The significance of single-cell transcriptome analysis in epididymis research.

As a crucial component of the male reproductive system, the epididymis plays multiple roles, including sperm storage and secretion of nutritive fluids for sperm development and maturation. The acquisition of fertilization capacity by sperm occurs during their transport through the epididymis. Compared with the testis, little has been realized about the importance of the epididymis. However, with the development of molecular biology and single-cell sequencing technology, the importance of the epididymis for male fertility should be reconsidered. Recent studies have revealed that different regions of the epididymis exhibit distinct functions and cell type compositions, which are likely determined by variations in gene expression patterns. In this research, we primarily focused on elucidating the cellular composition and region-specific gene expression patterns within different segments of the epididymis and provided detailed insights into epididymal function in male fertility.

Induced differentiation of primordial germ cell like cells from SOX9+ porcine skin derived stem cells.

Understanding the mechanisms behind porcine primordial germ cell like cells (pPGCLCs) development, differentiation, and gametogenesis is crucial in the treatment of infertility. In this study, SOX9+ skin derived stem cells (SOX9+ SDSCs) were isolated from fetal porcine skin and a high-purity SOX9+ SDSCs population was obtained. The SOX9+ SDSCs were induced to transdifferentiate into PGCLCs during 8 days of cultured. The results of RNA-seq, western blot and immunofluorescence staining verified SDSCs have the potential to transdifferentiate into PGCLCs from aspects of transcription factor activation, germ layer differentiation, energy metabolism, and epigenetic changes. Both adherent and suspended cells were collected. The adherent cells were found to be very similar to early porcine primordial germ cells (pPGCs). The suspended cells resembled late stage pPGCs and had a potential to enter meiotic process. This SDSCs culture-induced in vitro model is expected to provide suitable donor cells for stem cell transplantation in the future.

Aflatoxin B1 affects porcine alveolar macrophage growth through the calcium signaling pathway mediated by the ceRNA regulatory network.

BACKGROUND: Aflatoxin B1 (AFB1), one of the most prevalent contaminants in human and animal food, impairs the immune system, but information on the mechanisms of AFB1-mediated macrophage toxicity is still lacking. METHODS AND RESULTS: In this study, for the first time, we employed whole transcriptome sequencing technology to explore the molecular mechanism by which AFB1 affects the growth of porcine alveolar macrophages (PAM). We found that AFB1 exposure reduced the proliferative capacity of PAM and prevented cell cycle progression. Based on whole transcriptome analysis, RT-qPCR, ICC and RNAi, we verified the role and regulatory mechanism of the competing endogenous RNA (ceRNA) network in the process of AFB1 exposure affecting the growth of PAM. CONCLUSIONS: We found that AFB1 induced MSTRG.43,583, MSTRG.67,490, MSTRG.84,995, and MSTRG.89,935 to competitively bind miR-219a, miR-30b-3p, and miR-30c-1-3p, eliminating the inhibition of its target genes CACNA1S, RYR3, and PRKCG. This activated the calcium signaling pathway to regulate the growth of PAM. These results provide valuable information on the mechanism of AFB1 exposure induced impairment of macrophage function in humans and animals.

Identification of unique transcriptomic signatures through integrated multispecies comparative analysis and WGCNA in bovine oocyte development.

BACKGROUND: Cattle (Bos taurus) are a major large livestock, however, compared with other species, the transcriptional specificity of bovine oocyte development has not been emphasised. RESULTS: To reveal the unique transcriptional signatures of bovine oocyte development, we used integrated multispecies comparative analysis and weighted gene co-expression network analysis (WGCNA) to perform bioinformatic analysis of the germinal follicle (GV) and second meiosis (MII) gene expression profile from cattle, sheep, pigs and mice. We found that the expression levels of most genes were down-regulated from GV to MII in all species. Next, the multispecies comparative analysis showed more genes involved in the regulation of cAMP signalling during bovine oocyte development. Moreover, the green module identified by WGCNA was closely related to bovine oocyte development. Finally, integrated multispecies comparative analysis and WGCNA picked up 61 bovine-specific signature genes that participate in metabolic regulation and steroid hormone biosynthesis. CONCLUSION: In a short, this study provides new insights into the regulation of cattle oocyte development from a cross-species comparison.

Aflatoxin B1 disrupts testicular development via the cell cycle-related Ras/PI3K/Akt signaling in mice and pig.

Aflatoxins B1 (AFB1), a type I carcinogen widely present in the environment, not only poses a danger to animal husbandry, but also poses a potential threat to human reproductive health, but its mechanism is still unclear. To address this question, multi-omics were performed on porcine Sertoli cells and mice testis. The data suggest that AFB1 induced testicular damage manifested as decreased expression of GJA1, ZO1 and OCCLUDIN in mice (p < 0.01) and inhibition of porcine Sertoli cell proliferation. Transcriptomic analysis suggested changes in noncoding RNA expression profiles that affect the cell cycle-related Ras/PI3K/Akt signaling pathway after AFB1 exposure both in mice and pigs. Specifically, AFB1 caused abnormal cell cycle of testis with the characterization of decreased expressions of CCNA1, CCNB1 and CDK1 (p < 0.01). Flow cytometry revealed that the G2/M phase was significantly increased after AFB1 exposure. Meanwhile, AFB1 downregulated the expressions of Ras, PI3K and AKT both in porcine Sertoli cell (p < 0.01) and mice testis (p < 0.01). Metabolome analysis verified the alterations in the PI3K/Akt signaling pathway (p < 0.05). Moreover, the joint analysis of metabolome and microbiome found that the changes of metabolites were correlated with the expression of flora. In conclusion, we have demonstrated that AFB1 impairs testicular development via the cell cycle-related Ras/PI3K/Akt signaling.

Naringin regulates intestinal microorganisms and serum metabolites to promote spermatogenesis.

Naringin (NAR) is a dihydroflavonoid with various biological activities and pharmacological effects, especially natural antioxidant activity. To gain a better understanding of the effects of NAR on the reproductive system, especially spermatogenesis, we employed western blotting, immunofluorescence, immunohistochemistry, metabolomics and microbiomics to comprehensively dissect the impact of NAR on spermatogenesis. NAR promotes germ cell proliferation and testicular development, and promotes the secretion of sex hormones. Microbiomic and metabonomic analysis showed that NAR improved intestinal microflora and cooperated with serum metabolites to regulate spermatogenesis. Therefore, NAR is beneficial for male reproduction by regulating intestinal microorganisms and serum metabolism.

Identification of nucleotide polymorphisms in the key promoter region of chicken annexins A2 gene associatied with egg laying traits.

Annexin A2 (ANXA2) is a member of the A subfamily of a multifunctional calcium dependent membrane phospholipid binding protein family. The mRNA expression of ANXA2 is consistent with ovary function and egg laying in chickens. In this study, six nucleotide polymorphisms in the key promoter region of chicken ANXA2 gene (-2861 bp to -1394 bp), i.e.,: g.-2337 indel (GT), g.-2255 C > T, g. -2248 A > G, g.-2188 A > G, g.-2169 G > A, g.-2160 A > C, were identified. Their distributions in populations of Xinyang Brown, Recessive White Rock, Wenchang and Wenshang Barred chickens were analyzed. In the Recessive White Rock chicken population, CAA, CAG and TGG were three major haplotypes. Association analysis indicated that the individuals with diplotype TGG/TGG laid more eggs at 32 weeks, and the individual with diplotype CAG/TGG laid at the earlier age. Luciferase activity assay showed that mutation from C to T at -2255 increased trascriptional activity of chicken ANXA2, which is consistent with its effect on egg laying traits.

Cryopreservation of porcine skin-derived stem cells using melatonin or trehalose maintains their ability to self-renew and differentiate.

Porcine skin-derived stem cells (pSDSCs) are a type of adult stem cells (ASCs) that retain the ability to self-renew and differentiate. Currently, pSDSCs research has entered an intense period of development; however there has been no research regarding methods of cryopreservation. In this paper, we explored an efficient cryopreservation method for pSDSCs. Our results demonstrated that cryopreserving 50 µm diameter pSDSCs aggregates resulted in a lower apoptosis rate and a greater ability to proliferate to form larger spherical cell aggregates than during single-cell cryopreservation. To further optimize the cryopreservation method, we added different concentrations of melatonin (N-acetyl-5-methoxytryptamine, MLT) and trehalose (d-trehalose anhydrous, TRE) to act as cryoprotectants (CPAs) for the pSDSCs. After comparative experiments, we found that the cryopreservation efficiency of 50 mM TRE was superior. Further experiments demonstrated that the reason why 50 mM TRE improved cryopreservation efficiency was that it reduced the intracellular oxidative stress and mitochondrial damage caused by cryopreservation. Taken together, our results suggest that cryopreserving 50 µm diameter pSDSCs aggregates in F12 medium with 10% dimethyl sulfoxide (DMSO) and 50 mM TRE promotes the long-term storage of pSDSCs.

Comparative Transcriptomics Uncover the Uniqueness of Oocyte Development in the Donkey.

The donkey is an important domestic animal, however the number of donkeys world-wide is currently declining. It is therefore important to protect their genetic resources and to elaborate the regulatory mechanisms of donkey reproduction, particularly, oocyte development. Here, we adopted comparative transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) to uncover the uniqueness of donkey oocyte development compared to cattle, sheep, pigs, and mice, during the period from germinal vesicle (GV) to metaphase II (MII). Significantly, we selected 36 hub genes related to donkey oocyte development, including wee1-like protein kinase 2 (WEE2). Gene Ontology (GO) analysis suggested that these genes are involved in the negative regulation of cell development. Interestingly, we found that donkey specific differentially expressed genes (DEGs) were involved in RNA metabolism and apoptosis. Moreover, the results of WGCNA showed species-specific gene expression patterns. We conclude that, compared to other species, donkey oocytes express a large number of genes related to RNA metabolism to maintain normal oocyte development during the period from GV to MII.

YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/ß-catenin signaling pathway.

Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of SDSCs are a hot topic. In this paper, we explore the link between the transcriptional regulator yes-associated protein (YAP) and the fate of porcine SDSCs (pSDSCs). We found that lysophosphatidylcholine (LPC) activates YAP, promotes pSDSCs pluripotency, and counteracts transdifferentiation of pSDSCs into porcine primordial germ cell-like cells (pPGCLCs). YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Oct4 and Sox2. The overexpression of YAP prevented the differentiation of pSDSCs, and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/ß-catenin signaling pathway. When an activator of the Wnt/ß-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP, the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939, an inhibitor of the Wnt/ß-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Taken together, our results suggested that YAP and the Wnt/ß-catenin signaling pathway interact to regulate the fate of pSDSCs.

The proliferation role of LH on porcine primordial germ cell-like cells (pPGCLCs) through ceRNA network construction.

BACKGROUND: The transdifferentiation of skin-derived stem cells (SDSCs) into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells research in recent years. This technology provides a new theoretical basis for the treatment of human infertility. However, the transdifferentiation efficiency of SDSCs to PGCLCs is very low, and scientists are still exploring ways to improve this efficiency or promote the proliferation of PGCLCs. This study aims to investigate the molecular mechanism of luteinising hormone (LH) to enhance porcine PGCLCs (pPGCLCs) proliferation. RESULTS: In this study, we dissected the proliferation regulatory network of pPGCLCs by whole transcriptome sequencing, and the results showed that the pituitary-secreted reproductive hormone LH significantly promoted the proliferation of pPGCLCs. We combined whole transcriptome sequencing and related validation experiments to explore the mechanism of LH on the proliferation of pPGCLCs, and found that LH could affect the expression of Hippo signalling pathway-related mRNAs, miRNAs and lncRNAs in pPGCLCs. CONCLUSIONS: For the first time, we found that LH promotes pPGCLCs proliferation through the competing endogenous RNA (ceRNA) regulatory networks and Hippo signalling pathway. This finding may help to elucidate the molecular mechanism by which LH promotes pPGCLCs proliferation.

Establishment of lncRNA-mRNA network in bovine oocyte between germinal vesicle and metaphase II stage.

Long non-coding RNA (lncRNA), a type of non-protein coding transcripts with lengths exceeding 200 nucleotides, is reported to be widely involved in many cellular and developmental processes. However, few roles of lncRNA in oocyte development have been defined. In this study, to uncover the effect of lncRNA during oocyte maturation, bovine germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes underwent RNA sequencing. Results revealed a wealth of candidate lncRNAs, which might participate in the biological processes of stage-specific oocytes. Furthermore, their trans- and cis-regulatory effects were investigated in-depth by using bioinformatic software. Functional enrichment analysis of target genes showed that these lncRNAs were likely involved in the regulation of many key signaling pathways during bovine oocyte maturation from GV to MII stage, as well as multiple lncRNA-mRNA networks. One novel lncRNA (MSTRG.19140) was particularly interesting, as it appeared to mediate the regulation of oocyte meiotic resumption, progesterone-mediated oocyte maturation, and cell cycle. Therefore, this study enhanced insights into the regulation of molecular mechanisms of bovine oocyte maturation from a lncRNA-mRNA network perspective.