Single-cell sequencing reveals the transcriptional alternations of 17ß-estradiol suppressing primordial follicle formation in neonatal mouse ovaries.

Estrogen has been implicated in multiple biological processes, but the variation underlying estrogen-mediated primordial follicle (PF) formation remains unclear. Here, we show that 17ß-estradiol (E2) treatment of neonatal mice led to the inhibition of PF formation and cell proliferation. Single-cell RNA sequencing (scRNA-seq) revealed that E2 treatment caused significant changes in the transcriptome of oocytes and somatic cells. E2 treatment disrupted the synchronised development of oocytes, pre-granulosa (PG) cells and stromal cells. Mechanistically, E2 treatment disrupted several signalling pathways critical to PF formation, especially down-regulating the Kitl and Smad1/3/4/5/7 expression, reducing the frequency and number of cell communication. In addition, E2 treatment influenced key gene expression, mitochondrial function of oocytes, the recruitment and maintenance of PG cells, the cell proliferation of somatic cells, as well as disordered the ovarian microenvironment. This study not only revealed insights into the regulatory role of estrogen during PF formation, but also filled in knowledge of dramatic changes in perinatal hormones, which are critical for the physiological significance of understanding hormone changes and reproductive protection.

Estrogen influences the transzonal projection assembly of cumulus-oocyte complexes through G protein-coupled estrogen receptor during goat follicle development.

Estrogen is an important hormone that plays a role in regulating follicle development and oocyte maturation. Transzonal projections (TZPs) act as communication bridges between follicle somatic cells and oocytes, and their dynamic changes are critical for oocyte development and maturation. However, the roles and mechanisms of estrogen in regulating TZPs during follicular development are not yet understood. We found that the proportion of oocytes spontaneously resuming meiosis increases as the follicle grows, which is accompanied by rising estrogen levels in follicles and decreasing TZPs in cumulus-oocyte complex. To further explore the effect of elevated estrogen levels on TZP assembly, additional estrogen was added to the culture system. The increased estrogen level significantly decreased the mRNA and protein expression levels of TZP assembly-related genes. Subsequent research revealed that TZP regulation by estrogen was mediated by the membrane receptor GPER and downstream ERK1/2 signaling pathway. In summary, our study suggests that estrogen may regulate goat oocyte meiosis arrest by decreasing TZP numbers via estrogen-mediated GPER activation during follicle development.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis.

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

Intermittent fasting improves the oocyte quality of obese mice through the regulation of maternal mRNA storage and translation by LSM14B.

Obesity has significant repercussions for female reproductive health, including adverse effects on oocyte quality, fertility, embryo development and offspring health. Here, we showed that intermittent fasting (IF) has several notable effects on follicular development, oocyte development and maturation and offspring health in obese mice. IF treatment prevents obesity-associated germline-soma communication defects, mitochondrial dysfunction, oxidative damage, apoptosis, and spindle/chromosomal disruption. RNA-sequencing analysis of oocytes from normal diet (ND), high-fat diet (HFD), and HFD + IF mice indicated that IF treatment improved mitochondrial oxidative phosphorylation function and mRNA storage and translation, which was potentially mediated by the Smith-like family member 14 B (LSM14B). Knockdown of LSM14B by siRNA injection in oocytes from ND mice recapitulates all the translation, mitochondrial dysfunction and meiotic defect phenotypes of oocytes from HFD mice. Remarkably, the injection of Lsm14b mRNA into oocytes from HFD mice rescued the translation, mitochondrial dysfunction and meiotic defect phenotypes. These results demonstrated that dysfunction in the oocyte translation program is associated with obesity-induced meiotic defects, while IF treatment increased LSM14B expression and maternal mRNA translation and restored oocyte quality. This research has important implications for understanding the effects of obesity on female reproductive health and offers a potential nonpharmacological intervention to improve oocyte quality and fertility in obese individuals.

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells.

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor ß (Esr2/ERß) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERß and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERß and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERß and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERß and GPER in cumulus cells.

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 Relationship between Mastitis and Antimicrobial Peptide S100A7 Expression in Dairy Goats.

S100A7 is an inflammation-related protein and plays an essential role in host defenses, yet there is little research about the relationship between mastitis and S100A7 expression in dairy goats. Here, according to the clinical diagnosis of udders, SCC, and bacteriological culture (BC) of milk, 84 dairy goats were grouped into healthy goats (n = 25), subclinical mastitis goats (n = 36), and clinical mastitis goats (n = 23). The S100A7 concentration in subclinical mastitis goats was significantly upregulated than in healthy dairy goats (p = 0.0056) and had a limited change with clinical mastitis dairy goats (p = 0.8222). The relationship between log10 SCC and S100A7 concentration in milk was positive and R = 0.05249; the regression equation was Y = 0.1446 × X + 12.54. According to the three groups, the log10 SCC and S100A7 were analyzed using the receiver operating characteristics (ROC) curve; in subclinical mastitis goats, the area under the ROC curve (AUC) of log10 SCC was 0.9222 and p < 0.0001, and the AUC of S100A7 concentration was 0.7317 and p = 0.0022, respectively; in clinical mastitis goats, the AUC of log10 SCC was 0.9678 and p < 0.0001, and the AUC of S100A7 concentration was 0.5487 and p = 0.5634, respectively. In healthy goats, S100A7 was expressed weakly in the alveolus of the mammary gland of healthy goats while expressed densely in the collapsed alveolus of mastitis goats. Moreover, S100A7 expression increased significantly in mastitis goats than in healthy dairy goats. In this research, results showed the effects of mastitis on the S100A7 expression in the mammary gland and S100A7 concentration in milk and the limited relationship between SCC and mastitis, which provided a new insight into S100A7's role in the host defenses of dairy goats.

E2/ER signaling mediates the meiotic arrest of goat intrafollicular oocytes induced by follicle-stimulating hormone.

The increased production of high-quality oocytes lies at the heart of the search to accelerate the reproduction of high-quality breeding livestock using assisted reproductive technology. Follicle-stimulating hormone (FSH) maintains the arrest of oocyte meiosis during early follicular development in vivo and promotes the synchronous maturation of nucleus and cytoplasm to improve oocyte quality. However, the mechanism by which FSH maintains meiotic arrest in oocytes is still not fully understood. Oocytes spontaneously resume meiosis once released from the arrested state. In this study, we isolated goat antral follicles with a diameter of 2.0-4.0 mm, cultured them in vitro either with or without added FSH, and finally collected the oocytes to observe their meiotic state. The results showed that FSH effectively inhibited the meiotic recovery of oocytes in follicles [4 h: control (n = 84) vs. with FSH (n = 86), P = .0115; 6 h: control (n = 86) vs. FSH (n = 85), P = 0.0308; and 8 h: control (n = 95) vs. FSH (n = 101), P = 0.0039]. FSH significantly inhibited the downregulation of natriuretic peptide receptor 2 (NPR2) expression and cyclic guanosine monophosphate (cGMP) synthesis during follicular culture in vitro (P < 0.05). Further exploration found that FSH promoted the synthesis of 17ß-estradiol (E2) (P = .0249 at 4 h and P = .0039 at 8 h) and maintained the expression of the estrogen nuclear receptor ERß, but not the estrogen nuclear receptor ERα during follicle culture in vitro (P = .0190 at 2 h, and P = .0100 at 4 h). In addition, E2/ER (estrogen nuclear receptors ERα and ERß) mediated the inhibitory effect of FSH on the downregulation of NPR2 expression and cGMP synthesis, ultimately preventing the meiotic recovery of oocytes (P < .05). In summary, our study showed that FSH-induced estrogen production in goat follicles, and the E2/ER signaling pathway, both mediated meiotic arrest in FSH-induced goat oocytes.

Obtaining a greater number of high-quality oocytes to accelerate the reproduction of high-quality breeding livestock using artificial-assisted reproductive technology remains a pressing problem in animal husbandry and requires further research into the mechanism of oocyte maturation. We investigated the regulatory action of follicle-stimulating hormone (FSH) on the meiosis of oocytes during goat follicle culture in vitro. We found that FSH promoted 17ß-estradiol (E2) synthesis and that E2/ER (estrogen nuclear receptors ERα and ERß)-mediated FSH regulation of the CNP/NPR2 (C-type natriuretic peptide/natriuretic peptide receptor 2) signaling pathway and oocyte meiosis in goat follicles. This study provided an improved theoretical foundation for the increased production of high-quality oocytes using in vitro culture methods.

C-type natriuretic peptide improves maternally aged oocytes quality by inhibiting excessive PINK1/Parkin-mediated mitophagy.

The overall oocyte quality declines with aging, and this effect is strongly associated with a higher reactive oxygen species (ROS) level and the resultant oxidative damage. C-type natriuretic peptide (CNP) is a well-characterized physiological meiotic inhibitor that has been successfully used to improve immature oocyte quality during in vitro maturation. However, the underlying roles of CNP in maternally aged oocytes have not been reported. Here, we found that the age-related reduction in the serum CNP concentration was highly correlated with decreased oocyte quality. Treatment with exogenous CNP promoted follicle growth and ovulation in aged mice and enhanced meiotic competency and fertilization ability. Interestingly, the cytoplasmic maturation of aged oocytes was thoroughly improved by CNP treatment, as assessed by spindle/chromosome morphology and redistribution of organelles (mitochondria, the endoplasmic reticulum, cortical granules, and the Golgi apparatus). CNP treatment also ameliorated DNA damage and apoptosis caused by ROS accumulation in aged oocytes. Importantly, oocyte RNA-seq revealed that the beneficial effect of CNP on aged oocytes was mediated by restoration of mitochondrial oxidative phosphorylation, eliminating excessive mitophagy. CNP reversed the defective phenotypes in aged oocytes by alleviating oxidative damage and suppressing excessive PINK1/Parkin-mediated mitophagy. Mechanistically, CNP functioned as a cAMP/PKA pathway modulator to decrease PINK1 stability and inhibit Parkin recruitment. In summary, our results demonstrated that CNP supplementation constitutes an alternative therapeutic approach for advanced maternal age-related oocyte deterioration and may improve the overall success rates of clinically assisted reproduction in older women.

Immunolocalization of antibacterial peptide S100A7 in mastitis goat mammary gland and lipopolysaccharide induces the expression and secretion of S100A7 in goat mammary gland epithelial cells via TLR4/NFκB signal pathway.

The antimicrobial peptide S100A7, with antimicrobial activities for a broad spectrum of bacteria, has attracted more and more attention for the prevention and treatment of mastitis. However, there is little information about the expression and regulation mechanism of S100A7 in mastitis goats. This study revealed that S100A7 was mainly expressed in the stratified squamous epithelium of teat skin and streak canal, and S100A7 was present weakly in the healthy goat alveolus yet densely in the mastitis goat collapsed alveolus. Goat mammary epithelial cells (MECs) were isolated and treated with 2.5, 5, 10 and 20 µg/mL lipopolysaccharide (LPS) respectively for a different time, S100A7 mRNA expression and protein secretion were upregulated significantly with LPS treatment for 3 h, and the secretion level of S100A7 descended after 48 h treatment for all of these four groups. Moreover, after treatment with LPS, the mRNA levels of Toll-like receptor 4 (TLR4) and MyD88 were up-regulated, and the phosphorylation of p65 was up-regulated markedly. However, adding TLR4 inhibitor TAK-242 or/and NF-κB inhibitor QNZ significantly suppressed the phosphorylation of p65, and then inhibited the expression and secretion of S100A7 induced by LPS treatment. In conclusion, LPS induced the expression and secretion of S100A7 in goat MECs via TLR4/NF-κB signaling pathway.

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.

Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing.

BACKGROUND: Rewriting the genomes of living organisms has been a long-standing aim in the biological sciences. The revelation of the CRISPR/Cas9 technology has revolutionized the entire biological field. Since its emergence, this technology has been widely applied to induce gene knockouts, insertions, deletions, and base substitutions. However, the classical version of this system was imperfect for inducing or correcting desired mutations. A subsequent development generated more advanced classes, including cytosine and adenine base editors, which can be used to achieve single nucleotide substitutions. Nevertheless, these advanced systems still suffer from several limitations, such as the inability to edit loci without a suitable PAM sequence and to induce base transversions. On the other hand, the recently emerged prime editors (PEs) can achieve all possible single nucleotide substitutions as well as targeted insertions and deletions, which show promising potential to alter and correct the genomes of various organisms. Of note, the application of PE to edit livestock genomes has not been reported yet. RESULTS: In this study, using PE, we successfully generated sheep with two agriculturally significant mutations, including the fecundity-related FecBB p.Q249R and the tail length-related TBXT p.G112W. Additionally, we applied PE to generate porcine blastocysts with a biomedically relevant point mutation (KCNJ5 p.G151R) as a porcine model of human primary aldosteronism. CONCLUSIONS: Our study demonstrates the potential of the PE system to edit the genomes of large animals for the induction of economically desired mutations and for modeling human diseases. Although prime-edited sheep and porcine blastocysts could be generated, the editing frequencies are still unsatisfactory, highlighting the need for optimizations in the PE system for efficient generation of large animals with customized traits.

Sulforaphane prevents LPS-induced inflammation by regulating the Nrf2-mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis.

BACKGROUND: Mastitis not only deteriorates the composition or quality of milk, but also damages the health and productivity of dairy goats. Sulforaphane (SFN) is a phytochemical isothiocyanate compound with various pharmacological effects such as anti-oxidant and anti-inflammatory. However, the effect of SFN on mastitis has yet to be elucidated. This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide (LPS)-induced primary goat mammary epithelial cells (GMECs) and a mouse model of mastitis. RESULTS: In vitro, SFN downregulated the mRNA expression of inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6), inhibited the protein expression of inflammatory mediators (cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS)) while suppressing nuclear factor kappa-B (NF-κB) activation in LPS-induced GMECs. Additionally, SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation, up-regulating antioxidant enzymes expression, and decreasing LPS-induced reactive oxygen species (ROS) production in GMECs. Furthermore, SFN pretreatment promoted the autophagy pathway, which was dependent on the increased Nrf2 level, and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response. In vivo, SFN effectively alleviated histopathological lesions, suppressed the expression of inflammatory factors, enhanced immunohistochemistry staining of Nrf2, and amplified of LC3 puncta LPS-induced mastitis in mice. Mechanically, the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis. CONCLUSIONS: These results indicate that the natural compound SFN has a preventive effect on LPS-induced inflammation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis, which may improve prevention strategies for mastitis in dairy goats.

Gut Microbiota and Acylcarnitine Metabolites Connect the Beneficial Association between Estrogen and Lipid Metabolism Disorders in Ovariectomized Mice.

Decreased estrogen level is one of the main causes of lipid metabolism disorders and coronary heart disease in women after menopause. Exogenous estradiol benzoate is effective to some extent in alleviating lipid metabolism disorders caused by estrogen deficiency. However, the role of gut microbes in the regulation process is not yet appreciated. The objective of this study was to investigate the effects of estradiol benzoate supplementation on lipid metabolism, gut microbiota, and metabolites in ovariectomized (OVX) mice and to reveal the importance of gut microbes and metabolites in the regulation of lipid metabolism disorders. This study found that high doses of estradiol benzoate supplementation effectively attenuated fat accumulation in OVX mice. There was a significant increase in the expression of genes enriched in hepatic cholesterol metabolism and a concomitant decrease in the expression of genes enriched in unsaturated fatty acid metabolism pathways. Further screening of the gut for characteristic metabolites associated with improved lipid metabolism revealed that estradiol benzoate supplementation influenced major subsets of acylcarnitine metabolites. Ovariectomy significantly increased the abundance of characteristic microbes that are significantly negatively associated with acylcarnitine synthesis, such as Lactobacillus and Eubacterium ruminantium group bacteria, while estradiol benzoate supplementation significantly increased the abundance of characteristic microbes that are significantly positively associated with acylcarnitine synthesis, such as Ileibacterium and Bifidobacterium spp. The use of pseudosterile mice with gut microbial deficiency greatly facilitated the synthesis of acylcarnitine due to estradiol benzoate supplementation and also alleviated lipid metabolism disorders to a greater extent in OVX mice. IMPORTANCE Our findings establish a role for gut microbes in the progression of estrogen deficiency-induced lipid metabolism disorders and reveal key target bacteria that may have the potential to regulate acylcarnitine synthesis. These findings suggest a possible route for the use of microbes or acylcarnitine to regulate disorders of lipid metabolism induced by estrogen deficiency.

Human umbilical cord mesenchymal stem cells conditioned medium exerts anti-tumor effects on KGN cells in a cell density-dependent manner through activation of the Hippo pathway.

BACKGROUND: The conditioned medium from human umbilical cord mesenchymal stem cells (UCMSCs-CM) provides a new cell-free therapy for tumors due to its unique secretome. However, there are many contradictory reports about the effect of UCMSCs-CM on tumor cells. The loss of contact inhibition is a common characteristic of tumor cells. A relationship between the effect of UCMSCs-CM on tumor cells and contact inhibition in tumor cells is rarely concerned. Whether the effect of UCMSCs-CM on tumor cells is affected by cell density? Here, we explored the effect of UCMSCs-CM on granulosa tumor cell line (KGN) cells at low or high density. METHODS: Growth curve and CCK8 assay were used to assess cell proliferation and viability. Scratch wound and matrigel invasion assay were implicated to detect cell motility of KGN cells. UCMSCs-CM effects on cell cycle, apoptosis and pathway-related proteins were investigated by flow cytometry, TUNEL assay, western blot and immunofluorescence analysis respectively. RESULTS: In growth curve analysis, before KGN cells proliferated into confluence, UCMSCs-CM had no effect on cell proliferation. However, once the cells proliferate to contact each other, UCMSCs-CM significantly inhibited proliferation. Meanwhile, when KGN cells were implanted at high density, UCMSCs-CM could induce cell cycle arrest at G1 phase, inhibit cell migration, invasion and promote apoptosis. While it had no similar effect on KGN cells implanted at low density. In mechanism, the UCMSCs-CM treatment activated the Hippo pathway when KGN cells were implanted at high density. Consistently, the MST1/2 inhibitor, XMU-MP-1, inhibited the activation of the Hippo pathway induced by UCMSCs-CM treatment and accordingly declined the anti-tumor effect of UCMSCs-CM on KGN cells. CONCLUSIONS: The effect of UCMSCs-CM on tumor cells is affected by cell density. UCMSCs-CM exerted anti-tumor effect on KGN cells by activating Hippo pathway to restore contact inhibition. Our results suggest that UCMSCs-CM is a promising therapeutic candidate for GCT treatment.

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.

Sulforaphane Suppresses H2O2-Induced Oxidative Stress and Apoptosis via the Activation of AMPK/NFE2L2 Signaling Pathway in Goat Mammary Epithelial Cells.

Oxidative stress in high-yielding dairy goats adversely affects lactation length, milk quality, and the economics of dairy products. During the lactation period, goat mammary epithelial cells (GMECs) are often in a state of disordered metabolic homeostasis primarily caused by the overproduction of reactive oxygen species (ROS). Sulforaphane (SFN), an electrophilic compound that is enriched in broccoli, is a promising antioxidant agent for future potential clinical applications. The objective of the present study was to investigate the function of SFN on hydrogen peroxide (H2O2)-induced oxidative damage in primary GMECs and the underlying molecular mechanisms. Isolated GMECs in triplicate were pretreated with SFN (1.25, 2.5, and 5 µM) for 24 h in the absence or presence of H2O2 (400 µM) for 24 h. The results showed that SFN effectively enhanced superoxide dismutase (SOD) activity, elevated the ratio of glutathione (GSH)/glutathione oxidized (GSSG), and reduced H2O2-induced ROS and malondialdehyde (MDA) production and cell apoptosis. Mechanically, SFN-induced nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) translocation to the nucleus through the activation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway coupled with inhibition of the caspase apoptotic pathway. In addition, GMECs were transfected with NFE2L2 small interfering RNA (NFE2L2 siRNA) for 48 h and/or treated with SFN (5 µM) for 24 h before being exposed to H2O2 (400 µM) for 24 h. We found that knockdown of NFE2L2 by siRNA abrogated the preventive effect of SFN on H2O2-induced ROS overproduction and apoptosis. Taken together, sulforaphane suppressed H2O2-induced oxidative stress and apoptosis via the activation of the AMPK/NFE2L2 signaling pathway in primary GMECs.

Intermittent fasting reverses the declining quality of aged oocytes.

Decreased oocyte quality and compromised embryo development are particularly prevalent in older females, but the aging-related cellular processes and effective ameliorative approaches have not been fully characterized. Intermittent fasting (IF) can help improve health and extend lifespan; nevertheless, how it regulates reproductive aging and its mechanisms remain unclear. We used naturally aged mice to investigate the role of IF in reproduction and found that just one month of every-other-day fasting was sufficient to improve oocyte quality. IF not only increased antral follicle numbers and ovulation but also enhanced oocyte meiotic competence and embryonic development by improving both nuclear and cytoplasmic maturation in maternally aged oocytes. The beneficial effects of IF manifested as alleviation of spindle structure abnormalities and chromosome segregation errors and maintenance of the correct cytoplasmic organelle reorganization. Moreover, single-cell transcriptome analysis showed that the positive impact of IF on aged oocytes was mediated by restoration of the nicotinamide adenine dinucleotide (NAD+)/Sirt1-mediated antioxidant defense system, which eliminated excessive accumulated ROS to suppress DNA damage and apoptosis. Collectively, these findings suggest that IF is a feasible approach to protect oocytes against advanced maternal age-related oxidation damage and to improve the reproductive outcomes of aged females.

Estradiol Regulates the Expression and Secretion of Antimicrobial Peptide S100A7 via the ERK1/2-Signaling Pathway in Goat Mammary Epithelial Cells.

S100A7 has received extensive attention in the prevention and treatment of mastitis across a broad spectrum, yet there is a little information about its mechanism, especially in the immunomodulatory effects of estrogen. In the present study, based on the milk bacteriological culture (BC) of 30 dairy goats, the concentration of both estrogen and S100A7 in the BC-positive samples was not significantly different than in the BC-negative samples; the estrogen abundance in subclinical and clinical mastitis samples also showed only a limited difference; compared with healthy samples, the S100A7 abundance in subclinical mastitis samples differed little, while it was significantly decreased in clinical mastitis samples. Moreover, the relationship between estrogen and S100A7 was positive, and the regression equation was y = 0.3206x + 23.459. The goat mammary epithelial cells (gMECs) were isolated and treated with 1, 10, 100 nM E2 and/or 5 µg/mL lipopolysaccharide (LPS), respectively, for 6 h. Compared with control samples, 5 µg/mL LPS, 10 nM E2 and 100 nM E2 markedly induced S100A7 expression and secretion. More than separated treatment, the cooperation of LPS and E2 also significantly increased S100A7 expression, rather than S100A7 secretion. The p-ERK was up-regulated markedly with 100 nM E2 treatment, while the expression of p-JNK, p-p38 and p-Akt had little effect. The G protein-coupled estrogen receptor 1(GPER1) agonist G1 markedly induced S100A7 expression and secretion in gMECs, and the estrogen nuclear receptor antagonist ICI and GPER1 antagonist G15 significantly repressed this process. In conclusion, E2 binds to nuclear and membrane receptors to regulate the expression and secretion of S100A7 via the ERK1/2-signaling pathway in gMECs.