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A suitable additive for fish sperm storage in vitro is necessary for artificial reproduction. In this study, we evaluated the effects of different concentrations (100, 200, 400, and 800 µmol/L) of metformin (Met) on Schizthorax prenanti and Onychostoma macrolepis sperm under storage in vitro for 72 h. Compared with the control group, 400 µmol/L Met was more effective at improving the quality and fertilization capacity of Schizothorax prenanti sperm by increasing the adenosine triphosphate (ATP) content within the sperm. Further study found that metformin stabilized the ATP level by enhancing the glucose uptake in Schizothorax prenanti sperm, and this effect might be associated with the activation of AMP-activated protein kinase (AMPK) in sperm. In this study, we also found that the glucose could be absorbed by the sperm of Schizothorax prenanti, which was mainly accumulated in the midpiece of Schizothorax prenanti sperm, where mitochondria were located. In addition, Compound C(CC) significantly inhibited the beneficial effects of Met on the quality and glucose uptake capacity of Schizothorax prenanti sperm by inhibiting AMPK phosphorylation. These results revealed that AMPK played an important role in vitro sperm storage, and Met maintained ATP content, and increased the storage time of Schizothorax prenanti sperm in vitro for 72 h, possibly due to Met enhanced glucose uptake capacity of sperm by activating AMPK. Similarly, the beneficial effects of Met on Schizothorax prenanti sperm were also found in O. macrolepis sperm, suggesting that Met may hold great promise for the practice of storing fish in vitro.

Chinese forest musk deer (FMD,Moschus berezovskii) glands have the ability to secrete musk, which plays an important role in attracting females during the breeding season. Sebaceous glands (SGs) are exocrine skin glands associated with hair follicles that continuously release a mixture of sebum, lipids and cellular debris, by holocrine secretion. Both the musk glands and the skin tissues of the FMD contain abundant sebaceous glands, andSox9, Caveolin1, andAndrogen receptor(AR) are all involved in the regulation of sebum secretion by the sebaceous glands. However, there are fewer studies on the correlation between skin and musk glands and the expression ofSox9, Caveolin1, andARin the musk glands and skin tissue of FMD. To address this gap, we analyzed biochemical data from FMD skin tissues and musk glands using transcriptomic data, hematoxylin-eosin (HE) staining, western blotting (WB), immunohistochemistry (IHC), tissue dissection, and RT-qPCR. Anatomical results show that only adult male FMD had complete glandular part and sachets, while 4-month-old FMD do not have well-developed sachets. Transcriptomic data showed that 88.24% of genes were co-expressed in the skin and musk glands tissues of FMD. The WB, IHC, and RT-qPCR results showed that the genes involved in regulating sebum secretion,Sox9, Caveolin1, andARwere expressed in the skin tissues and musk glands. In summary, skin tissues and musk glands tissue have a strong correlation, andSox9, Caveolin1, andARmay play important roles in skin tissues and musk glands tissue.

Epididymal specific proteins play a crucial role in sperm maturation. Some of the post-translational modified proteins are transported from the caput to the cauda of the epididymis through exosomes which regulate the function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expressed specifically in the caput epididymis, localizes on the sperm; however, the regulatory ways in which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce the fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at the 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes.

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle-associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

Sperm are susceptible to excessive reactive oxygen species (ROS). Spermine and spermidine are secreted in large amounts by the prostate and potent natural free radical scavengers and protect cells against redox disorder. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. Seven mature and fertile Duroc boars (aged 15 to 30 mo) were used in this study. In experiment 1, spermine and spermidine (3.6 ± 0.3 and 3.3 ± 0.2 mmol/L, respectively) were abundant in seminal plasma, and the content of polyamine decreased (P0.05) after preservation at 17 °C for 7 d or incubation at 37 °C for 6 h. In experiment 2, using labeling of spermine or spermidine by conjugation with fluorescein isothiocyanate and ultra-high-performance liquid chromatography, we found that the accumulation of spermine or spermidine in sperm was inhibited by quinidine and dl-tetrahydropalmatine (THP, organic cation transporters [OCT] inhibitors, P0.05), but not mildronate and l-carnitine (organic cation/carnitine transporter [OCTN] inhibitors, P0.05). In experiment 3, the addition of spermine or spermidine (0.5 mmol/L) in the extender resulted in higher motility, plasma membrane and acrosome integrity, and lower ROS level after preservation in vitro at 17 °C for 7 d (P0.05). In experiment 4, in the condition of oxidative stress (treatment with H2O2 at 37 °C for 2 h), the addition of spermine (1 mmol/L) or spermidine (0.5 mmol/L) in extender increased activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase; reduced glutathione and oxidized glutathione ratio (P0.05); and alleviate oxidative stress-induced lipid peroxidation, DNA damage, mitochondrial membrane potential (ΔΨm) decline, adenosine triphosphate depletion, and intracellular calcium concentration ([Ca2+]i) overload (P0.05), thereby improving boar sperm motility, the integrity of plasma membrane and acrosome (P0.05) in vitro. These data suggest that spermine and spermidine alleviate oxidative stress via the antioxidant capacity, thereby improving the efficacy of boar semen preservation.Boar semen preservation and artificial insemination are widely used in the pig industry. Although preservation in vitro prolongs sperm lifespan, reactive oxidative species (ROS) also accumulate in sperm with the increased preservation period. ROS over-accumulation would impair motility, the integrity of plasma membrane and acrosome, mitochondrial function, and eventually lead to infertility. Spermine and spermidine are secreted in large amounts by the prostate and are potent natural free radical scavengers. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. We found for the first time that organic cation transporters mediated polyamines uptake in sperm cells, and that extracellular polyamines decreased during preservation in vitro. The addition of polyamines increased the activities of glutathione-related antioxidant enzymes and reduced glutathione and oxidized glutathione ratio, and alleviate oxidative stress-induced mitochondrial dysfunction, lipid peroxidation, and DNA damage, thereby maintaining sperm quality in vitro. These data suggest that spermine and spermidine alleviate oxidative stress, thereby improving the efficacy of boar semen preservation.

The Onychostoma macrolepis (O. macrolepis) is a rare and endangered wild species. Their endangered extinction might be due to their low fertility. To further illustrate the molecular mechanism of gonad development of the male and female O. macrolepis, the present study carried out de novo testicular and ovarian transcriptome sequencing. By comparing ovary and testis, 30,869 differentially expressed unigenes (9870 in female, 20999 in male) were identified. In addition, KEGG and GO analysis suggested that the Hedgehog signaling pathway have important roles in testis maintenance and spermatogenesis, whereas the Hippo signaling pathway and Wnt signaling pathway are likely to participate in ovary maintenance. RT-qPCR analysis results were consistent with transcriptome sequencing that all of gender differentiation-related genes (FOXL2, GDF9, WNT4, CYP19A1, SOX9 and GATA4), temperature-enriched genes (NOVA1, CTGF and NR4A1), clock-related genes (PER2, PER3, CRY1, CRY2, BMAL1 and CIPC) were significantly differential expression in testis compared with ovaries. Taken together, these results revealed a potential molecular mechanism that low fertility of the O. macrolepis might strong correlate with the gonadal dyssynchrony development of the male and female, which might provide theoretical basis and technical support for artificial reproduction and germplasm resource protection of the O. macrolepis.

The late overwintering period and breeding period are two important developmental stages of testis in Onychostoma macrolepis. Small non-coding RNAs (sncRNAs) are well-known regulators of biological processes associated with numerous biological processes. This study aimed to elucidate the roles of four sncRNA classes (microRNAs [miRNAs], Piwi-interacting RNAs [piRNAs], tRNA-derived small RNAs [tsRNAs], and rRNA-derived small RNAs [rsRNAs]) across testes in the late overwintering period (in March) and breeding period (in June) by high-throughput sequencing. The testis of O. macrolepis displayed the highest levels of piRNAs and lowest levels of rsRNAs. Compared with miRNAs and tsRNAs in June, tsRNAs in March had a higher abundance, while miRNAs in March had a much lower abundance. Bioinformatics analysis identified 1,362 and 1,340 differentially expressed miRNAs and tsRNAs, respectively. Further analysis showed that miR-200-1, miR-143-1, tRFi-Lys-CTT-1, and tRFi-Glu-CTC-1 could play critical roles during the overwintering and breeding periods. Our findings provided an unprecedented insight to reveal the epigenetic mechanism underlying the overwintering and reproduction process of male O. macrolepis.

Cell division cycle 25A (CDC25A), a member of the CDC25 family of phosphatases, is required for progression from G1 to the S phase of the cell cycle. CDC25A provides an essential function during early embryonic development in mice, suggesting that it plays an important role in growth and development. In this study, we used mathematical expectation (ME) methods to identify a 20-bp insertion/deletion (indel) polymorphism of CDC25A gene in Shaanbei White Cashmere (SBWC) goats. We also investigated the association between this 20-bp indel and growth-related traits in SBWC goats. Association results showed that the indel was related to growth traits (height at hip cross, cannon circumference, and cannon circumference index) in SBWC goats. The height at hip cross of individuals with insertion/insertion (II) genotype was higher than those with insertion/deletion (ID) genotype (P=0.02); on the contrary, the cannon circumference and cannon circumference index of individuals with ID genotype were superior when compared with those with II genotype (P=0.017 and P=0.009). These findings suggest that the 20-bp indel in the CDC25A gene significantly affects growth-related traits, and could be utilized as a candidate marker for marker-assisted selection (MAS) in the cashmere goat industry.

e-polylysine (e-PL) has potent antibacterial effects and is often used in the food industry. However, no studies have clarified the antibacterial effects of e-PL during storage of boar semen. In this study, boar semen samples were diluted with BTS buffer supplemented with different concentrations (0, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28 g/L) of e-PL and different combinations of e-PL plus gentamicin during liquid storage at 17 °C for 5 days. Bacterial concentrations, bacterial community compositions, sperm quality parameters, and in vitro fertilization (IVF) were evaluated in order to analyze the antibacterial effects of these parameters during boar semen preservation. The results indicated that the optimum concentration of e-PL was 0.16 g/L, which significantly improved sperm quality parameters, including sperm motility, plasma membrane integrity, mitochondrial membrane potential, and acrosome integrity, and changed bacterial proliferation and composition (P

Intramuscular fat (IMF), which is modulated by the number and size of intramuscular preadipocytes, plays a key role in pork quality. DNA (cytosine-5)-methyltransferase 3A (DNMT3A), an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, is involved in the management of diverse intracellular processes. However, the physiological functions of DNMT3A in proliferation and differentiation of porcine intramuscular preadipocytes have not been clearly established. Here, we found that DNMT3A significantly promoted the proliferation, while inhibited the differentiation of intramuscular preadipocytes. We demonstrated that overexpression of DNMT3A promoted the expression of cell proliferation markers but significantly decreased the expression of p21 to repress cell proliferation by the methylation of p21 promoter. Moreover, overexpression of DNMT3A decreased lipid accumulation and significantly down-regulated the levels of adipogenic marker genes including PPARg (Peroxisome proliferator-activated receptor gamma), SREBP-1c (Sterol regulatory element-binding protein 1c), and aP2 (FABP4, fatty acid binding protein 4) through the methylation of PPARg promoter. The blocking effect of DNMT3A on adipogenesis can be rescued by rosiglitazone treatment. Collectively, these findings illustrated the essential role of DNMT3A in the proliferation and differentiation of porcine intramuscular preadipocytes, and provide a potential target to improve pork quality.

Melatonin (MLT) is a well-known antioxidant and can improve sperm quality during storage in many species. In the present study, the effects of different concentration of MLT (0.1, 0.5, 1 and 5 μM) on paddlefish sperm quality and ATP content in sperm were evaluated during semen storage at 4 °C. Compared with other groups, 0.5 μM MLT supplement in semen could be more effective to improve the plasma membrane integrity, maintain the steady reactive oxygen species (ROS) level and inhibit decrease of mitochondrial membrane potential of the non-activated sperm during whole storage. To the activated sperm, the total motility, progressive motility, beat cross frequency of sperm in semen with 0.5 μM MLT was significant higher (p

The Onychostoma macrolepis (O. macrolepis) is a rare and endangered fishery species inhabiting the river of Qinling Mountains and some flowing freshwaters in China. The declining population of O. macrolepis caused by asynchrony of male and female development prompted us to focus on genetic regulation of its reproduction. In this study, high-throughput RNA-sequencing technology was applied to assemble and annotate the transcriptome of O. macrolepis testis and ovary. The results showed that a number of 338089335 (ovary:163216500, testis:174872835) raw sequences were obtained. After non-redundant analysis, a number of 207826065 (ovary:102334008, testis:105492057) high quality reads were obtained and predicted as unigenes, in which 201,038,682 unigenes were annotated with multiple databases. Taking the ovarian transcriptome as a control, comparative transcriptome analysis showed that 9918 differentially expressed genes (DEGs) up-regulated in the testis and 13,095 DEGs down-regulated. Many DEGs were involved with sex-related GO terms and KEGG pathways, such as oocyte maturation, gonadal development, steroid biosynthesis pathways, MAPK signaling pathway and Wnt signaling pathway. Finally, the expression patterns of 19 unigenes were validated by using quantitative real-time polymerase chain reaction (qRT-PCR). This study illustrates a potential molecular mechanism on the unsynchronized male and female development of the O. macrolepis during the reproduction period in June and provides a theoretical basis for future artificial reproduction.

BackgroundSpermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and juxtacrine testicular cross-talk . Sertoli cells (SCs) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors. However, many of the molecular pathways of SCs action remain controversial and unclear. Recently, research has focused on exosomes as an important mechanism of intercellular communication. ResultsW e found that the exosomes derived from SCs ( SC-Exos ) significantly inhibited the apoptosis of the primary spermatogonial stem cells (SSCs). Total of 1016 miRNAs in SCs and 556 miRNAs in SC-Exos were detected using microRNA (miRNA) high-throughput sequencing. Further, 294 miRNAs were differentially expressed between SCs and SC-Exos. Based on the GO and KEGG analyses, the target genes of 37 (high-expressed in exosomes and RPM>1000) selected miRNAs were involved in multiple biological pathw ays, including the MAPK signaling pathway and PI3K/AKT signaling pathway. And miR-10b is one of the top ten exosomes with relatively large enrichment of microRNA. In addition, the overexpression of miR-10b down-regulated expression of the target KLF4 to reduce spermatogonial apoptosis in SSCs or C18-4 cell line. ConclusionsThe study indicated a large number of small RNAs loaded in exosomes was secreted form the donor SCs to target spermatogonial regulated the apoptosis. And miR-10b inhibits the apoptosis of spermatogonia through the target gene KLF4.

The development of the ovary of Onychostoma macrolepis undergoes distinct annual cyclic changes in which small non-coding RNAs (sncRNAs) could play vital roles. In this study, four sncRNA classes in the ovary, including miRNA, piRNAs, tsRNA, and rsRNAs, were systematically profiled by high-throughput sequencing. In adult ovaries of O. macrolepis, 247 miRNAs and 235 tsRNAs were identified as differentially expressing in the late overwintering period (in March) and breeding period (in June). Some up-regulated sncRNAs in March, such as miR-125-1 and tRFi-Lys-CTT-1, could be involved in inhibiting biomolecule metabolism and enhancing stress tolerance during the overwintering period. Compared with the level expression of sncRNAs in March, some sncRNAs were up-regulated in June, such as miR-146-1 and tRFi-Gly-GCC-1, and could be involved in influencing molecular synthesis and metabolism, enhancing oocyte proliferation and maturation, accelerating ovarian development, and increasing fertilization of oocytes by regulating related target mRNAs. The results suggested that sncRNAs in the ovary of Onychostoma macrolepis not only reflect characteristics of the fish's physiology at different developmental periods, but also directly affect ovarian development and oocyte maturation during the breeding period. In conclusion, these results significantly advance our understanding of the roles of sncRNA during overwintering and reproduction periods, and provide a novel perspective for uncovering characteristics of the special overwintering ecology and reproductive physiology of an atypical cavefish.

Musk secreted by Chinese forest musk deer (FMD; Moschus berezovskii) is a highly valuable ingredient in the fields of perfumery and medicine, and the main factor affecting the production of musk is the androgen level of male FMD. To clarify whether the musk gland of FMD can synthesize androgen, we compared and analyzed the expression patterns of steroid hormone biosynthesis-related genes in the musk gland and testis of FMD by RNA-seq and RT-qPCR. We obtained 33,308 and 38,602 unigenes from the musk gland and testis, respectively, and 26,780 co-expressed unigenes. Analysis of co-expressed genes revealed that 12,647 genes were annotated to 11,484 Gene Ontology terms and 10,941 genes were annotated to 6120 pathways, including several pathways important in metabolic and synthetic activity. Next, 21 steroid hormone synthesis-related genes were screened from the transcriptome of the musk gland of 4-month-old FMD. The expression levels of three key genes of steroid hormone biosynthesis (CYP11A1, CYP17A1, and HSD3B) in the musk gland differed from their expression levels in the testis based on RT-qPCR. Furthermore, immunohistochemistry indicated that CYP11A1, CYP17A1, and HSD3B were localized in the glandular tubular columnar cells of the musk gland. These results suggested that the musk gland of male FMD has the potential to locally synthesize steroid hormone and thus plays a critically important role in musk secretion.

Melatonin (MLT) is an efficient antioxidant that protects spermatozoa against damages caused by oxidative stress. In this study, to maintain good function of Onychostoma macrolepis spermatozoa during semen preservation invitro at 4°C, different concentrations of MLT (0.5, 1 and 2μM) were added to the semen. After storage (0, 24, 48 and 72h), 1μM MLT in semen markedly improved sperm quality, as reflected by better plasma membrane integrity, the relative steady level of reactive oxygen species (ROS) and slower rate of decrease in mitochondrial membrane potential. Activated spermatozoa in semen with 1μM MLT had higher kinematic performance (i.e. percentage of motile and progressive spermatozoa and the beat cross frequency; P

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2′-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G(2)-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3′-UTR of serum response factor (SRF) and Wnt1 mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of SRF and Wnt1 during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by SRF overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear β-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/β-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/β-catenin signaling pathways in myogenesis.

Chlorpyrifos (CPF) is the most frequently applied insecticide. Aside from effects on the neuronal cholinergic system, previous studies suggested a potential relationship between CPF exposure and male infertility; however, the molecular mechanism remains elusive. The aim of this study was to investigate the toxic effect of CPF on testicular cells and the potential mechanism via in vitro and in vivo experiments. The cytotoxic effects of CPF on mouse-derived spermatogonial cell lines (GC-1), Sertoli cell lines (TM4) and Leydig cell lines (TM3) were assessed by a CCK-8 assay, flow cytometry, a TUNEL assay, quantitative RT-PCR, and Western blotting. Exposure to CPF (10-50 μM) for 12 or 24 h resulted in significant death in all three testicular cell lines. The number of TUNEL-positive apoptotic cells were dose-dependent and increased with raised CPF concentrations. Further investigation indicated that CPF induced cell-cycle arrest and then promoted cell apoptosis. Additionally, CPF increased reactive-oxygen-species (ROS) production and lipid peroxidation (MDA) and reduced mitochondrial-membrane potential. The mechanism of cell apoptosis induced by CPF involved an increase in phosphorylated-AMP-activated-protein-kinase (p-AMPK) levels in the tested cells. In vivo, the expression of steroid-hormone-biosynthesis-related genes in testis, spleen, and lung in F0 and F1 mice were downregulated when there was intraperitoneal injection or dietary supplementation of CPF. This study provides a potential molecular mechanism of CPF-induced toxicity in testicular cells and a theoretical basis for future treatment of male infertility.

This study focuses on the effect of outer membrane vesicles (OMVs) in gram-negative bacteria on boar sperm function during in vitro storage. In the 40 ejaculates collected from Guangzhong Black boar, six gram-negative bacterial species were detected by 16S rDNA sequencing, of which Proteus mirabilis was the main contaminating bacterium. The OMVs of P. mirabilis were isolated by gradient ultracentrifugation. To reveal the effect of OMVs on boar sperm, different OMV concentrations were added to the Modena medium during sperm storage at 17 °C. Even after 3 days of storage, it was noted that low OMV dose (5 μg/mL) in the extender did not significantly reduce sperm quality as compared with that in the control semen samples; however, sperm motility and sperm morphology were significantly altered in the extender owing to a high OMV dose (10 μg/mL). The relative ROS level successively increased with OMV dose in sperm samples and storage time. Meanwhile, OMVs dramatically elevated the mitochondrial potential of sperm. OMVs could bind with the sperm membrane to further influence the capacity of sperm-oocyte binding; they also increased the expression of LC3 and caspase 3 and decreased that of anti-apoptosis-related protein, Bcl2, in sperm. It was concluded that OMVs of P. mirabilis influenced the function of boar sperm by inducing sperm membrane reconstruction as well as autophagy and apoptosis of sperm.

Abnormal embryos of Chinese giant salamander (Andrias davidianus, CGS) were observed in an imitating ecologic breeding system at 20°C. The aim of the present study was to investigate the relationship of bacterial infection on the early embryonic development of CGS. The ratio of abnormal embryos at 10 days after embryos incubated was 35.0% ± 2.1%, 35.6% ± 2.3% and 34.7% ± 3.4% in six breeding system farms of Hanzhong city in the years 2014, 2015, and 2016, respectively. However, in the standard imitating ecology breeding mesocosm, the proportion of abnormal embryos was about 5%. Six bacteria species in the egg water of the early-dead embryos and eight bacteria species in the gut of healthy CGSs were detected and identified by PCR and 16S rRNA gene sequence homology analysis. All bacteria species in the egg water were also found in the cloaca contents. Cetobacterium somerae and Hafnia alvei, which individually can cause embryo death, were first isolated from egg water and gut of CGSs. Further, the egg jelly membrane and the egg water of embryos did not inhibit bacteria survival and the bacteria could individually lead to CGS larva death. These results suggest that bacteria in the eggs of CGS may derive from the gut and that high-velocity flow of water through nest may decrease bacterial infection of egg in the imitating ecologic culture system.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), which is extensively expressed in vertebrates, is a deubiquitinating enzymes that inhibits the degradation of proteins by reversing ubiquitination modification. Herein, a 1087-bp sequence encoding UCHL1 was identified from the Chinese giant salamander (CGS; Andrias davidianus). The coding sequences (CDS) of UCHL1 encoded a putative poly peptide of 222 amino acids. The CGS UCHL1 isoforms were more related to their human and mouse counterparts. The phylogenic tree of vertebrate UCHL1 indicated that CGS UCHL1 has the closest relationship with human UCHL1 (up to 73.99 %). Before the gonads of male CGSs matured, the peak level of UCHL1 expression in testes appeared in 3-year-old CGSs according to RT-qPCR and western blot. In adult testes, the level of UCHL1 protein was lower in the breeding period than in the post-breeding period, whereas the level of UCHL1 protein in interstitial fluid of adult CGS testes was higher during the breeding period than during the post-breeding period. In testicular seminiferous lobules in the developmental stage of CGSs, immunohistochemistry displayed three kinds of localizing patterns of UCHL1, including nuclear localization at half year old, cytoplasmic localization from one year to three years old, and extracellular localization in adult. In testicular seminiferous lobules of adult CGS, the different developmental germ cells were separated by cysts containing UCHL1 protein, but UCHL1 did not localize on the mature sperm. The results showed that extracellular UCHL1 loaded on exosomes, as a component of the homogeneous germ cell cysts, could regulate the synchronous development of sperm in testes of adult CGS.

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle–associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

Hydroxysteroid 17-Beta Dehydrogenase 3 (Hsd17b3), primarily expressed in Leydig cells (LCs) of the mammalian testes, is essential for testosterone biosynthesis and male fertility. The aim of our study was to profile the expression, splice variants (SV) and novel insertion/deletion (indel) of Hsd17b3 in boars. Quantitative analysis showed that the expression level of Hsd17b3 in the testis was significantly highest. Among different testicular cell types, the Hsd17b3 mRNA expression level of LCs was significantly higher than that of SSCs (spermatogonial stem cells) and SCs (Sertoli cells). Furthermore, the SV was firstly identified in pigs and it was highly expressed in LCs comparing with SSCs and SCs. In addition, two mutations were identified in pig Hsd17b3 gene promotor and intron, respectively, which were associated with male reproductive traits (P 0.05). In conclusion, both transcripts of Hsd17b3 gene were highly expressed in pig testes and LCs; the two novel indel variants of Hsd17b3 gene can be used as potential DNA makers for the marker-assisted selection in pigs. All these findings would enrich the study of Hsd17b3 gene in pig genetic breeding.

Cell division cycle 25A (CDC25A), a member of the CDC25 family of phosphatases, is required for progression from G1 to the S phase of the cell cycle. CDC25A provides an essential function during early embryonic development in mice, suggesting that it plays an important role in growth and development. In this study, we used mathematical expectation (ME) methods to identify a 20-bp insertion/deletion (indel) polymorphism of CDC25A gene in Shaanbei White Cashmere (SBWC) goats. We also investigated the association between this 20-bp indel and growth-related traits in SBWC goats. Association results showed that the indel was related to growth traits (height at hip cross, cannon circumference, and cannon circumference index) in SBWC goats. The height at hip cross of individuals with insertion/insertion (II) genotype was higher than those with insertion/deletion (ID) genotype (P=0.02); on the contrary, the cannon circumference and cannon circumference index of individuals with ID genotype were superior when compared with those with II genotype (P=0.017 and P=0.009). These findings suggest that the 20-bp indel in the CDC25A gene significantly affects growth-related traits, and could be utilized as a candidate marker for marker-assisted selection (MAS) in the cashmere goat industry.

Skeletal muscle is the dominant executant in locomotion and regulator in energy metabolism. Embryonic myogenesis and postnatal muscle growth are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms. MicroRNAs (miRNAs), a family of non-coding RNA of 22 nucleotides in length, post-transcriptionally regulates expression of mRNA by pairing the seed sequence to 3' UTR of target mRNA. Increasing evidence has demonstrated that miRNAs are important regulators in diverse myogenic processes. The profiling of miRNA expression revealed that miR-432 is more enriched in the longissimus dorsi of 35-day-old piglets than that of adult pigs. Our gain of function study showed that miR-432 can negatively regulate both myoblast proliferation and differentiation. Mechanically, we found that miR-432 is able to down-regulate E2F transcription factor 3 (E2F3) to inactivate the expression of cell cycle and myogenic genes. We also identified that phosphatidylinositol 3-kinase regulatory subunit (P55PIK) is another target gene of miR-432 in muscle cells. downregulation of P55PIK by miR-432 leads to inhibition of P55PIK-mediated PI3K/AKT/mTOR signaling pathway during differentiation. The blocking effect of miR-432 on this pathway can be rescued by insulin treatment. Taken together, our findings identified microRNA-432 as a potent inhibitor of myogenesis which functions by targeting E2F3 and P55PIK in muscle cells.

// Jian Du 1 , Jian Shen 1 , Yuanxian Wang 1 , Chuanying Pan 1 , Weijun Pang 1 , Hua Diao 2 and Wuzi Dong 1 1 College of Animal Science and Technology, Northwest AF boar sperm quality; capacitation; liquid storage; Pathology Section Received : May 20, 2016 Accepted : July 28, 2016 Published : August 16, 2016 Abstract Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity ( p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro . Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity.

Sex determining region Y-box 9 (SOX9), an important member of the SRY- type HMGbox (SOX) gene family, plays an important role in the regulation of mammalian reproduction, including sex differentiation during the embryonic development stage and spermatogenesis after birth. To explore the roles of polymorphism and expression of the SOX9 gene in the development of testes, we analyzed the indel of SOX9 in pigs and the corresponding expression level of the SOX9 gene in 7-day and 5-month-old porcine Sertoli cells. Results revealed that the DD haplotype of SOX9 gene as well as the ID genotype were significantly associated with larger testicular weight, while the II haplotype was closely related to the smaller testicular weight. More importantly, the SOX9 gene expression of ID genotyped group was significantly higher than that in II genotyped group. Our results first revealed that the indel polymorphism and expression of SOX9 were significantly associated with pig reproduction traits indicating the critical roles of SOX9 gene in testes development. The study provides a new clue for understanding the regulation of animal reproductive activities.

Chinese giant salamander iridovirus (CGSIV) is a large double-stranded DNA virus that infects Chinese giant salamanders (CGSs) and is responsible for a high mortality rate of CGSs under certain conditions. It is generally believed that CGSIV is a horizontally transmitting virus that affects lower vertebrates. Exosomes from tissues and cells affect the mechanism of viral infections. UCHL1, a deubiquitinating enzyme, is indirectly involved in virus propagation via cytokine and chemokine suppression. In our study, a few CGSIVs were detected in the testis of the special symptom CGSs using PCR and immunofluorescence analysis. The exosomes originating in the testicular fluid was isolated and identified using the Nanosight NS300 system and scanning electron microscopy. The UCHL1-loaded exosomes may resist CGSIV entry by fusing with and remodeling CGSIV. UCHL1 in the primary testicular fibroblasts was maintained at a stable level to inhibit the infection and replication of CGSIV by secreting and sorting exosomes. These data provided a new insight into CGSIV being a type of horizontally transmitting virus.

Lysine-specific demethylase 1B (KDM1B) which plays a crucial role in regulating methylation status at lysine 4 of histone 3 is important for male fertility. The aim of this study was to explore the KDM1B mRNA expression profiles and to identify novel genetic variants of the pig KDM1B gene, as well as to determine the association between these variants and testis measurement traits in male piglets. The KDM1B mRNA expression profiles indicated that this gene widely expressed in all tested organs. In addition, a novel 17-bp deletion (NC_010449.4:g.31142_31159delCATGGATAGTAGTTGCT) within KDM1B gene was found. Notably, this deletion sequence was inconsistent with the prediction by NCBI. Association analysis revealed that the 17-bp indel locus was significantly associated with the testis weight in 40-day-old Large White pigs (P

Sialic acid (SA), which usually occupies the terminal position of oligosaccharide chains in mammalian spermatozoa, has important functions in fertilization. Compared with other methods, such as lectin probing, boronic acid could recognize and bind SA with a higher affinity and specificity at pH 6.9. In this study, two boronic acid carriers, 3-aminophenylboronic acid-labeled fluorescent latex (CML-APBA) and magnetic beads (CMM-APBA were applied to explore surface sialylation profile and sialoglycoproteins of the boar sperm. There are three binding sections of CML-APBA on the head of ejaculated sperm: acrosomal region, equatorial segment and the head posterior, which are the major regions undergoing sialylation. After capacitationin vitro, two major binding patterns of CML-APBA exists on sperm head. On some spermatozoa, sialylation exists on the equatorial segment and the posterior head, whilst on other spermatozoa, sialylation occurs on the acrosomal region and equatorial segment. Flow cytometry analysis suggested that the level of sialylation on boar sperm membrane decreases after capacitation. Furthermore, using CMM-APBA, we pulled down sialylated proteins from spermatozoa. Among them, two decapacitation factors associating on sperm surface, AWN and PSP-1, were identified. The levels of the two proteins reduced during capacitation, which might contribute to the decrease of sialylation on boar sperm surface.

The outbreak of Chinese Giant Salamander (Andrias davidianus, CGS) Iridovirus (CGSIV) caused massive death of CGSs. However, some CGSs with low level of CGSIV usually could survive. In our study, major capsid protein (MCP) DNA replicates of CGSIV in shedding skin were employed to assess the relative content of CGSIV in the living CGSs by qPCR. Furthermore, the examinations of autophagy and apoptosis in CGSs in vivo and in the primary renal cells in vitro were performed, respectively. The results showed that the relative contents of CGSIV in the shedding skin could reflect those in liver, spleen, and kidney of the CGSs. In these tissues of the CGSs with low-level replicates of CGSIV, there were not obviously macroscopic lesions. But the irregularly-shaped vesicles perhaps involving in autophagosome were observed by transmission electron microscopy (TEM). The LC3B protein displayed uneven distribution by Immunohistochemistry and the level mRNA of Atg5 was higher in these tissues than that in the tissues of healthy CGSs using qRT-PCR. Meanwhile, the apoptosis also appeared in these tissues by TUNEL staining and higher level mRNA of type I IFN were detected in these tissues using qRT-PCR. Further, both the expression level of LC3B II protein and Atg5 mRNA increased significantly at 2h after the virus infected the primary renal cells from the health CGSs in vitro. In addition, apoptosis and type I IFN mRNA began to increase significantly at 4h after the virus infected the renal cells. It was suggested that autophagy may be a pivotal role for survival of CGSIV in the CGSs during early infection and the rapid proliferation of CGSIV could be inhibited by innate immune response and apoptosis.

The aim of this study was to characterise the molecular structure of the oestrogen receptor ERα and to evaluate the effect of bisphenol A (BPA) on ERα expression during sexual development of the Chinese giant salamander (Andrias davidianus). The ERα cDNA of A. davidianus includes an open reading frame of 1755bp (encoding 584 amino acids), a 219-bp 5′ untranslated region (UTR) and a 611-bp 3′UTR. A polyadenylation signal was not found in the 3′UTR. Amino acid sequence analysis showed high homology between ERα of A. davidianus and that of other amphibians, such as Andrias japonicas (99.66% identity) and Rana rugose (81.06% identity). In 3-year-old A. davidianus, highest ERα expression was observed in the liver and gonads. During different developmental stages in A. davidianus (from 1 to 3 years of age), ERα expression in the testes increased gradually. ERα was localised in the epithelial cells of seminiferous lobules and in interstitial cells. ERα-positive cells were more abundant in the interstitial tissue during testicular development. ERα was located in the nucleus of oocytes during ovary development. We found that the sex of 6-month-old A. davidianus larvae could not be distinguished anatomically. The sex ratio did not change after larvae were treated with 10μM BPA for 1 month. However, BPA treatment reduced bodyweight and ERα expression in the gonads in male larvae.

Pluripotent mesenchymal stem-like cell lines were established from lungs of 3-4 months old aborted fetus. The cells present the high ex vivo expansion potential of MSC, a typical fibroblast-like morphology and proliferate up to 15 passages without displaying clear changes in morphology. Immunological localization and flow cytometry analyses showed that these cells are positive for OCT4, c-Kit, CD11, CD29, CD44, telomerase, CD106, CD105, CD166, and SSEA1, weakly expression or negative for SSEA1, SSEA3, SSEA4, CD34, CD105 and CD106. These cells can give rise to the adipogenic as evidenced by accumulation of lipid-rich vacuoles within cells identified by Oil-red O when they were induced with 0.5 mM isobutylmethylxanthine, 200 microM indomethacin, 10(-6)M dexamethasone, and 10 microg/ml of insulin in high-glucose DMEM. Osteogenic lineage cells were generated in 0.1 microM dexamethasone, 50 microg/ml ascorbic acid, 10 mM beta-glycerophosphate, which are shaped as the osteoblastic morphology, expression of alkaline phosphatase (AP), and the formation of a mineralized extracellular matrix identified by Alizarin Red staining. Neural cells are observed when the cultures were induced with 2-mercapometal, which are positive for nestin, NF-100, MBP and GFAP. Additionally, embryoid bodies (EBs) and sperm like cells are obtained in vitro differentiation of these lung MSCs induced with 10(-5)M retinoic acid (RA). These results demonstrated that these MSCs are pluripotent and may provide an in vitro model to study germ-cell formation and also as a potential source of sperms for male infertility.

Mesenchymal stem cells derived from bone marrow are a well characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. Reports have shown that murine and human embryonic stem cells can differentiate into primordial germ cells and then to early gametes. Evidence has indicated that some adult stem cells also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of human mesenchymal stem cells into germ cells. This study investigated the ability of retinoic acid and testicular extracts to induce human bone marrow stem cells (hBMSC) to differentiate into male germ cells. It was found that a small population of hBMSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers OCT4, STELLA, NANOG and VASA, and male germ-ceil-specific markers such as DAZL, TH2, c-kit, beta(1)-integrin, ACR, PRMl, FSHR, STRA8 and SCP3, as analysed by reverse transcription-polymerase chain reaction and immunohistochemistry. These results demonstrated that hBMSC may differentiate into male germ cells and the same could be used as a potential source of cells for reproductive toxicological studies.

To the Editor: The Chinese giant salamander (Andreas davidianus) is one of the world’s largest amphibian species and is often referred to as a living fossil. They primarily inhabit drainage basins of the Yangtze River, the Yellow River, and the Pearl River in the People’s Republic of China (1). Because of habitat loss, pollution, and overharvesting, the population of wild Chinese giant salamanders has dropped sharply (2,3). As a result, the Chinese giant salamander is artificially farmed in mesocosms for research and conservation. The mesocosms (ambient temperature 96% homology with the corresponding sequences of the 5 iridovirus strains. Additionally, neighbor-joining tree analysis showed that the virus was clustered in 1 lineage with frog virus 3, soft-shelled turle iridovirus, and tiger frog virus (Technical Appendix Figure 2 ). These results suggest that the high mortality rates in Chinese giant salamanders were caused by a virus in the iridovirus family. The iridoviruses are carried in the bodies of vertebrates such as gopher tortoises (Gopherus polyphemus) (5), Chinese forest frogs (Rana dybowskii) (6), and fish (7,8). Iridoviruses are thought to be transmitted horizontally in lower vertebrates such as bullfrogs (7,9,10). In addition, some iridovirus infections may be chronic or conditional (7). In this study, the virus was isolated from the liver and spleen of 30 sick (n = 7) or dead (n = 23) salamanders that were farmed in ditch mesocosms, where ambient temperatures were unusually high (>25°C) at the time of the epidemic. Although the virus also was isolated from animals living in cooler cave mesocosms (ambient temperature 25°C) and the associated stress on the animals may have increased disease in ditch-dwelling Chinese giant salamanders. This seems particularly likely, given the absence of clinical signs of disease in infected salamanders that lived in the cooler cave mesocosms (i.e.

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male's life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitro cell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2 was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.