Melatonin regulates the antioxidant capacity of sheep granulosa cells through a novel uORF-Nrf2aa mediated Nrf2/KEAP1 pathway.

Ovarian dysfunction stands as a prevalent contributor to female infertility, with its etiology intertwined with genetic, autoimmune, and environmental factors. Within the ovarian follicles, granulosa cells (GCs) represent the predominant cell population. Alterations in GCs, notably oxidative stress (OS) and the consequential surge in reactive oxygen species (ROS), play pivotal roles in the orchestration of ovarian function. Nrf2aa, a newly identified upstream open reading frame (uORF), is situated within the 5' untranslated region (5'UTR) of sheep Nrf2 mRNA and is regulated by melatonin, a crucial intrafollicular antioxidant. In this study, we have noted that Nrf2aa has the capacity to encode a peptide and exerts a negative regulatory effect on the translation efficiency (TE) of the Nrf2 CDs region. Further in vitro experiments, we observed that interfering with Nrf2aa can enhance the cellular functionality of GCs under 3-np-induced oxidative stress, while overexpressing Nrf2aa has the opposite effect. Furthermore, overexpression of Nrf2aa counteracts the rescuing effect of melatonin on the cellular functions of GCs under oxidative stress conditions, including estrogen secretion, proliferation, apoptosis, and many more. Finally, we confirmed that Nrf2aa, by regulating the expression of key proteins in the Nrf2/KEAP1 signaling pathway, further modulates the antioxidant levels in GCs.

FTO demethylates regulates cell-cycle progression by controlling CCND1 expression in luteinizing goat granulosa cells.

In mammals, N6-methyladenosine (m6A) stands out as one of the most abundant internal mRNA modifications and plays a crucial role in follicular development. Nonetheless, the precise mechanism by which the demethylase FTO regulates the progression of the goat luteinizing granulosa cells (LGCs) cycle remains to be elucidated. In our study, we primarily assessed the protein and mRNA expression levels of genes using Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), cell proliferation via EdU, cell viability with CCK-8, and apoptosis and cell cycle progression through flow cytometry. Here, the results demonstrated that knockdown of FTO significantly enhanced apoptosis, impeded cell proliferation, and increased autophagy levels in goat LGCs. Furthermore, the silencing of FTO substantially reduced cyclin D1 (CCND1) expression through the recognition and degradation of YTHDF2, consequently prolonging the cell cycle progression. This study sheds light on the mechanism by which FTO demethylation governs cell cycle progression by controlling the expression of CCND1 in goat LGCs, underscoring the dynamic role of m6A modification in the regulation of cell cycle progression.

N6-methyladenosine methyltransferase METTL3 modulates the cell cycle of granulosa cells via CCND1 and AURKB in Haimen goats.

N6-methyladenosine (m6A) plays a crucial role in many bioprocesses across species, but its function in granulosa cells during oocyte maturation is not well understood in animals, especially domestic animals. We observed an increase in m6A methyltransferase-like 3 (METTL3) in granulosa cells during oocyte maturation in Haimen goats. Our results showed that knockdown of METTL3 disrupted the cell cycle in goat granulosa cells, leading to aggravated cell apoptosis and inhibition of cell proliferation and hormone secretion. Mechanistically, METTL3 may regulate the cell cycle in goat granulosa cells by mediating Aurora kinase B (AURKB) mRNA degradation in an m6A-YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) manner and participating in AURKB transcription via the Cyclin D1 (CCND1)-Retinoblastoma protein (RB)-E2F transcription factor 1 (E2F1) pathway. Overall, our study highlights the essential role of METTL3 in granulosa cells during oocyte maturation in Haimen goats. These findings provide a theoretical basis and technical means for understanding how RNA methylation participates in oocyte maturation through granulosa cells.

chi-miR-130b-3p regulates the ZEA-induced oxidative stress damage through the KEAP1/NRF2 signaling pathway by targeting SESN2 in goat GCs.

As a dominant mycotoxin, zearalenone (ZEA) has attracted extensive attention due to its estrogen-like effect and oxidative stress damage in cells. In order to find a way to relieve cell oxidative stress damage caused by ZEA, we treated goat granulosa cells (GCs) with ZEA and did a whole transcriptome sequencing. The results showed that the expression level of Sesterin2 (SESN2) was promoted extremely significantly in the ZEA group (p < .01). In addition, our research demonstrated that SESN2 could regulate oxidative stress level in GCs through Recombinant Kelch Like ECH Associated Protein 1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The overexpression of SESN2 could reduce the oxidative damage, whereas knockdown of SESN2 would aggravate the oxidative damage caused by ZEA. What's more, microRNA (miRNA) chi-miR-130b-3p can bind to SESN2 3'-untranslated region (3'UTR) to regulate the expression of SESN2. The mimics/inhibition of chi-miR-130b-3p would have an effect on oxidative damage triggered by ZEA in GCs as well. In summary, these results elucidate a new pathway by which chi-miR-130b-3p affects the KEAP1/NRF2 pathway in GCs by modulating SESN2 expression in response to ZEA-induced oxidative stress damage.

Melatonin protects Leydig cells from HT-2 toxin-induced ferroptosis and apoptosis via glucose-6-phosphate dehydrogenase/glutathione -dependent pathway.

HT-2 toxin is a mycotoxin commonly found in food and water that can have adverse effects on male reproductive systems, including testosterone secretion. Ferroptosis and apoptosis are two types of programmed cell death that have been implicated in the regulation of cellular functions. Melatonin, a powerful antioxidant with various physiological functions, has been shown to regulate testosterone secretion. However, the mechanisms underlying the protective effects of melatonin against HT-2 toxin-induced damage in testosterone secretion are not fully understood. In this study, we investigated the effects of HT-2 toxin on sheep Leydig cells and the potential protective role of melatonin. We found that HT-2 toxin inhibited cell proliferation and testosterone secretion of Leydig cells in a dose-dependent manner and induced ferroptosis and apoptosis through intracellular reactive oxygen species accumulation, leading to lipid peroxidation. Exposure of Leydig cells to melatonin in vitro reversed the defective phenotypes caused by HT-2 toxin via a glucose-6-phosphate dehydrogenase/glutathione-dependent mechanism. Interference of glucose-6-phosphate dehydrogenase disrupted the beneficial effect of melatonin on ferroptosis and apoptosis in HT-2 toxin-treated Leydig cells. Furthermore, similar results were observed in vivo in the testes of male mice injected with HT-2 toxin with or without melatonin treatment for 30 days. Our findings suggest that melatonin inhibits ferroptosis and apoptosis by elevating the expression of glucose-6-phosphate dehydrogenase to eliminate reactive oxygen species accumulation in HT-2 toxin-treated Leydig cells. These results provide fundamental evidence for eliminating the adverse effects of HT-2 toxin on male reproduction.

The function of the m6A methyltransferase METTL3 in goat early embryo development under hypoxic and normoxic conditions.

It has been reported that N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) plays an important role in zygote genome activation during embryonic development, but the effects of METTL3 under oxidative stress in the early development of goat embryos remain largely unknown. In this study, zygotes were monitored at 72 and 168 h after fertilization, and they developed to the 8-cell stage and blastocyst stage under hypoxic conditions and normoxic conditions. Single-cell transcriptome sequencing was performed at the 8-cell stage and the blastocyst stage in the goat embryos, the differentially expressed METTL3 was screened from the sequencing results. We found that microinjection of small interfering RNA (siRNA) against METTL3 caused developmental arrest, both 8-cell rates (37.45 ± 2.21% vs. 47.09 ± 1.38%; P < 0.01) and blastocyst rates of Si-METTL3 (12.17% ± 2.84 vs. 20.83 ± 3.61%; P < 0.01) in Si-METTL3 group were significantly decreased compared with that of control under hypoxic conditions, significant changes were found in the m6A-related genes and the expression levels of critical transcription factors, such as, NANOG, GATA3, CDX2 and SOX17, were decreased. This study revealed the key role of METTL3 in the regulation of embryonic development under oxidative stress, and laid the foundation for further study of the crucial mechanism of oxidative stress during the early embryonic development of goats.

Facile preparation of iron oxide doped Fe-MOFs-MW as robust peroxydisulfate catalyst for emerging pollutants degradation.

A novel catalyst (Fe-MOFs-MW) was facilely synthesized under microwave-assisted with NaOH as modulator for activating peroxydisulfate (PDS). The accelerated nucleation process was confirmed by Johnson-Mehl-Avrami (JMA) model. There were abundant reactive sites on prepared Fe-MOFs-MW while maintaining high Space-Time-Yield value up to 2300 kg/m3·d. Degradation performance of Fe-MOFs-MW as PDS catalyst on sulfamethoxazole (SMX) removal was evaluated. Results indicated that Fe-MOFs-MW with more Fe element anchored (10%) exhibited excellent catalytic capacity for PDS. Besides, the fantastic stability and reusability were confirmed through recycle experiment. After recycled for 4 times, the removal efficiency of SMX and TOC was 88% and 31.3% compared to 98% and 38% without recycling, respectively. An accurate prediction model on the degradation effect with water matrices coexisted was established by response surface methodology (RSM) method. Moreover, SO4·-, O2·- and ·OH were confirmed as the main reactive species through chemical quenching and EPR tests. The mechanism of Fe-MOFs-MW/PDS process mainly based on electron circulation theory was proposed. As the robust PDS catalyst, facile prepared Fe-MOFs-MW was promising in the treatment of emerging pollutants.

Effect of PPARGC1A on the development and metabolism of early rabbit embryos in vitro.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a central regulator of mitochondrial biogenesis and metabolism, and its expression is closely related to embryo development. To gain insights into the possible mechanisms of PPARGC1A during early embryogenesis, the development potential, mitochondrial biogenesis, and the culture medium metabolomics of embryos were evaluated when PPARGC1A overexpressed or suppressed in rabbit zygotes. Results showed that different PPARGC1A levels in rabbit zygotes could affect blastocyst percentage, and the expressions of mitochondrial biogenesis and metabolic-related genes, as well as the glutathione and adenosine triphosphate levels during early embryo development. In addition, compared with the controls, 12 and 10 different metabolites involved in carbohydrate, amino acid, and fatty acid metabolism were screened in the 5 day's spent culture medium of PPARGC1A overexpressed and suppressed embryos by gas chromatography-mass spectrometer, respectively. Consistent with these metabolite changes, the transcriptions of genes encoding glucose transporters and fatty acid biosynthetic proteins in the embryos from different groups were regulated by PPARGC1A during rabbit embryo development. Taken together, these data provide evidence that PPARGC1A may regulate early rabbit embryo development through mitochondrial biogenesis and metabolism.

Induction of goat bone marrow mesenchymal stem cells into putative male germ cells using mRNA for STRA8, BOULE and DAZL.

Bone mesenchymal stem cells (BMSCs) have the capacity to differentiate into germ cells (GCs). This study was conducted to develop a non-integrated method of using RNA transfection to derive putative male GCs from goat BMSCs (gBMSCs) in vitro by overexpressing STRA8, BOULE and DAZL. The gBMSCs were induced by co-transfection these three mRNAs together (mi-SBD group) or sequential transfection according to their expression time order in vivo (mi-S + BD group). After transfection, a small population of gBMSCs transdifferentiated into early germ cell-like cells and had the potential to enter meiosis. These cells expressed primordial germ cell specific genes STELLA, C-KIT and MVH, as well as premeiotic genes DAZL, BOULE, STRA8, PIWIL2 and RNF17. Importantly, the expression level of meiotic marker synaptonemal complex protein 3 significantly increased in these transfected two groups compared with control cells by qRT-PCR, immunofluorescence and western blot analysis (P < 0.05). Moreover, the protein expression of MVH was significantly higher in mi-S + BD group than that in mi-SBD group (P < 0.05). In addition, compared with control group, the methylation rate of imprinted gene H19 decreased in these two transfected group (P < 0.05), and the rate was significantly lower in mi-S + BD group compared with mi-SBD group (P < 0.05). This study helps to understand the mechanisms of action of key genes in GCs differentiation and also provides a novel system for in vitro induction of male GCs from stem cells.

Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos.

Vitamin D receptor expression and potential role of vitamin D on cell proliferation and steroidogenesis in goat ovarian granulosa cells.

This study aimed to investigate the expression of the vitamin D receptor (VDR) in goat follicles and to determine the effects of Vit D3 supplementation on goat granulosa cells (GCs) function linked to follicular development. The results demonstrated that VDR was prominently localized in GCs, with expression increasing with follicle diameter. Addition of Vit D3 (1α,25-(OH)2VD3; 10 nM) to GCs caused an increase in VDR and in steroidogenic acute regulator (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression. Additionally, Vit D3 increased the cyclic adenosine monophosphate (cAMP), estradiol (E2), and progesterone (P4) levels, while it decreased anti-müllerian hormone receptor (AMHR) and follicle-stimulating hormone receptor (FSHR) mRNA expression (P < 0.05). Addition of FSH remarkably increased E2, P4, and cAMP levels (P < 0.05), and Vit D3 further enhanced the E2 and cAMP levels in the presence of FSH (P < 0.05). Vit D3 significantly induced the mRNA expression of CDK4 and CyclinD1, and downregulated P21 gene expression (P < 0.05). In addition, Vit D3 significantly decreased reactive oxygen species (ROS) production and increased the mRNA and protein expression of superoxide dismutase 2 (SOD2) and catalase (CAT) (P < 0.05). In conclusion, VDR is expressed in GCs of the goat ovaries and Vit D3 might play an important role in GCs proliferation by regulating cellular oxidative stress and cell cycle-related genes. Meanwhile, Vit D3 enhances the E2 and P4 output of GCs by regulating the expression of 3ß-HSD and StAR and the level of cAMP, which regulate steroidogenesis, supporting a potential role for Vit D3 in follicular development.

Effects of NRF1 on steroidogenesis and apoptosis in goat luteinized granulosa cells.

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

Effect of PGC-1α overexpression or silencing on mitochondrial apoptosis of goat luteinized granulosa cells.

During goat follicular development, abnormal expression of peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1α) in granulosa cells (GCs) may contribute to follicular atresia with unknown regulatory mechanisms. In this study, we investigate the effect of ectopic expression or interference of PGC-1α on cell apoptosis of goat first passage granulosa cells (FGCs) in vitro. The results indicate that PGC-1α silencing by short hairpin RNA (shRNA) in goat FGCs significantly reduced mitochondrial DNA (mtDNA) copy number (P < 0.05), changed mitochondria ultrastructure, and induced cell apoptosis (P < 0.05). The transcription and translation levels of the apoptosis-related genes BCL-2-associated X protein (BAX), caspase 3, and caspase 9 were significantly up-regulated (P < 0.05, respectively). Moreover, the ratio of BAX/B-cell lymphoma 2 (BCL-2) was reduced (P < 0.05), and the release of cytochrome c (cyt c) and lactate dehydrogenase (LDH) was significantly enhanced (P < 0.05, respectively) in PGC-1α interference goat FGCs. Furthermore, the expression of anti-oxidative related genes superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase (CAT) was down-regulated (P < 0.05, respectively) and the activity of glutathione/glutathione disulfide (GSH/GSSG) was inhibited (P < 0.05). While enforced expression of PGC-1α increased the levels of genes involved in the regulation of mitochondrial function and biogenesis, and enhanced the anti-oxidative and anti-apoptosis capacity. Taken together, our results reveal that lack of PGC-1α may lead to mitochondrial dysfunction and disrupt the cellular redox balance, thus resulting in goat GCs apoptosis through the mitochondria-dependent apoptotic pathway.

Using cysteine/cystine to overcome oxidative stress in goat oocytes and embryos cultured in vitro.

Assisted reproductive techniques expose gametes to excessive concentrations of reactive oxygen species. The present study aimed to evaluate the effects of oxidative stress on apoptosis in goat oocytes and embryonic development. The results demonstrated that the addition of 100 µM hydrogen peroxide (H2O2) into media produces an oxidative environment during oocyte maturation. The number of cumulus cells positive for terminal deoxynucleotidyl transferase UTP nick end labeling, and the activity of caspase 3 in mature oocytes were increased, compared with the control group (P<0.05). In addition, the expression levels of mitochondrial regulators, including peroxisome proliferator­activated receptor Î³ coactivator-1 α (PGC-1α) and nuclear respiratory factor­1 (NRF­1) were increased in the oxidative oocytes, compared with those in the control group (P<0.05). The ratio of the proapoptotic gene, B cell lymphoma (Bcl-2)-associated X protein (BAX), to the anti­apoptotic gene, BCL­2, was higher in the H2O2 group, compared with the control group (P<0.05). To overcome oxidative stress in oocytes and embryos cultured in vitro, 200 µM cysteine and 200 µM cystine were added to the media, thereby increasing the concentration of intracellular glutathione (GSH) and assisting in maintaining the redox state of the cells. In conclusion, cysteine and cystine reduced the oxygen tension caused by H2O2, thereby providing a novel strategy for optimizing in vitro embryonic development systems.

A novel fluorescence reporter system for the characterization of dairy goat mammary epithelial cells.

Goat mammary epithelial cells (GMECs) are a useful model to understand the physiological function of mammary glands and to assess the efficiency of mammary-specific vectors. The aim of this study was to develop an effective and convenient way to evaluate the secretory capacity of GMECs in primary culture. In this study, we developed a reporter system using fluorescent proteins driven by the CSN2 (Capra hircus beta-casein) gene promoter to detect the secretory capacity of GMECs. Additionally, we evaluated the efficiency of the reporter system by determining the expression of cytoskeletal proteins and beta-casein protein. The results suggest that this reporter system provides an easy, convenient and effective method to detect the function of milk synthesis in GMECs. Primary cultures of GMECs were homogeneous and retained the function of milk synthesis, prompting their usefulness as a model for further studies.

Isolation, characterization, and gene modification of dairy goat mesenchymal stem cells from bone marrow.

Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an attractive cell type for therapy models and for nuclear transfer transgenesis. To date, MSCs from various species have been studied, but only a limited amount of information regarding dairy goat MSCs (gMSCs) is available. The objectives of this study were to isolate, induce the multilineage mesenchymal differentiation, and investigate the gene modification efficiency of gMSCs, thereby initiating further research on these cells. The gMSCs isolated from bone marrow grew, attached to plastic with a typical fibroblast-like morphology, and expressed the mesenchymal surface marker CD44, CD29, CD90, and CD166, but not the hematopoietic marker CD45. Furthermore, the gMSCs expressed the transcription factors Oct-4 and Nanog, which have been shown to be critical for stem cell self-renewal and pluripotency. The multilineage differentiation potential of gMSCs was revealed by their ability to undergo adipogenic and osteogenic differentiation when exposed to specific inducing conditions. Transient transduction of gMSCs with a plasmid containing the GFP gene resulted in higher transfection rate compared with fetal fibroblasts (FFs). Furthermore, cell colonies with stable genetic modifications were obtained when gMSCs were transfected with a mammary-specific expression vector containing human lysosomal acid beta-glucosidase gene (hGCase). In conclusion, these results demonstrated that typical mesenchymal stem cells were isolated from dairy goat bone marrow, possessed the characteristics of pluripotent stem cells, and had the potential of specific genetic modifications for gene therapy and producing transgenic goats.

Effect of different levels of short-term feed intake on folliculogenesis and follicular fluid and plasma concentrations of lactate dehydrogenase, glucose, and hormones in Hu sheep during the luteal phase.

This study investigated the effects of short-term food restriction or supplementation on folliculogenesis and plasma and intrafollicular metabolite and hormone concentrations. Ewes were randomly assigned to three groups: the control group received a maintenance diet (M) while the supplemented group and restricted group received 1.5×M and 0.5×M respectively on days 6-12 of their estrous cycle. Estrus was synchronized by intravaginal progestogen sponges for 12 days. On days 7-12, blood samples were taken. After slaughter, the ovarian follicles were classified and the follicular fluid was collected. Compared with restriction, supplementation shortened the estrous cycle length, decreased the number of follicles 2.5-3.5 mm and follicular fluid estradiol (E2) concentration, increased the number of follicles>3.5 mm and plasma glucose, insulin and glucagon concentrations, and augmented the volume of follicles>2.5 mm. Restricted ewes had higher intrafollicular insulin concentration, but it was similar to that of supplemented ewes. Compared with follicles≤2.5 mm, the intrafollicular glucose and E2 concentrations were increased and the testosterone, insulin, and glucagon concentrations and lactate dehydrogenase (LDH) activity were decreased in follicles>2.5 mm. Only in restricted ewes were intrafollicular LDH and testosterone concentrations in follicles≤2.5 mm not different from those in follicles≤2.5 mm. In conclusion, the mechanism by which short-term dietary restriction inhibits folliculogenesis may involve responses to intrafollicular increased E2, testosterone, and LDH levels in late-stage follicles. This may not be due to the variation of intrafollicular insulin level but rather due to decreased circulating levels of glucose, insulin, and glucagon.