Berberine enhances autophagic flux by activating the Nrf2 signaling pathway in bovine endometrial epithelial cells to resist LPS-induced apoptosis.

Berberine exerts many beneficial effects on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (BEECs). Recently, we also found that berberine shows significant antiapoptotic and autophagy-promoting activities, but the underlying mechanism has not been elucidated. This research explored the association between the antiapoptotic and autophagy-promoting activities of berberine in LPS-treated BEECs. BEECs were first preconditioned with an inhibitor of autophagic flux (chloroquine [CQ]) for 1 h, treated with berberine for 2 h, and then incubated with LPS for 3 h. Cell apoptosis was assessed by flow cytometry, and autophagy activities were assessed by immunoblot analysis of LC3II and p62. The results indicated that the antiapoptotic activity of berberine was notably inhibited in LPS-treated BEECs after preconditioning with CQ for 1 h. Furthermore, to determine whether berberine promoted autophagy by activating the nuclear factor-erythroid 2 related factor 2 (Nrf2) signaling pathway, we assessed autophagy in LPS-treated BEECs after preconditioning with a signaling pathway inhibitor of Nrf2 (ML385). The results indicated that the enhanced autophagy activity induced by berberine was partially reversed in LPS-treated BEECs after the Nrf2 signaling pathway was disturbed by ML385. In conclusion, berberine enhances autophagic flux to allow resistance to LPS-induced apoptosis by activating the Nrf2 signaling pathway in BEECs. The present study may provide new insight into the antiapoptotic mechanism of berberine in LPS-induced BEECs.

Baicalin inhibits oxidative injures of mouse uterine tissue induced by acute heat stress through activating the Keap1/Nrf2 signaling pathway.

Heat stress effect the physiological functions of body, and reproductive system is one of the most sensitive. It's imperative to find out suitable measures to alleviate harmful effects of heat stress. Baicalin is well-known with its antioxidative property. To examine whether Baicalin could reduce oxidative injures of uterine tissue in heat-stressed mice. The mice were divided into four groups: control (Con), Baicalin (Bai), heat stress (H) and heat stress plus Baicalin (H + Bai). The oxidative damage of uterine tissue was detected by ELISA, H&E staining, tunnel assay and immunohistochemical staining. The protein/mRNA expressions of Keap1/Nrf2 related factors were detected by Western blot or QPCR. The results showed that mice heat-stressed at 41 °C for 2 h induced macroscopic changes, significantly increased MDA content and reduced activities of antioxidant enzymes including SOD, CAT and GSH-Px of the uterine tissue. Compared with Con group, heat stress up-regulated caspase-3 and caspase-9, enhanced the apoptosis of endometrial epithelial and glandular epithelial cells, improved the HO-1 mRNA/protein and NQO1 protein expressions, while down-regulated the mRNA/protein of Keap1. Compared with H group, antioxidant enzyme activities, Nrf2 protein and Nrf2, NQO1 and GCLC mRNA expressions were significantly increased in the H + Bai group. While the uterine epithelial cells apoptosis, MDA contents, caspase-3, caspase-9 and Keap1 protein and HO-1 mRNA expressions were decreased in the H + Bai group of mice compared with that in H group. Briefly, acute heat stress causes oxidative injures and apoptosis of mouse uterine tissue and Baicalin protects uterine tissue from the damages possibly through Keap1/Nrf2 signaling pathway.

Tanshinone IIa alleviates LPS-induced oxidative stress in dairy cow mammary epithelial cells by activating the Nrf2 signalling pathway.

OBJECTIVE: Mastitis is the most prevalent disease in dairy cows worldwide. Evidence has emerged that oxidative stress plays a crucial role in the development of mastitis. This study aimed to investigate the antioxidative effects of tanshinone IIa (Tan IIa) on LPS-induced oxidative stress in dairy cow mammary epithelial cells (CMECs). METHODS AND RESULTS: We examined the levels of ROS and MDA in LPS-treated CMECs after supplementation with Tan IIa using detection kits and found that Tan IIa significantly inhibited the upregulation of these factors. In addition, we also found that Tan IIa significantly reversed the decrease in mitochondrial membrane potential induced by LPS. Moreover, Tan IIa improved the activities of antioxidant enzymes, which were decreased by LPS. Finally, we examined the probable pathway in which Tan IIa exerted its antioxidant effects using qPCR and western blotting and found that Tan IIa significantly activated the Keap1/Nrf2 signalling pathway. CONCLUSION: These results suggest that Tan IIa might become a possible therapeutic agent for the treatment of dairy cow mastitis by weakening oxidative stress induced by LPS in CMECs.

Association of sexually transmitted infection with semen quality in men from couples with primary and secondary infertility.

This study aims to compare the prevalence of sexually transmitted infections (STIs) with semen quality in men from couples with primary and secondary infertility. Semen samples were collected from 133 men who requested fertility evaluation. Seminal tract infection with Ureaplasma spp. (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and herpes simplex virus-2 (HSV-2) was assessed by PCR-based diagnostic assays. Among all patients, the prevalence of STIs was higher in men from couples with primary infertility than that in men from couples with secondary infertility (39.7% vs 21.7%, P = 0.03). The prevalence of UU was 28.8% and 13.3% in men from couples with primary and secondary infertility, respectively. Men from couples with primary infertility were more likely to be positive for UU than men from couples with secondary infertility (P = 0.04). Regarding the UU subtype, the prevalence of Ureaplasma urealyticum (Uuu) and Ureaplasma parvum (Uup; including Uup1, Uup3, Uup6, and Uup14) did not differ between the two groups. No associations between the prevalence rates of MH, MG, and CT were found in men from either infertility group. A lower sperm concentration was associated with STI pathogen positivity in men with primary infertility according to the crude model (P = 0.04). The crude and adjusted models showed that semen volume (both P = 0.03) and semen leukocyte count (both P = 0.02) were independently associated with secondary infertility. These findings suggest the importance of classifying the type of infertility during routine diagnosis of seminal tract infections.

Baicalin enhances the thermotolerance of mouse blastocysts by activating the ERK1/2 signaling pathway and preventing mitochondrial dysfunction.

Heat stress causes oxidative damage and induces excessive cell apoptosis and thus affects the development and/or even causes the death of preimplantation embryos. The effects of baicalin on the developmental competence of heat-stressed mouse embryos were investigated in this experiment. Two-cell embryos were cultured in the presence of baicalin and subjected to heat stress (42 °C for 1 h) at their blastocyst stage followed by continuous culture at 37 °C until examination. The results showed that heat stress (H group) increased reactive oxygen species (ROS) production, apoptosis and even embryo death, along with reductions in both mitochondrial activity and membrane potential (ΔΨm). Both heat stress (H group) and inhibition of the ERK1/2 signaling pathway (U group) led to significantly reduced expression levels of the genes c-fos, AP-1 and ERK2, and the phosphorylation of ERK1/2 and c-Fos, along with significantly increased c-Jun mRNA expression and phosphorylation levels. These negative effects of heat stress on the ERK1/2 signaling pathway were neutralized by baicalin treatment. To explore the signal transduction mechanism of baicalin in improving embryonic tolerance to heat stress, mitochondrial quality and apoptosis rate in the mouse blastocysts were also examined. Baicalin was found to up-regulate the expression of mtDNA and TFAM mRNA, increased mitochondria activity and ΔΨm, and improved the cellular mitochondria quality of mouse blastocysts undergoing heat stress. Moreover, baicalin decreased Bax transcript abundance in blastocyst, along with an increase in the blastocyst hatching rate, which were negatively affected by heat stress. Our findings suggest that baicalin improves the developmental capacity and quality of heat-stressed mouse embryos via a mechanism whereby mitochondrial quality is improved by activating the ERK1/2 signaling pathway and inducing anti-cellular apoptosis.

Tanshinone â ¡A inhibits the lipopolysaccharide-induced epithelial-mesenchymal transition and protects bovine endometrial epithelial cells from pyolysin-induced damage by modulating the NF-κB/Snail2 signaling pathway.

Mixed infection with Escherichia coli and Trueperella pyogenes (T. pyogenes) leads to purulent endometritis, but the underlying molecular mechanisms remain unclear. The aim of this study was to investigate the effect of tanshinone ⅡA (Tan ⅡA) on E. coli and T. pyogenes -induced purulent endometritis and explore the underlying mechanism. First, lipopolysaccharide (LPS) isolated from E. coli and bacteria-free filtrates (BFFs) isolated from T. pyogenes were used to induce a model of bovine endometrial epithelial cell (bEEC) damage in vitro. bEECs were pretreated with or without Tan ⅡA for 2 h, before LPS and BFFs were introduced to induce damage to investigate the protective effect of Tan IIA. Then, the cytolytic activity and inflammatory response in bEECs were examined using CCK-8, LDH and RT-qPCR assays. Furthermore, we confirmed the molecular mechanism by which Tan ⅡA reversed the damaged phenotypes in LPS- and BFFs-induced bEECs via the NF-κB/Snail2 pathway using qPCR and Western blotting. Tan ⅡA significantly decreased the cytolytic activity and inflammatory response in LPS- and BFFs-induced bEECs. In addition, Tan ⅡA reversed the dysregulation of E-cadherin, N-cadherin and vimentin. Moreover, Tan ⅡA significantly inhibited the activation of the NF-κB signaling pathway and decreased the expression level of Snail2, which is the main regulator of the epithelial-mesenchymal transition (EMT). In summary, Tan ⅡA inhibits the LPS-induced EMT and protects bEECs from pyolysin-induced damage by modulating the NF-κB/Snail2 signaling pathway.

Berberine attenuates the inflammatory response by activating the Keap1/Nrf2 signaling pathway in bovine endometrial epithelial cells.

Endometritis is a common inflammatory disease that disturbs the rapid development of dairy farming. In the present study, we investigated the anti-inflammatory effects of berberine on the LPS-induced inflammatory response in bovine endometrial epithelial cells (bEECs) and the participation of the Keap1/Nrf2 signaling pathway in this process. Berberine treatment significantly reduced the LPS-induced expression levels of CRP, IL-1ß, IL-6, and TNF-α in bEECs. The Nrf2 signaling pathway in these cells was also activated by berberine. We further evaluated the effects of Nrf2 activators and inhibitors on the downregulation of proinflammatory cytokines. The activator of Nrf2 significantly inhibited the production of these proinflammatory cytokines that was induced by LPS. However, an inhibitor of Nrf2 only partially inhibited the anti-inflammatory effects of berberine on the LPS-induced inflammatory response in bEECs. In conclusion, our findings suggest that berberine exerts anti-inflammatory effects partially by activating the Keap1/Nrf2 signaling pathway.

HDAC3 controls male fertility through enzyme-independent transcriptional regulation at the meiotic exit of spermatogenesis.

The transition from meiotic spermatocytes to postmeiotic haploid germ cells constitutes an essential step in spermatogenesis. The epigenomic regulatory mechanisms underlying this transition remain unclear. Here, we find a prominent transcriptomic switch from the late spermatocytes to the early round spermatids during the meiotic-to-postmeiotic transition, which is associated with robust histone acetylation changes across the genome. Among histone deacetylases (HDACs) and acetyltransferases, we find that HDAC3 is selectively expressed in the late meiotic and early haploid stages. Three independent mouse lines with the testis-specific knockout of HDAC3 show infertility and defects in meiotic exit with an arrest at the late stage of meiosis or early stage of round spermatids. Stage-specific RNA-seq and histone acetylation ChIP-seq analyses reveal that HDAC3 represses meiotic/spermatogonial genes and activates postmeiotic haploid gene programs during meiotic exit, with associated histone acetylation alterations. Unexpectedly, abolishing HDAC3 catalytic activity by missense mutations in the nuclear receptor corepressor (NCOR or SMRT) does not cause infertility, despite causing histone hyperacetylation as HDAC3 knockout, demonstrating that HDAC3 enzyme activity is not required for spermatogenesis. Motif analysis of the HDAC3 cistrome in the testes identified SOX30, which has a similar spatiotemporal expression pattern as HDAC3 during spermatogenesis. Depletion of SOX30 in the testes abolishes the genomic recruitment of the HDAC3 to the binding sites. Collectively, these results establish the SOX30/HDAC3 signaling as a key regulator of the transcriptional program in a deacetylase-independent manner during the meiotic-to-postmeiotic transition in spermatogenesis.

Aucubin ameliorates the LPS-induced inflammatory response in bovine endometrial epithelial cells by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway.

Cows are susceptible to pathogenic bacterial infection after pregnancy, leading to inflammation of the endometrium. Aucubin (AU) has been proven to exhibit highly effective anti-inflammatory activity, but its ability to protect against endometritis in dairy cows remains unclear. Therefore, the goal of the present study was to evaluate the protective effect of AU on the LPS-induced inflammatory response of bovine endometrial epithelial cells (BEECs). After pre-treating BEECs with AU (10, 20 and 50 µM) for 6 hr, the cells were stimulated with LPS for 3 hr. Subsequently, BEECs apoptosis was analysed by flow cytometry, the expression of pro-inflammatory cytokine mRNA was detected by qRT-PCR, and changes in NF-κB and Keap1/Nrf2 signalling were analysed by western blotting and immunofluorescence analyses. The results showed that AU can reduce TNF-α, IL-1ß, IL-6, COX-2 and iNOS mRNA expression in BEECs and reduce cell apoptosis. Furthermore, AU significantly reduced the level of NF-κB p65 and IκB phosphorylation and inhibited the nuclear translocation of NF-κB p65. AU also activated the Keap1/Nrf2 pathway, promoting the nuclear transfer of Nrf2 and increasing Keap1, Nrf2, HO-1 and NQO1 mRNA and protein levels. Taken together, these results indicate that AU ameliorates the LPS-induced inflammatory response by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway, which has a protective effect on BEECs.

Tanshinone IIA exhibits anti-inflammatory and antioxidative effects in LPS-stimulated bovine endometrial epithelial cells by activating the Nrf2 signaling pathway.

Endometritis is a major disease in productive bovines, and is also caused by conditional pathogens after delivery. The integrity and activity of bovine endometrial epithelial cells (bEECs) determine the development of endometritis. Tanshinone IIA, a compound purified from Salvia miltiorrhiza bunge, has been reported to have anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of tanshinone IIA in the LPS-induced inflammatory response of bEECs. The results showed that tanshinone IIA inhibited the mRNA expression levels of COX-2 and iNOS, and reduced the expression levels of IL-1ß, TNF-α, IL-6 and IL-8 induced by LPS. In addition, we found that tanshinone IIA inhibited the level of MDA, but increased the activities of CAT and SOD. To evaluate the anti-inflammatory mechanism of tanshinone IIA, we examined the activation of Nrf2. The results showed that the Nrf2 signaling pathway was significantly activated by tanshinone IIA. In conclusion, these results showed that tanshinone IIA exhibited anti-inflammatory and antioxidative effects by activating the Nrf2 signaling pathway.

Chlorogenic acid ameliorates mice clinical endometritis by activating Keap1/Nrf2 and inhibiting NFκB signalling pathway.

OBJECTIVES: Clinical endometritis is a common reproductive disorder in mammals that seriously endangers animal health and causes economic losses worldwide. This study aims to use lipopolysaccharide and Trueperella pyogenes exotoxin as modelling reagents (LC) to perfuse the mouse uterus in order to establish a model of clinical endometritis and to investigate the anti-inflammatory and antioxidant effects of chlorogenic acid (CGA). METHODS: In this study, five LC uterine perfusions were selected to model clinical endometritis. The anti-inflammatory and antioxidant effects of CGA were clarified. Through HE staining, proinflammatory cytokines, blood testing, NFκB and Keap1/Nrf2 signalling pathways and other index changes to explore the protection mechanism of CGA. KEY FINDINGS: After CGA treatment, the appearance, inflammatory damage and blood indicators of the mouse uterus returned to normal. Simultaneously, CGA could inhibit the activation of NFκB and reduce the release of inflammatory cytokines; CGA could also activate Keap1/Nrf2, promote the dissociation of Keap1 and Nrf2 and significantly increase the expression of the downstream genes HO-1 and NQO1. CONCLUSIONS: The above results together explain that five LC uterine perfusions can be used to establish a mouse model of clinical endometritis. CGA can treat clinical endometritis by activating Keap1/Nrf2 and inhibiting the NFκB signalling pathway.

Andrographolide attenuates inflammatory response induced by LPS via activating Nrf2 signaling pathway in bovine endometrial epithelial cells.

Endometritis is one of the main causes of bovine infertility, which causes serious economic losses to the industry. The endometrium is the first line of defense against invading microbial pathogens in the uterus. Andrographolide is the primary active component of A. paniculate, and has been shown to have anti-inflammatory and antioxidant effects. However, its effects on the LPS-induced signaling pathway in bovine endometrial epithelial cells (bEECs) have not been reported yet. The aim of this study was to investigate the anti-inflammatory effects and mechanism of andrographolide in the LPS-induced inflammatory response of bEECs. We found that andrographolide strongly reduced LPS-induced NO and iNOS expression. The production of cytokines that were upregulated by LPS was significantly suppressed. To investigate the anti-inflammatory mechanism of andrographolide, we examined the activation of Nrf2. The results shown that andrographolide inhibited the expression of Keap1 but increased the expression of Nrf2. The expression levels of target genes of Nrf2 including Ho-1 and Nqo-1 were increased by andrographolide. Taken together, these results suggest that andrographolide may serve as a candidate to protect against the LPS-induced inflammatory response by inducing Nrf2 activation.

Determination of uterine bacterial community in postpartum dairy cows with metritis based on 16S rDNA sequencing.

Metritis is a frequently occurring diseases in postpartum cows and is one of the important reasons for the infertility of dairy cows, accounting for 20-30% of dairy cow diseases and has serious implications for the dairy industry. It has been reported in the literature that the bacterial balance of genital tracts is directly related to the maintenance of physiological function and the development of various diseases of the reproductive system. By analyzing the changes in abundance and diversity of bacteria in the cow uterus from 1 to 35 days postpartum, the objective was to reveal the mechanism of metritis in cows and provide the basis for diagnosis, treatment and prevention of metritis in postpartum dairy cows. Uterine contents were taken from six cows (three healthy and three with metritis) on 1, 7, 14, 21 and 35 days after parturition. DNA genomes extracted from the samples were primed with 515F5'-GTGCCAGCMGCCGCGG-3' and 907R5'-CCGTCAATTCMTTRAGTTT-3' for PCR amplification of the V4+V5 regions of the 16S rDNA genes and construction of a gene library. The sequence of the bacterial structure of the cow uterine contents was analyzed using 16S rDNA high-throughput sequencing technology. A total of 30 samples were tested by PCR, and 29 samples qualified. The results of cluster analysis showed that except for one sample, the number of OTUs in the healthy cows was above 200, while in the cows with metritis, except for three samples, OTUs were below 200. The Chao1 and Shannon indices showed that the abundance of bacteria in the cow uterus was lower than that of healthy cows. Analysis of the relative abundance of bacteria in the cow uterus showed that there were six phyla present, including Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria and Tenericutes. There were 10 dominant genera in healthy cows, including Bacteroides, Clostridium sensu stricto 1, Escherichia-Shigella, Fusobacterium, Halomonas, Helcococcus, Porphyromonas, Prevotella 6, Rikenellaceae RC9 gut group and Streptococcus. There were nine dominant genera in cows with metritis, including Bacteroides, Caviibacter, Clostridium sensu stricto 1, Falsiporphyromonas, Fusobacterium, Halomonas, Helcococcus, Porphyromonas and Prevotella 7. Phylogenetic tree analysis showed that uterine contents from 29 samples could be separated into two clusters. Eleven samples from the cows with metritis were clustered with one sample from the healthy group, and 13 samples from the healthy cows were clustered together with four samples from the metritis group. Principal co-ordinate analysis showed that the points representing healthy cows and those representing the metritis group were concentrated in two distinct regions, which shows that there were significant differences in the structure evolution between healthy cows and cows with metritis. The above results indicate that bacterial diversity declines with time postpartum in healthy cows and is lower in cows with metritis, with characteristic changes in the relative abundances, including increases in Bacteroidetes and Fusobacteria, decreases in Firmicutes and Proteobacteria, increases in Porphyromonas, Bacteroides and Fusobacterium, and a decrease in Clostridium sensu stricto 1.

Dnajb8, a target gene of SOX30, is dispensable for male fertility in mice.

BACKGROUND: The DNAJ family of molecular chaperones maintains protein homeostasis in mitotic and postmeiotic cells, especially germ cells. Recently, we found that the transcription factor SOX30 initiates transcription of Dnajb8 during late meiosis and spermiogenesis in mouse testes. METHODS: We used the CRISPR/Cas9 system to generate Dnajb8 mutant mice and analyze the phenotype of the Dnajb8 mutants. RESULTS: Although Dnajb8 is an evolutionarily conserved gene, it is not essential for spermatogenesis and male fertility. We provide this phenotypic information, which could prevent duplicative work by other groups.

Biological and RNA regulatory function of MOV10 in mammalian germ cells.

BACKGROUND: RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells. RESULTS: MOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3'-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites. CONCLUSIONS: These data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis.

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, underlining the importance of post-transcriptional regulation of gene expression orchestrated by RBPs. To elucidate mechanistic roles of an RBP, crosslinking immunoprecipitation (CLIP) methodology can be used to capture its endogenous direct RNA targets and define the actual interaction sites. The enhanced CLIP (eCLIP) is a newly-developed method that offers several advantages over the conventional CLIPs. However, the use of eCLIP has so far been limited to cell lines, calling for expanded applications. Here, we employed eCLIP to study MOV10 and MOV10L1, two known RNA-binding helicases, in mouse testis. As expected, we find that MOV10 predominantly binds to 3' untranslated regions (UTRs) of mRNA and MOV10L1 selectively binds to Piwi-interacting RNA (piRNA) precursor transcripts. Our eCLIP method allows fast determination of major RNA species bound by various RBPs via small-scale sequencing of subclones and thus availability of qualified libraries, as a warrant for proceeding with deep sequencing. This study establishes an applicable basis for eCLIP in mammalian testis.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes.

Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids. Sox30 mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most Sox30 mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including Tnp1, Hils1, Ccdc54 and Tsks These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.

Characterization of the cervical bacterial community in dairy cows with metritis and during different physiological phases.

For the development of disease prevention and intervention strategies, a better understanding of the dynamics and interactions within cervical bacterial communities in both healthy cows and cows with metritis is required. Understanding the complexity and ecology of microorganisms in the vagina of dairy cows with metritis and during different physiological phases is critical for developing strategies to balance microorganism content. To gain deeper insight into fluctuations within the cervical microbiota, swab samples were collected from 40 Holstein dairy cows, and16S rDNA amplicon sequencing was used to analyze cervical bacterial diversity. Meanwhile, vaginal bacterial composition was analyzed during different physiological phases, including the formative (CF), gestational (CG), and postpartum (CP) stages, and in cows with metritis (CM). The results revealed a complex profile with extensive differences in the cervical bacterial composition. A total of 678,043clean 16S rDNA V4-V6 reads were gained, and 1877 Operational Taxonomic Units (OTUs) were observed after calculation. At both the phylum and genus levels, the top 10 bacteria by percentage were the same when comparing the CF, CG, and CP groups of cows, with some variation in abundance. At the phylum level, the cervical microbial community in the CF, CG, and CP groups included mainly Firmicutes, which accounted for 39.3%, 48.3%, and 49.6% of the total microbial composition of each group, respectively. However, the cervical bacterial community in the CM group consisted of mostly Bacteroidetes, which accounted for 72.6% of the total microbial composition. The second major bacterial community in the CF and CG groups of cows was Proteobacteria, which accounted for 28.3%and 30.1% of the total microbial compositions of these groups, respectively, while the second major bacterial community in the CP group was Bacteroidetes (23.5%). However, in the CM group, the second major bacterial community was Fusobacteria, which accounted for18.0% of the total microbial composition. At the genus level, the cervical bacterial community in the CM group of cows was dominated by Porphyromonas(44.4%) and Fusobacterium(12.1%), while Porphyromonas accounted for only 1.3%, 1.1%, and 1.4% of the total microbial compositions of the CF, CG, and CP groups, respectively. Likewise, Fusobacterium accounted for 2.3%, 0.7%, and 4.7% of the total microbial compositions of the CF, CG, and CP groups, respectively. The results demonstrate that cervical bacterial diversity decreases in cows with metritis and that the predominant bacterial genera are Porphyromonas and Fusobacterium. Cervical bacterial diversity was rich in all observed physiological phases, and the predominant bacterial phylum was Firmicutes. Pregnancy had little effect on the cervical bacterial community; however, there were increases in the abundances of pathogenic species in postpartum cows. Cervical bacterial diversity decreased in cows with metritis, however, due to the highly dynamic and complex course of metritis, the relationship between cervical bacterial diversity and metritis requires further investigation.