of early embryos. Immunofluorescence results suggested that HIF1α played a critical role in these progresses. RT-qPCR results showed that maintaining a stable expression of HIF1α in cumulus cells with 5% oxygen concentration enhanced glycolysis, expansion, and proliferation abilities, up-regulated the expression of development-related genes, and suppressed apoptosis level. Consequently, it improved the maturation efficiency and quality of oocytes, leading to improve developmental capacity of buffalo early embryos. Similar outcomes were also observed when embryos were cultured with 5% oxygen. Collectively, our study provided insights into the role of oxygen regulation during oocytes maturation and early embryo development, and could potentially improve the efficiency of human assisted-reproduction technology.

A retrospective study on epidemiological analysis of pre-hospital emergency care in Hangzhou, China.

Out-of-hospital cardiac arrest (OHCA) is a leading cause of global mortality, with numerous factors influencing the patient survival rate and prognosis. This study aimed to evaluate the OHCA epidemiology in China and elaborate on the current Hangzhou emergency system status. This retrospective analysis was based on the medical history system of the Hangzhou Emergency Center registered from 2015-2021. We provided a detailed description of OHCA characteristics and investigated the factors affecting the success rate of emergency treatment in terms of epidemiology, causes of onset, bystander rescue, and outcome factors. We included 9585 out-of-hospital cardiac arrest cases, of which 5442 (56.8%) had evidence of resuscitation. Patients with underlying diseases constituted the vast majority (80.1%); trauma and physicochemical factors accounted for 16.5% and 3.4%, respectively. Only 30.4% of patients (about 80.0% of bystanders witnessed) received bystander first aid. The outcome rate of emergency doctors dispatched by emergency centres was significantly higher than doctors dispatched by hospitals. Additionally, physician's first-aid experience, emergency response time, emergency telephone availability, initial heart rhythm, out-of-hospital defibrillation, out-of-hospital intubation, and using of epinephrine significantly can significantly improve the out-of-hospital return of spontaneous circulation in patients. All steps in pre-hospital care are important for patients, especially for bystander first aid and physician's first-aid experience. The popularity of first-aid training and the public emergency medical system are not potent enough. We should take those key factors into consideration when developing a pre-hospital care system for OHCA.

Beneficial effects of fibroblast growth factor 10 supplementation during in vitro maturation of buffalo cumulus-oocyte complexes.

Fibroblast growth factor 10 (FGF10) is an important regulator of the mammalian cumulus-oocyte complex that plays a crucial role in oocyte maturation. In this study, we investigated the effects of FGF10 supplementation on the in vitro maturation (IVM) of buffalo oocytes and its related mechanisms. During IVM, the maturation medium was supplemented with a range of concentrations of FGF10 (0, 0.5, 5, and 50 ng/mL) and the resulting effects were corroborated using aceto-orcein staining, TUNEL apoptosis assay, detection of Cdc2/Cdk1 kinase in oocytes, and real-time quantitative PCR. In matured oocytes, the 5 ng/mL-FGF10 treatment resulted in a significantly increased nuclear maturation rate, which increased the activity of maturation-promoting factor (MPF) and enhanced buffalo oocyte maturation. Furthermore, it treatment significantly inhibited the apoptosis of cumulus cells, while simultaneously promoting its proliferation and expansion. This treatment also increased the absorption of glucose in cumulus cells. Thus, our results indicate that adding an appropriate concentration of FGF10 to a maturation medium during IVM can be beneficial to the maturation of buffalo oocytes and improve the potential of embryo development.

METTL3-mediated m6A methylation regulates granulosa cells autophagy during follicular atresia in pig ovaries.

Follicular atresia is a normal physiological event in mammals, yet its mechanism remains to be studied. Granulosa cell (GC) autophagy is closely associated with follicular atresia. The N6-methyladenosine (m6A) modification is the most common post-transcriptional modification in eukaryotes, but its role in follicular atresia is still unknown. In this study, the possible relationship amongst follicular atresia, GC autophagy and m6A modification was studied. Our results showed that the level of autophagy in GCs increased with the degree of follicle atresia, whereas the overall m6A level decreased. Rapamycin treatment induced atresia in vitro cultured follicles, whereas 3-Methyladenine inhibited follicular atresia. Progressed atretic follicle (PAF) GCs had significantly lower METTL3 levels and significantly higher FTO levels than healthy follicle (HF) GCs. Differential gene expression analysis of GCs in PAF and HF by RNA sequencing was showed that the autophagy-related genes ULK1, ULK2, ATG2A, and ATG2B were significantly elevated in the PAF. In cultured GCs, overexpression of METTL3 significantly decreased the mRNA level of ULK1, as well as the autophagy level, whereas knockdown of METTL3 by RNAi significantly increased the mRNA level of ULK1, as well as the autophagy level. Our results indicate that m6A modification can regulate autophagy in GCs and play a role in the process of porcine follicular atresia.

Analysis of antitumor immunity in mouse glioma microenvironment through intercellular communication.

Objective: This study aimed to map the antitumor immunity in the glioma microenvironment by analyzing intercellular communication. Materials and Methods: The single-cell RNA-sequencing (scRNA-Seq) data were obtained from fresh mouse gliomas. Tumor cells were inferred by estimating genomic copy number profiles. CellMarker database was used to identify cell types. Intercellular communication was inferred using CellChat. Flow cytometry was used to detect the effect of microglia or stroma-educated monocytes on CD4+ T cell proliferation. Results: Mouse glioma contained at least eight cell populations, and T cells were the only infiltrating immunocytes. Whether in signal outgoing or signal incoming, intercellular communication could be divided into four patterns by which cell populations in the tumor microenvironment (TME) cooperate with each other. By analyzing the complex communication between brain cell populations and infiltrating T cells in TME, we found that the brain cell populations used 25 signaling pathways to connect to T cells, and T cells used 21 signaling pathways to connect to brain cell populations. We also found that microglia from normal mice and brain stroma-educated monocytes exhibited immunosuppressive activity against CD4+ T cell proliferation. Conclusions: We described the previously underestimated complex communication between infiltrating T cells and brain cell populations. Our data suggest that the tolerogenic property of glioma TME is related to the immune privilege of CNS.

Blastocyst Stage Affects the Isolation and Culture of Buffalo Naive/Primed Embryonic Stem-Like Cells.

Since embryonic stem cells (ESCs) were first identified, significant progress has been achieved. However, the establishment of buffalo ESCs (bESCs) is still unclear. This study was undertaken to explore the effect of the blastocyst stage on the isolation of bESCs. Firstly, our results indicated that the pluripotent genes were mainly expressed at the early stages of blastocyst, and the attachment and colony formation rates of bESCs derived from expanded blastocyst and hatched blastocyst were significantly higher than early blastocyst and blastocyst. In the meantime, bESCs showed positive alkaline phosphatase activity and expressed genes like OCT4, NANOG, SOX2, c-MYC, CDH1, KLF4, and TBX3. Immunofluorescence also confirmed the expression of OCT4, SOX2. Embryoid bodies expressing three marker genes were generated from the differentiation experiment, and fibroblast, epithelial, and neuron-like cells were induced. Moreover, naive-related genes KLF4, TBX3, primed-related genes FGF5, ACTA2 were expressed in the cells, but not REX-1. Immunofluorescence and western blot confirmed the FGF5 expression. Furthermore, bESCs could maintain pluripotency with the signal of LIF and bFGF. In summary, our results indicated that expanded blastocyst and hatched blastocyst are more suitable for bESCs isolation.

RNAi-mediated knockdown of Xist improves development of the female buffalo (Bubalus bubalis) nuclear transfer embryos.

Xist plays a critical role in the X-chromosome inactivation (XCI), an important epigenetic reprogramming of somatic cell nuclear transfer (SCNT) embryos. Modulation of Xist expression enhanced the developmental ability of mouse cloned embryos. However, the roles of Xist in buffalo SCNT embryos remain unknown. In this study, we investigated the methylation and expression status of Xist in different genders of buffalo donor cells and various stages (two-cell, eight-cell, morula and blastocyst) of in vitro fertilization (IVF) and SCNT embryos. The methylation of Xist in SCNT-♀ and SCNT-♂ embryos was aberrant hypomethylation compared with the buffalo foetal fibroblast (♀-BFF and ♂-BFF), IVF-♀ and IVF-♂ embryos. At the eight-cell stage, Xist expression was significantly higher in SCNT-♀ embryos compared with those in SCNT-♂, IVF-♀ and IVF-♂ embryos (P < 0.05). Meanwhile, no significant difference was found between IVF-♀ and IVF-♂ embryos (P > 0.05). Accordingly, we suppressed Xist expression by RNAi-Xist in SCNT-♀ embryos. Results showed that injection of Xist-shRNA significantly improved the morula and blastocyst rates (P < 0.05). These results indicated that correcting the abnormal expression of the Xist gene contributed to the development of SCNT-♀ embryos.

Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism.

Mammalian individuals differ in their somatic cell cloning efficiency, but the mechanisms leading to this variation is poorly understood. Here we found that high cloning efficiency buffalo fetal fibroblasts (BFFs) displayed robust energy metabolism, looser chromatin structure, high H3K9 acetylation and low heterochromatin protein 1α (HP1α) expression. High cloning efficiency BFFs had more H3K9ac regions near to the upstream of glycolysis genes by ChIP-seq, and involved more openness loci related to glycolysis genes through ATAC-seq. The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by qRT-PCR. Two key enzymes of glycolysis, PDKs and LDH, were confirmed to be associated with histone acetylation and chromatin openness of BFFs. Treatment of low cloning efficiency BFFs with PS48 (activator of PDK1) resulted in an increase in the intracellular lactate production and H3K9 acetylation, decrease in histone deacetylase activity and HP1α expression, less condensed chromatin structure and more cloning embryos developing to blastocysts. These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure, and can be improved by increasing glycolytic metabolism.

Hypoxia promotes steroidogenic competence of buffalo (Bubalus bubalis) theca cells.

Theca cells (TCs) play an important role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells aromatization to estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo TCs remain unclear. In the present study, the impacts of hypoxic conditions (5% oxygen) on androgen synthesis in buffalo TCs were examined. The results showed that hypoxia improved both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, and 3ß-HSD) and the secretion levels of testosterone in buffalo TCs. Hypoxic conditions promoted the sensitivity of buffalo TCs to LH. Furthermore, inhibition of PI3K/AKT signaling pathway reduced both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, and 3ß-HSD) and the secretion levels of testosterone in hypoxia-cultured buffalo TCs. Besides, inhibition of PI3K/AKT signaling pathway lowered the sensitivity of buffalo TCs to LH under hypoxic conditions. This study indicated that hypoxia enhanced the steroidogenic competence of buffalo TCs main through activating PI3K/AKT signaling pathway and subsequently facilitating the responsiveness of TCs to LH. This study provides a basis for further exploration of ovarian endocrine mechanism for steroidogenesis.

Granulosa cells affect in vitro maturation and subsequent parthenogenetic development of buffalo (Bubalus bubalis) oocytes.

Granulosa cells (GCs) play a crucial role in follicular development and atresia. Previous studies have showed that GCs in the form of monolayer influenced in vitro maturation (IVM) of oocytes. However, the effects of GCs in the form of conditioned medium and monolayer on IVM and development competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of GC-conditioned medium (GCCM) and monolayer GC on maturation efficiency and embryo development of buffalo oocytes after parthenogenetic activation (PA). Our results showed that GCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days and 20%) exerted significant negative effects on IVM rate (41.6% vs. 44.5%), but significantly enhanced embryo development (oocyte cleavage, 81.3% vs. 69.3%; blastocyst formation, 36.3% vs. 29.3%) of buffalo oocytes after PA compared with the control group. Furthermore, monolayer GC significantly reduced both maturation efficiency (40.2% vs. 44.5%) and embryo development (oocyte cleavage, 60.6% vs. 69.3%; blastocyst formation, 20.6% vs. 29.3%) of buffalo oocytes after PA compared to the control group. Our study indicated that GCs in the form of GCCM (2 days and 20%) and monolayer GC had different effects on IVM and subsequent parthenogenetic development of buffalo oocytes.

Transforming Growth Factor-ß1 Enhances Mesenchymal Characteristics of Buffalo (Bubalus bubalis) Bone Marrow-Derived Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that BMSCs were prone to malignant transformation of mesenchymal-to-epithelial transition in vitro, which can cause many barriers to further application of BMSCs. The transforming growth factor ß (TGF-ß) signaling pathway has been widely studied as the most important signaling pathway involved in regulating mesenchymal features of BMSCs. However, the effects of the TGF-ß signaling pathway on mesenchymal characteristics of buffalo BMSCs (bBMSCs) remain unclear. In the present study, the impacts of the growth factor, TGF-ß1, on cell proliferation, apoptosis, migration, and karyotype of bBMSCs were tested. Besides, the effects of TGF-ß1 on pluripotency, mesenchymal markers, and epithelial-to-mesenchymal transition (EMT)-related gene expression of bBMSCs were also examined. Results showed that the suitable concentration and time of TGF-ß1 treatment (2 ng/mL and 24 hours) promoted cell proliferation and significantly reduced cell apoptosis (p < 0.05) in bBMSCs. The cell migration capacity and normal karyotype rate of bBMSCs were significantly (p < 0.05) improved under TGF-ß1 treatment. The expression levels of pluripotency-related genes (Sox2 and Nanog) and mesenchymal markers (N-cadherin and Fn1) were significantly (p < 0.05) up-regulated under TGF-ß1 treatment. Furthermore, TGF-ß1 activated the EMT process, thereby contributing to significantly enhancing the expression levels of EMT-related genes (Snail and Slug) (p < 0.05), which in turn improved maintenance of the mesenchymal nature in bBMSCs. Finally, bBMSCs underwent self-transformation more easily and efficiently and exhibited more characteristics of mesenchymal stem cells under TGF-ß1 treatment. This study provides theoretical guidance for elucidating the detailed mechanism of the TGF-ß signaling pathway in mesenchymal feature maintenance of bBMSCs and is of significance to establish a stable culture system of bBMSCs.