Bromoacetic acid impairs mouse oocyte in vitro maturation through affecting cytoskeleton architecture and epigenetic modification.

As a major public health achievement, disinfection of drinking water significantly decreases outbreaks of waterborne disease, but produces drinking water disinfection by-products (DBPs) unfortunately. The haloacetic acids (HAAs) including bromoacetic acid (BAA), the second major class of DBPs, are considered as a global public health concern. BAA has been identified as cytotoxic, genotoxic, mutagenic, carcinogenic, and teratogenic in somatic cells. However, the toxic effects of BAA on oocyte maturation remain obscure. Herein, we documented that exposure to BAA compromised mouse oocyte maturation in vitro, causing blocked polar body extrusion (PBE). Meiotic progression analysis demonstrated that exposure to BAA induced the activated spindle assembly checkpoint (SAC) mediated metaphase I (MI) arrest in oocytes. Further study revealed that exposure to BAA resulted in the hyperacetylation of α-tubulin, disrupting spindle assembly and chromosome alignment, which is responsible for the activation of SAC. Besides, the organization of actin, the other major component of cytoskeleton in oocytes, was disturbed after BAA exposure. In addition, exposure to BAA altered the status of histone H3 methylation and 5 mC, indicative of the damaged epigenetic modifications. Moreover, we found that exposure to BAA induced DNA damage in a dose-dependent manner in oocytes. Collectively, our study evidenced that exposure to BAA intervened mouse oocyte maturation via disrupting cytoskeletal dynamics, damaging epigenetic modifications and inducing accumulation of DNA damage.

Zinc pyrithione exposure compromises oocyte maturation through involving in spindle assembly and zinc accumulation.

Zinc Pyrithione (ZPT), a Food and Drug Administration (FDA) approved chemical, is widely used for topical antimicrobials and cosmetic consumer products, including anti-dandruff shampoos. ZPT and its degraded byproducts have detected in large quantities in the environment, and identified to pose healthy risks on aquatic organisms and human. However, so far, knowledge about ZPT effects on female reproduction, particularly oocyte maturation and quality, is limited. Herein, we investigated the adverse impact of ZPT on mouse oocyte maturation and quality in vitro and found exposure to ZPT significantly compromises oocyte maturation. The results revealed that ZPT disturbed the meiotic cell cycle by impairing cytoskeletal dynamics, kinetochore-microtubule attachment (K-MT), and causing spindle assembly checkpoints (SAC) continuous activation. Further, we observed the microtubule-organizing centers (MTOCs) associated proteins p-MAPK and Aurora-A were disrupted in ZPT-treated oocytes, signified by decreased expression and abnormal localization, responsible for the severe cytoskeletal defects. In addition, ZPT exposure induced a significant increase in the levels of H3K9me2, H3K9me3, H3K27me1, and H3K27me3, suggesting the alterations of epigenetic modifications. Moreover, the accumulation of zinc ions (Zn2+) was observed in ZPT-treated oocytes, which was detrimental because overmuch intracellular Zn2+ disrupted oocyte meiosis. Finally, these above alterations impaired spindle organization and chromosome alignment in metaphase-II (MII) oocytes, indicative of damaged oocytes quality. In conclusion, ZPT exposure influenced oocyte maturation and quality via involvement in MTOCs-associated proteins mediated spindle defects, altered epigenetic modifications and zinc accumulation.

Bisphenol F exposure affects mouse oocyte in vitro maturation through inducing oxidative stress and DNA damage.

Bisphenol F (BPF), a substitute for bisphenol A (BPA), is progressively used to manufacture various consumer products. Despite the established reproductive toxicity of BPF, the underlying mechanisms remain to elucidate. This in-vitro study deep in sighted the BPF toxicity on mouse oocyte meiotic maturation and quality. After treating oocytes with BPF (300 µM), the oocyte meiotic progression was blocked, accentuated by a reduced rate in the first polar body extrusion (PBE). Next, we illustrated that BPF induced α-tubulin hyper-acetylation disrupted the spindle assembly and chromosome alignment. Concurrently, BPF resulted in severe oxidative stress and DNA damage, which triggered the early apoptosis in mouse oocytes. Further, altered epigenetic modifications following BPF exposure were proved by increased H3K27me3 levels. Concerning the toxic effects on spindle structure, oxidative stress, and DNA damage in mouse oocytes, BPF toxicity was less severe to oocyte maturation and spindle structure than BPA and induced low oxidative stress. However, compared with BPA, oocytes treated with BPF were more prone to DNA damage, indicating not less intense or even more severe toxic effects of BPF than BPA on some aspects of oocytes maturation. In brief, the present study established that like wise to BPA, BPF could inhibit meiotic maturation and reduce oocyte quality, suggesting it is not a safe substitute for BPA.

WDR62 regulates mouse oocyte meiotic maturation related to p-JNK and H3K9 trimethylation.

WDR62 (WD40-repeat protein 62) participates in diverse biological process, especially mitotic spindle organization via regulating centriole biogenesis and the function of centriole-associated protein. However, the role of WDR62 exerts in spindle assembly and meiotic progression control in oocytes lacking typical centrosomes remains obscure. In a previous study, we reported that WDR62 is involved in spindle migration and asymmetric cytokinesis in mouse oocyte meiosis. In the current study, another novel function of WDR62 regulating cell cycle progression through meiotic spindle formation during oocyte meiotic maturation was found. Knockdown of WDR62 through siRNA microinjection disrupted the meiotic cell cycle and induced metaphase-I (MI) arrest coupled with severe spindle abnormality, chromosome misalignment, and aneuploid generation. Moreover, WDR62 depletion induced defective kinetochore-microtubule attachments (K-MT) and activated spindle assembly checkpoint (SAC), which could trigger the arrest of meiotic progression. Further study demonstrated that depletion of WDR62 was associated with an aberrant location of p-JNK and reduced its expression level; concomitantly, status of H3K9 trimethylation was also altered. In addition, phenotypes similar to WDR62 depletion were observed during the function-loss analysis of p-JNK using a specific inhibitor (SP600125), which signifies that WDR62 is important for spindle organization and meiotic progression, and this function might be via its regulation of p-JNK. In conclusion, this study revealed that WDR62 functions in multiple ways during oocyte meiotic maturation, which could be related to p-JNK and H3K9 trimethylation.

FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro.

Fibroblast growth factor 2 (FGF2), a member of FGF family, binds with FGF receptors (FGFR) to initiate biological functions in various somatic cells. However, little is known regarding the role of FGF2/FGFR on oocyte meiosis. In this study, we investigated expression patterns and functions of FGF2/FGFR during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs). Among four FGFRs, Ffgr1 was the most abundant in COCs. The transcripts for Fgf2 and Ffgr1 in COCs increased during IVM. Ffgr1 was present in oocytes and cumulus cells, while Fgf2 was present in only cumulus cells. Treatment of COCs with the selective FGFR inhibitor SU5402 blocked oocyte meiotic progression and downregulated expression of Bmp15 and Gdf9. In contrast, supplement of FGF2 promoted oocyte meiotic progression and upregulated Bmp15 and Gdf9 expression. Inhibition of FGFR with SU5402 reduced cumulus expansion and expressions of Ptx3, Has2 and Tnfaip6. Treatment with FGF2 increased Ptx3 and Has2 expression. Inhibition of FGFR had no effect on meiotic progression of denuded oocytes (DOs). However, co-culture of DOs with COCs or supplementation with FGF2 promoted meiotic progression of DOs. Inhibition of FGF2/FGFR signaling also downregulated Ffgr1 expression, while supplemental FGF2 upregulated Fgfr1 expression. Furthermore, inhibition of FGFR in COCs interrupted the c-Mos/MAPK pathway and maturation-promoting factor (MPF), as indicated by downregulation of oocyte c-mos and Ccnb1 transcripts, respectively. Overall, this study suggests that FGF2 produced by cumulus cells, activates a FGF2/FGFR autocrine/paracrine loop within COCs to regulate cumulus expansion and oocyte meiosis. These findings reveal a novel role for FGF2/FGFR signaling during in vitro maturation of COCs.

Dihydropyridine Enhances the Antioxidant Capacities of Lactating Dairy Cows under Heat Stress Condition.

Heat stress (HS), a nonspecific response to environmental heat, can seriously affect dairy cow health. Feed additives may alleviate HS in dairy cows by improving rumen fermentation efficacy, stimulating feed consumption, enhancing vasodilation, and/or improving antioxidant capacity. The temperature-humidity index (THI) indicates that spring is a non-HS season, and summer is an HS season. HS results in the decrease in dairy cow antioxidant capacities. Our results indicated the decrease in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidation (T-AOC) levels and the increase in malondialdehyde (MDA) level during HS season. Meanwhile, antioxidant indexes (SOD, GSH-Px, and T-AOC) were positively correlated with milk yield (p < 0.01), whereas MDA exhibited a significant negative correlation with milk yield (p < 0.05). In addition, the effects of dihydropyridine (DHP) on antioxidant capacity and ruminal microbial communities in dairy cows under HS were investigated. During summer, dairy cows were randomly assigned into two groups under HS, including a standard diet (S-ND) group and standard diet with 3 g/day/cow DHP (S-D) group. DHP treatment significantly restored SOD and GSH-Px levels under HS. Denaturing gradient gel electrophoresis results indicated that the DHP altered ruminal bacterial community mainly composed Proteobacteria and Firmicutes in dairy cows under HS. Our results suggest that DHP can enhance the antioxidant abilities of dairy cows with favorable effects on ruminal microbial communities under HS, further alleviating HS on dairy cows.

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells.

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase-3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.

[Observation of curative effect of hemorrhoids lotion on pain, edema and bleeding after anorectal surgery].

The purpose of this study was to evaluate the value of Chinese herbal fumigation in the postoperative anal disease. The authors randomly divided 348 patients into treatment group and control group with 174 cases in each group. The treatment group was given to the Chinese herbal medicine hemorrhoids lotion for fumigation based on conventional anti infective therapy, routine dressing change and relaxing bowel. The control group was given to 1 000 mL 1: 5 000 potassium permanganate solution for sitz bath, fumigation based on conventional anti infective therapy, routine dressing change and relaxing bowel. The pain score, edema score, bleeding score, granulation tissue growth score and wound healing time of two groups were compared after operation. The results showed that the postoperative 6 h pain scores were higher in the two groups, the postoperative 3,5,7 d pain scores gradually decreased, the difference was statistically significant (P < 0.05). The difference of postoperative 6 h pain scores was no significant difference between the two groups, while postoperative 3,5,7 d pain scores in the treatment group were significantly lower than those in the control group (P < 0.05). 7 days after operation, anal margin of edema score and blood in the stool score in the treatment group were lower than those in control group, meat medicine growth score was higher than that of the control group, the difference had statistical meaning (P < 0.05). The healing time of two groups was respectively (13.89 + 2.78), (18.45 + 1.65) d (P < 0.05). This study suggested that Chinese herbal fumigation and washing could reduce the pain degree of patients, the anal margin of edema, and the blood in the stool, also could promote granulation tissue growth and shorten the time of wound healing, deserve the clinical expansion.

Polymorphisms in luteinizing hormone receptor and hypothalamic gonadotropin-releasing hormone genes and their effects on sperm quality traits in Chinese Holstein bulls.

Genes of hypothalamic-pituitary-gonadal axis play a key role in male reproductive performance. This study evaluated the polymorphisms of luteinizing hormone receptor (LHR) and hypothalamic gonadotropin-releasing hormone (GnRH) genes and their effects on sperm quality traits including semen volume per ejaculate (VOL), sperm density (SD), fresh sperm motility (FSM), thawed sperm motility (TSM), acrosome integrity rate (AIR), and abnormal sperm rate (ASR) collected from 205 Chinese Hostein bulls. The study bulls consisted of 205 mature Chinese Holstein, 27 Simmental, 28 Charolais, and 14 German yellow cattle. One single nucleotide polymorphism (SNP) (A883G) in exon 2 of GnRH and two SNPs (A51703G and G51656T) in intron 9 of LHR were identified in 274 bulls. Analysis of variance in 205 Chinese Holstein bulls showed that age had significant effect on both SD and FSM (P < 0.01), and ASR (P < 0.05). With regards to genotype and its interaction with age, only the SNP of G51656T in LHR gene had significant effect on SD (P < 0.05, P < 0.01; respectively). The association result showed that bulls with AG genotype had higher FSM than bulls with AA and GG genotype in LHR at 51,703 locus (P < 0.10), and bulls with GG genotype had higher SD than bulls with TT genotype in LHR at G51656T locus (P < 0.10). Phenotypic correlation among the traits revealed that significant negative correlations were observed between ASR and AIR (r = -0.736, P < 0.01), ASR and AIR (r = -0.500, P < 0.01). There were moderate positive correlations between VOL and SD (r = 0.422, P < 0.01), as well as FSM (r = 0.411, P < 0.01). In conclusion, LHR may be a potential marker for sperm quality of SD and FSM.

Polymorphisms in the 5' upstream region of the FSH receptor gene, and their association with superovulation traits in Chinese Holstein cows.

To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, variation in the bovine follicle stimulating hormone receptor (FSHR) gene was investigated using PCR-single-strand conformational (PCR-SSCP) and DNA sequencing. One single nucleotide polymorphism (SNP) of G-278A located in the 5' upstream region of bovine FSHR gene was found in 118 Chinese Holstein cows treated for superovulation. Two SNPs of G-278A (GU253337) and A-320T (rs43676359) were analyzed. In polymorphic locus -278, all cows without superovulation response were mutations with genotypes of CD and DD. Cows with CC genotype had a significant increase in the total number of ova (TNO) (P<0.01), and produced more transferable embryos (NTE) than those of the CD and DD genotypes (P<0.01). At this locus, the additive effects seemed to be highly significant (P<0.01) and allele C was associated with an increase in the TNO and NTE. These results indicated that the FSHR is a potential marker for superovulation response and can be used as a predictor for superovulation in Chinese Holstein cows.

A review of research progress of FecB gene in Chinese breeds of sheep.

FecB gene is a major gene responsible for high prolificacy firstly identified in Booroola Merino sheep. Subsequently, many other aspects of the FecB including endocrinology, fetal and postnatal growth were studied. A forced PCR-RFLP method was performed to screen some Chinese breeds or strains of sheep to determine if FecB gene is responsible for their high prolificacies. The FecB gene was present in some Chinese prolific breeds of sheep, such as Huyang, Small Tail Han (STH), Cele, Duolang sheep and Chinese Merino prolific strains, but absent in the low prolific sheep breeds such as Mongolia, Chinese Merino, Tan, Xinjiang, Hulunbeier, Inner Mongolia Fine Wool and Northeastern Half-fuzz Sheep. It has been confirmed that FecB gene was associated with high prolificacy in some Chinese breeds or strains of sheep. Moreover, introducing FecB gene to some low prolific breeds of sheep by crossbreeding system can improve the reproductive traits.

None of polymorphism of ovine fecundity major genes FecB and FecX was tested in goat.

The polymorphism of mutation Q249R in BMPR-IB gene (FecB) and loci FecX(I), FecX(H), FecX(G), FecX(B) in BMP15 gene was analyzed by forced PCR-RFLP method in 550 individuals from 6 flocks or breeds of goats with litter size varied from 1.4 to 2.7 including Boer (209), Haimen (128), second generation of Boer goat crossed with Huanghuai goat (82), Huanghuai (71), Nubi (37) and Matou (23) goat. None of mutations was detected in these goat breeds and their crossbreed. These results suggest that fecundity of goat is not linked to the same loci in BMPR-IB and BMP15 as sheep. Therefore, it is necessary to seek for other genes or loci in order to develop marker assistance selection techniques and study the prolific mechanism of the goat.

[Correlation analysis between 9 microsatellite markers and fecundity trait of Boer goat].

Five microsatellite markers BM1329, BM143, OarHH55, TGLA68, and LSCV043, which were closely linked to the fecundity gene FecB and FecX in ovis aries sheep, were chosen based on their high conservatism in the close species and another 4 microsatellite markers ETH225, INRA063, BM1225, and MAF0214 were selected to analyze their correlation with the litter size of Boer goat. All the 9 microsatellite loci in Boer goat were high polymorphism locus (PIC > 0.5). Twelve alleles were found to be significantly correlate with litter size of Boer goat. Three of them, i.e., 120 bp and 108 bp of BM143, 183 bp of ETH225, were positively correlated to first parity litter size of Boer goat. Eight alleles, i.e., 216 bp of BM1329, 110 bp of BM143, 255 bp and 239 bp of BM1225, 175 bp, 177 bp and 189 bp of INRA063, and 163 bp of ETH225, had negative correlation with first parity litter size. One allele, i.e., 115 bp of TGLA68, were positively correlated to second parity litter size.

[Hae RFLP of INHA and its relationship to goat litter size].

The polymorphism in a fragment within the coding region of the inhibin a subunit ( INHA ) gene was studied in 323 heads of Matou, Nubi, Boar and Haimen goats by PCR-SSCP, PCR-RFLP and sequencing. A new mutation G284A (Accession number: L28815) was identified, which could be detected by Hae digestion of a PCR product spanning this site. Hae PCR-RFLP analysis indicated that allele G was dominant. Association studies indicated that the effect of INHA genotypes on litter size was greatest for GG, followed by AG and AA genotypes. Thus, INHA may be a major gene controlling the prolificacy of goat, and allele G is positively correlated with litter size.