Exploring novel function of Gpx5 antioxidant activity: Assisting epididymal cells secrete functional extracellular vesicles.

Glutathione peroxisomal-5 (Gpx5) promotes the elimination of H2O2 or organic hydrogen peroxide, and plays an important role in the physiological process of resistance to oxidative stress (OS). To directly and better understand the protection of Gpx5 against OS in epididymal cells and sperm, we studied its mechanism of antioxidant protection from multiple aspects. To more directly investigate the role of Gpx5 in combating oxidative damage, we started with epididymal tissue morphology and Gpx5 expression profiles in combination with the mouse epididymal epithelial cell line PC1 (proximal caput 1) expressing recombinant Gpx5. The Gpx5 is highly expressed in adult male epididymal caput, and its protein signal can be detected in the sperm of the whole epididymis. Gpx5 has been shown to alleviate OS damage induced by 3-Nitropropionic Acid (3-NPA), including enhancing antioxidant activity, reducing mitochondrial damage, and suppressing cell apoptosis. Gpx5 reduces OS damage in PC1 and maintains the well-functioning extracellular vesicles (EVs) secreted by PC1, and the additional epididymal EVs play a role in the response of sperm to OS damage, including reducing plasma membrane oxidation and death, and increasing sperm motility and sperm-egg binding ability. Our study suggests that GPX5 plays an important role as an antioxidant in the antioxidant processes of epididymal cells and sperm, including plasma membrane oxidation, mitochondrial oxidation, apoptosis, sperm motility, and sperm-egg binding ability.

Paternal obesity induces subfertility in male offspring by modulating the oxidative stress-related transcriptional network.

BACKGROUND/OBJECTIVE: The effects of fathers' high-fat diet (HFD) on the reproductive health of their male offspring (HFD- F1) remain to be elucidated. Parental obesity is known to have a negative effect on offspring fertility, but there are few relevant studies on the effects of HFD-F1 on reproductive function. METHODS: We first succeeded in establishing the HFD model, which provides a scientific basis in the analysis of HFD-F1 reproductive health. Next, we assessed biometric indices, intratesticular cellular status, seminiferous tubules and testicular transcriptomic homeostasis in HFD-F1. Finally, we examined epididymal (sperm-containing) apoptosis, as well as antioxidant properties, motility, plasma membrane oxidation, DNA damage, and sperm-egg binding in the epididymal sperm. RESULTS: Our initial results showed that HFD-F1 mice had characteristics similar to individuals with obesity, including higher body weight and altered organ size. Despite no major changes in the types of testicular cells, we found decreased activity of important genes and noticed the presence of abnormally shaped sperm at seminiferous tubule lumen. Further analysis of HFD-F1 testes suggests that these changes might be caused by increased vulnerability to oxidative stress. Finally, we measured several sperm parameters, these results presented HFD-F1 offspring exhibited a deficiency in antioxidant properties, resulting in damaged sperm mitochondrial membrane potential, insufficient ATP content, increased DNA fragmentation, heightened plasma membrane oxidation, apoptosis-prone and decreased capacity for sperm-oocyte binding during fertilization. CONCLUSION: HFD- F1 subfertility arises from the susceptibility of the transcriptional network to oxidative stress, resulting in reduced antioxidant properties, motility, sperm-egg binding, and elevated DNA damage. Schematic representation of the HFD-F1 oxidative stress susceptibility to subfertility. Notably, excessive accumulation of ROS surpasses the physiological threshold, thereby damaging PUFAs within the sperm plasma membrane. This oxidative assault affects crucial components such as mitochondria and DNA. Consequently, the sperm's antioxidant defense mechanisms become compromised, leading to a decline in vitality, motility, and fertility.

Arginine Biosynthesis Mediates Wulingzhi Extract Resistance to Busulfan-Induced Male Reproductive Toxicity.

Busulfan, an indispensable medicine in cancer treatment, can cause serious reproductive system damage to males as a side effect of its otherwise excellent therapeutic results. Its widespread use has also caused its accumulation in the environment and subsequent ecotoxicology effects. As a Chinese medicine, Wulingzhi (WLZ) has the effects of promoting blood circulation and improving female reproductive function. However, the potential effects of WLZ in male reproduction and in counteracting busulfan-induced testis damage, as well as its probable mechanisms, are still ambiguous. In this study, busulfan was introduced in a mouse model to evaluate its production of the testicular damage. The components of different WLZ extracts were compared using an untargeted metabolome to select extracts with greater efficacy, which were further confirmed in vivo. Here, we demonstrate abnormal spermatogenesis and low sperm quality in busulfan-injured testes. The WLZ extracts showed a strong potential to rehabilitate the male reproductive system; this effect was more prominent in room-temperature extracts. Additionally, both water and ethanol WLZ extracts at room temperature alleviated various busulfan-induced adverse effects. In particular, WLZ recovered spermatogenesis, re-activated arginine biosynthesis, and alleviated the increased oxidative stress and inflammation in the testis, ultimately reversing the busulfan-induced testicular injury. Collectively, these results suggest a promising approach to protecting the male reproductive system from busulfan-induced adverse side effects, as well as those of other similar anti-cancer drugs.

A comparative study of dietary amino acid patterns: unveiling growth, composition, and molecular signatures in juvenile Onychostoma macrolepis.

The wild Onychostoma macrolepis, a species under national class II protection in China, lacks a specific compound feed for captive rearing. Understanding the dietary amino acid pattern is crucial for optimal feed formulation. This study aimed to investigate the effects of the four different dietary amino acid patterns, i.e., anchovy fishmeal protein (FMP, control group) and muscle protein (MP), whole-body protein (WBP), fish egg protein (FEP) of juvenile Onychostoma macrolepis, on the growth performance, body composition, intestinal morphology, enzyme activities, and the expression levels of gh, igf, mtor genes in juveniles. In a 12-week feeding trial with 240 juveniles (3.46±0.04g), the MP group demonstrated superior outcomes in growth performance (FBW, WGR, SGR), feed utilization efficiency (PER, PRE, FCR). Notably, it exhibited higher crude protein content in whole-body fish, enhanced amino acid composition in the liver, and favorable fatty acid health indices (AI, TI, h/H) in muscle compared to other groups (P < 0.05). Morphologically, the MP and FMP groups exhibited healthy features. Additionally, the MP group displayed significantly higher activities of TPS, ALP, and SOD, along with elevated expression levels of gh, igf, mtor genes, distinguishing it from the other groups (P < 0.05). This study illustrated that the amino acid pattern of MP emerged as a suitable dietary amino acid pattern for juvenile Onychostoma macrolepis. Furthermore, the findings provide valuable insights for formulating effective feeds in conserving and sustainably farming protected species, enhancing the research's broader ecological and aquacultural significance.

Seasonal modulation of the testis transcriptome reveals insights into hibernation and reproductive adaptation in Onychostoma macrolepis.

The Onychostoma macrolepis have a unique survival strategy, overwintering in caves and returning to the river for reproduction in summer. The current knowledge on the developmental status of its testes during winter and summer is still undiscovered. We performed RNA-seq analysis on O. macrolepis testes between January and June, using the published genome (NCBI, ASM1243209v1). Through KEGG and GO enrichment analysis, we were able to identify 2111 differentially expressed genes (DEGs) and demonstrate their functions in signaling networks associated with the development of organism. At the genomic level, we found that during the overwintering phase, genes associated with cell proliferation (ccnb1, spag5, hdac7) were downregulated while genes linked to testicular fat metabolism (slc27a2, scd, pltp) were upregulated. This indicates suppression of both mitosis and meiosis, thereby inhibiting energy expenditure through genetic regulation of testicular degeneration. Furthermore, in January, we observed the regulation of autophagy and apoptosis (becn1, casp13), which may have the function of protecting reproductive organs and ensuring their maturity for the breeding season. The results provide a basis for the development of specialized feed formulations to regulate the expression of specific genes, or editing of genes during the fish egg stage, to ensure that the testes of O. macrolepis can mature more efficiently after overwintering, thereby enhancing reproductive performance.

Analysis of morphology, histology characteristics, and circadian clock gene expression of Onychostoma macrolepis at the overwintering period and the breeding period.

Fish typically adapt to their environment through evolutionary traits, and this adaptive strategy plays a critical role in promoting species diversity. Onychostoma macrolepis is a rare and endangered wild species that exhibits a life history of overwintering in caves and breeding in mountain streams. We analyzed the morphological characteristics, histological structure, and expression of circadian clock genes in O. macrolepis to elucidate its adaptive strategies to environmental changes in this study. The results showed that the relative values of O. macrolepis eye diameter, body height, and caudal peduncle height enlarged significantly during the breeding period. The outer layer of the heart was dense; the ventricular myocardial wall was thickened; the fat was accumulated in the liver cells; the red and white pulp structures of the spleen, renal tubules, and glomeruli were increased; and the goblet cells of the intestine were decreased in the breeding period. In addition, the spermatogenic cyst contained mature sperm, and the ovaries were filled with eggs at various stages of development. Throughout the overwintering period, the melano-macrophage center is located between the spleen and kidney, and the melano-macrophage center in the cytoplasm has the ability to synthesize melanin, and is arranged in clusters to form cell clusters or white pulp scattered in it. Circadian clock genes were identified in all organs, exhibiting significant differences between the before/after overwintering period and the breeding period. These findings indicate that the environment plays an important role in shaping the behavior of O. macrolepis, helping the animals to build self-defense mechanisms during cyclical habitat changes. Studying the morphological, histological structure and circadian clock gene expression of O. macrolepis during the overwintering and breeding periods is beneficial for understanding its unique hibernation behavior in caves. Additionally, it provides an excellent biological sample for investigating the environmental adaptability of atypical cavefish species.

Maslinic acid supplementation prevents di(2-ethylhexyl) phthalate-induced apoptosis via PRDX6 in peritubular myoid cells of Chinese forest musk deer.

Chinese forest musk deer (FMD), an endangered species, have exhibited low reproductive rates even in captivity due to stress conditions. Investigation revealed the presence of di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, in the serum and skin of captive FMDs. Feeding FMDs with maslinic acid (MA) has been observed to alleviate the stress response and improve reproductive rates, although the precise molecular mechanisms remain unclear. Therefore, this study aims to investigate the molecular mechanisms underlying the alleviation of DEHP-induced oxidative stress and cell apoptosis in primary peritubular myoid cells (PMCs) through MA intake. Primary PMCs were isolated and exposed to DEHP in vitro. The results demonstrated that DEHP significantly suppressed antioxidant levels and promoted cell apoptosis in primary PMCs. Moreover, interfering with the expression of PRDX6 was found to induce excessive reactive oxygen species (ROS) production and cell apoptosis in primary PMCs. Supplementation with MA significantly upregulated the expression of PRDX6, thereby attenuating DEHP-induced oxidative stress and cell apoptosis in primary PMCs. These findings provide a theoretical foundation for mitigating stress levels and enhancing reproductive capacity of in captive FMDs.

Comparative transcriptome provides insights into differentially expressed genes between testis and ovary of Onychostoma macrolepis in reproduction period.

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.

Testicular miRNAs and tsRNAs provide insight into gene regulation during overwintering and reproduction of Onychostoma 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.

Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG-derived small RNAs present in mature spermatozoa.

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.

Palmitoylated GLB1L4 transfers via exosomes to maintain sperm function in rat epididymis.

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.

sRNAPrimerDB: comprehensive primer design and search web service for small non-coding RNAs.

MOTIVATION: Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. RESULTS: The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531 306 and 2 941 669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. AVAILABILITY AND IMPLEMENTATION: sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Melatonin regulates ATP content and fertilising capacity of Onychostoma macrolepis spermatozoa by inhibiting ROS accumulation during semen storage in vitro.

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<0.05) and longer duration of sperm motility (P<0.05) compared with spermatozoa in semen withother MLT concentrations. Furthermore, 1µM MLT maintained higher ATP concentrations in spermatozoa during semen storage and significantly improved the fertilising capacity of spermatozoa after 72h semen storage compared with the other MLT concentrations. To expand wild resources of O. macrolepis, 1µM MLT can be used as a semen additive to maintain better sperm function and enhance sperm fertilising capacity in artificial insemination (AI).

MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1.

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 G2-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 ß-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/ß-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/ß-catenin signaling pathways in myogenesis.

Molecular characterisation of oestrogen receptor ERα and the effects of bisphenol A on its expression during sexual development in the Chinese giant salamander (Andrias davidianus).

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.

Exploring boar sperm sialylation during capacitation using boronic acid-functionalized beads.

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 capacitation in 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.

Bacteria in Abnormal Eggs of Chinese Giant Salamanders (Andrias davidianus) May Derive from Gut.

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.

Novel 17-bp Deletion in KDM1B Gene is Significantly Associated with Testis Weight in Male Piglet.

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 < 0.05). Furthermore, through bioinformatics analysis, transcriptional factor heat shock factor-1 could combine the 17-bp sequence. These results not only extend the genetic variations of the pig KDM1B gene but also contribute to implementing marker-assisted selection in pig breeding.

Comparative microRNAome analysis of the testis and ovary of the Chinese giant salamander.

MicroRNAs (miRNAs) are 18-24 nucleotides non-coding RNAs that regulate gene expression by post-transcriptional suppression of mRNA. The Chinese giant salamander (CGS, Andrias davidianus), which is an endangered species, has become one of the important models of animal evolution; however, no miRNA studies on this species have been conducted. In this study, two small RNA libraries of CGS ovary and testis were constructed using deep sequencing technology. A bioinformatics pipeline was developed to distinguish miRNA sequences from other classes of small RNAs represented in the sequencing data. We found that many miRNAs and other small RNAs such as piRNA and tsRNA were abundant in CGS tissue. A total of 757 and 756 unique miRNAs were annotated as miRNA candidates in the ovary and testis respectively. We identified 145 miRNAs in CGS ovary and 155 miRNAs in CGS testis that were homologous to those in Xenopus laevis ovary and testis respectively. Forty-five miRNAs were more highly expressed in ovary than in testis and 21 miRNAs were more highly expressed in testis than in ovary. The expression profiles of the selected miRNAs (miR-451, miR-10c, miR-101, miR-202, miR-7a and miR-499) had their own different roles in other eight tissues and different development stages of testis and ovary, suggesting that these miRNAs play vital regulatory roles in sexual differentiation, gametogenesis and development in CGS. To our knowledge, this is the first study to reveal miRNA profiles that are related to male and female CGS gonads and provide insights into sex differences in miRNA expression in CGS.

MicroRNA-432 targeting E2F3 and P55PIK inhibits myogenesis through PI3K/AKT/mTOR signaling pathway.

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.