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The branched-chain amino acids (BCAAs: leucine, isoleucine and valine) are essential for animal growth and metabolic health. However, the effect of valine on male reproduction and its underlying molecular mechanism remain largely unknown. Here, we showed that l-valine supplementation (0.30% or 0.45%, water drinking for 3 weeks) did not change body and testis weights, but significantly altered morphology of sertoli cells and germ cells within seminiferous tubule, and enlarged the space between seminiferous tubules within mouse testis. l-valine treatment (0.45%) increased significantly the Caspase3/9 mRNA levels and CASPASE9 protein levels, therefore induced apoptosis of mouse testis. Moreover, gene expression levels related to autophagy (Atg5 and Lamb3), DNA 5 mC methylation (Dnmt1, Dnmt3a, Tet2 and Tet3), RNA m6A methylation (Mettl14, Alkbh5 and Fto), and m6A methylation binding proteins (Ythdf1/2/3 and Igf2bp1/2) were significantly reduced. Protein abundances of ALKBH5, FTO and YTHDF3 were also significantly reduced, but not for ATG5 and TET2. Testis transcriptome sequencing detected 537 differentially expressed genes (DEGs, 26 up-regulated and 511 down-regulated), involved in multiple important signaling pathways. RT-qPCR validated 8 of 9 DEGs (Cd36, Scd1, Insl3, Anxa5, Lcn2, Hsd17b3, Cyp11a1, Cyp17a1 and Agt) to be decreased significantly, consistent with RNA-seq results. Taken together, l-valine treatment could disturb multiple signaling pathways (autophagy and RNA methylation etc.), and induce apoptosis to destroy the tissue structure of mouse testis., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Alternative polyadenylation (APA) affects the composition of cis-elements in 3'-untranslated region (3'-UTR), to regulate gene expression and localization, and subsequently the downstream biological processes. Acute heat stress could change rapidly the cellular transcriptome, however the underlying molecular changes are less explored. Here, we systematically catalogued the global 3'-UTRome dynamics, by analyzing our previously reported transcriptome sequencing data of porcine immature Sertoli (iST) cells before (Control group), acute heat stress treatment at 43 °C for 0.5h (HS0.5 group), and 36h recovery culture (HS0.5-R36h group) after acute heat stress treatment. After three group comparisons (HS0.5 vs. Control, HS0.5-R36 vs. HS0.5, and HS0.5-R36 vs. Control), DaPars (dynamic analysis of alternative polyadenylation) identified 639, 464 and 290 mRNAs, and APAtrap (a tool to identify APA sites and detect changes of APA site usage) identified 713, 518 and 321 mRNAs, with significantly different 3'-UTRs (Padj.≤0.05), respectively. These genes with different 3'-UTR patterns were mainly enriched in P53, glycolysis/gluconeogenesis, HIF-1, apoptosis, PI3K-Akt and AMPK signaling pathways. Further analysis identified that average 3'-UTR lengths of Acss2, Inpp1 and Nr1h4 were more than 140 nt longer (HS0.5-R36 vs. HS0.5), and contained different cis-elements (PAS, CPE and microRNA binding sites). Moreover, Hsp70.2, Inhbb and Dhrs were identified to have extremely different 3'-UTR abundances. Further 3'RACE assays validated several 3'-UTRs of Nr1h4, and RT-qPCR confirmed the abundance changes of different 3'-UTR isoforms for Nr1h4 and Hsp70.2. Our findings provide useful information and resources to further uncover the molecular role of 3'-UTR, in regulating the response of porcine iST cells to acute heat stress., Competing Interests: Declaration of competing interest The authors declare that there are no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Heat shock protein 90 (HSP90), as a molecular chaperone, regulates hundreds of protein clients under both physiological and stress conditions in eukaryotic cells. However, the functional role of HSP90 in mammalian male reproduction remains largely unknown. Here, we aimed to investigate the function and effect of HSP90AA1 on the basic and reproductive function of pig immature Sertoli cells (iSCs). We first confirmed that the transfection of pBI-CMV3-HSP90AA1 vector into porcine iSCs for 24 h significantly increased mRNA and protein levels of HSP90AA. Moreover, HSP90AA1 over-expression significantly increased cell viability and the PLK2 mRNA abundance, promoted lactate production via elevating the LDHA activity, and inhibited the secretion of anti-Mullerian hormone and estradiol. In comparison, HSP90AA inhibition by allylamino-17-demethoxygeldanamycin (17-AAG) (2 μM) treatment of pig iSCs for 36 h had a totally contrasting effect, i.e. significantly reduced cell viability, promoted cell apoptosis via modulating expression of genes related to cell cycle and apoptosis (CCNB1, CCN1, PLK2, PTMA, YBX3 and CASP3), suppressed lactate production via dropping LDHA activity, but increased the secretion of anti-Mullerian hormone and estradiol. Taken together, our findings demonstrated that HSP90AA1 could regulate positively cell viability and lactate production, but negatively the secretion of reproductive hormones (anti-Mullerian hormone and estradiol). However, the detailed molecular mechanism of HSP90AA1 remains to be investigated., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Somatic Sertoli cells constitute the microenvironment and produce essential substances, to support male germ cell development and maturation in testis. We previously found that ascorbic acid treatment of porcine immature Sertoli cells enhances its proliferation and secretion of reproductive hormones and metabolites, and reprograms the global transcriptome. Proteomics is a powerful tool to systematically profile the underlying protein changes. Here, by employing the TMT-based quantitative proteomics method, we identified 96 and 64 significantly up- and down-regulated proteins in porcine immature Sertoli cells treated by ascorbic acid, respectively. Gene enrichment (GO and KEGG) and protein-protein interaction analyses revealed important molecular pathways (dioxygenase activity, sterol biosynthetic process, PI3K-Akt, negative regulation of peptide hormone secretion, extracellular matrix etc.). Further validation of three proteins, HMGCS1 (cholesterol synthesis), P4HA1 (glycolysis) and KDM5A (demethylation of histone 3 at lysine 4), confirmed their significant differential abundance, respectively. Taken together, our findings show that ascorbic acid can alter multiple important protein molecules and related signaling pathways, which could explain partially phenotypic changes (proliferation, apoptosis, nucleic acid methylation, lactate and reproductive hormone secretion) of porcine immature Sertoli cells as induced by ascorbic acid., Competing Interests: Declaration of competing interest The authors declare that there are no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sertoli cells (SCs), the only somatic constituent of the testicular seminiferous epithelium, are vital to spermatogenesis. We previously found that vitamin C (ascorbic acid, AA) can reprogram the transcriptome, and promote the proliferation and reproductive function of pig immature SCs (iSCs). However, the global change of microRNAs (miRNA) expression and its effect on pig iSCs as induced by vitamin C treatment is still unknown. Here, we performed small RNA sequencing on pig iSCs after 250 μM AA2P (l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate) treatment for 36h. Total number of detected miRNAs ranged from 326 to 335 known, and 400-570 novel miRNAs. Of the top ten highly expressed miRNAs, we found that 8 were common (miR-21-5p, let-7i-5p, miR-30a-5p, let-7f-5p, let-7g, miR-100, miR-10a-5p and miR-30d), which were predicted to target mRNAs involved in cell development and differentiation. We identified 78 differentially expressed (DE) miRNAs (|log2 (Fold Change)|≥1; Padj.<0.05), including 7 known and 71 novel miRNAs. We further selected 13 highly and stably expressed DE miRNAs (4 up-regulated: miR-184, novel-miR-610, novel-miR-316 and novel-miR-1274; 9 down-regulated: miR-222, miR-221-5p, miR-221-3p, miR-210, miR-146b, miR-146a-5p, novel-miR-182, novel-miR-1088 and novel-miR-1016), and performed integrated analysis on the miRNA-mRNA regulatory network. DE mRNAs negatively targeted by these 13 DE miRNAs were enriched in multiple GO and KEGG signaling pathways (e.g. pyruvate and steroid metabolic processes, developmental process in reproduction, response to oxidative stress, Glycolysis/Gluconeogenesis and HIF-1). We validated 8 DE miRNAs and their 12 DE mRNA targets, most of them showed expression patterns consistent with (mi)RNA-seq results. Taken together, our findings demonstrate that vitamin C could induce the global change of miRNAs, which possibly regulate cell proliferation, energy metabolism and male reproduction as induced by vitamin C treatment on pig iSCs., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Chloroquine (CQ) could function as a lysosomotropic agent to inhibit the endolysosomal trafficking in the autophagy pathway, and is widely used on malarial, tumor and recently COVID-19. However, the effect of CQ treatment on porcine immature Sertoli cells (iSCs) remains unclear. Here we showed that CQ could reduce iSC viability in a dose-dependent manner. CQ treatment (20 μM) on iSCs for 36h could elevate oxidative stress, damage mitochondrial function and promote apoptosis, which could be partially rescued by melatonin (MT) (10 nM). Transcriptome profiling identified 1611 differentially expressed genes (DEGs) (776 up- and 835 down-regulated) (20 μM CQ vs. DMSO), mainly involved in MAPK cascade, cell proliferation/apoptosis, HIF-1, PI3K-Akt and lysosome signaling pathways. In contrast, only 467 (224 up- and 243 down-regulated) DEGs (CQ + MT vs. DMSO) could be found after MT (10 nM) addition, enriched in cell cycle, regulation of apoptotic process, lysosome and reproduction pathways. Therefore, the partial rescue effects of MT on CQ treatment were confirmed by multiple assays (cell viability, ROS level, mitochondrial function, apoptosis, and mRNA levels of selected genes). Collectively, CQ treatment could impair porcine iSC viability by deranging the signaling pathways related to apoptosis and autophagy, which could be partially rescued by MT supplementation., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sertoli cells, important constituents of the somatic niche, supports the growth and development of spermatogonia. Heat stress (HS), among multiple intrinsic and external factors, can induce physiological and biochemical changes in Sertoli cells. However, the underlying molecular mechanism remains largely unclear. Here, we showed that acute heat stress (43 °C, 0.5 h) could reduce cell viability, promote apoptosis, and increase the lactate production of porcine immature Sertoli cells (iSCs) cultured in vitro. Then, transcriptome sequencing identified 126 immediately and 3372 prolonged responded differentially expressed genes (DEGs) after acute heat stress (43 °C, 0.5 h) (HS0.5), and 36 h recovery culture following heat stress (HS0.5-R36), respectively. Enrichment analyses found different signaling pathways: immediate changes including cell response to heat, regulation of cellular response to stress, heat shock protein binding, chaperon-mediated protein folding, and sterol biosynthetic process, but prolonged changes mainly involving cell cycle, regulation of apoptotic process/cell proliferation, reproductive process, P53, PI3K-Akt and Glycolysis/Gluconeogenesis. Furthermore, transcriptional patterns of 9 DEGs (Dnajb1, Traf6, Insig1, Gadd45g, Hdac6, Fkbp4, Serpine1, Pfkp and Galm), and 6 heat shock proteins (HSPs) (Hspa6, Hspb1, Hspd1, HSP90aa1, HSP90ab1 and Hsph1) were validated, as well as the protein pattern of HSP90AA1 via immunostaining and western blot. Taken together, heat stress could initiate immediate changes of heat shock-related genes, and reprogram transcriptome and signaling pathways affecting the viability, apoptosis and metabolite production of pig iSCs., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Coenzyme Q10 (CoQ10) is essential to many fundamental biological processes. However, the effect of CoQ10 on meiotic maturation of pig oocytes still remains elusive. In the present study we aimed to understand the effects of CoQ10 on porcine oocyte maturation, by supplementing different concentrations of CoQ10 (25, 50 and 100 μM) into the maturation medium. We showed that CoQ10 at 50 μM had better capacity to promote the nuclear maturation of pig oocytes derived from both small and large antral follicles. Though the cleavage and blastocyst rates of parthenotes stayed stable, 50 μM CoQ10 treatment could accelerate the development of parthenotes to blastocyst stage, and increase the average cell number of blastocyst. For cumulus-oocyte complexes from large antral follicles categorized by the brilliant cresyl blue (BCB) test, 50 μM CoQ10 treatment could specifically promote the nuclear maturation of poor-quality oocytes in the BCB-negative group. Mitochondrial function of oocytes treated by 50 μM CoQ10 could be boosted, through increasing the levels of mitochondrial membrane potential, ATP production and CoQ6, and changing the pattern of mitochondrial distribution as well. Moreover, 50 μM CoQ10 treatment suppressed the level of reactive oxygen species and reduced the percentage of oocytes with early apoptosis signal. Taken together, CoQ10 could improve the meiotic maturation of pig oocytes, especially for poor-quality oocytes, mainly through enhancing mitochondrial function and suppressing oxidative stress to reduce apoptosis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Vitamin C (ascorbic acid, AA) can regulate antioxidation and affect many cellular processes. However, the effect of AA on the reproduction of male animals remains less explored. Here, we showed that by supplementing exogenous AA to porcine immature Sertoli cells (iSCs), AA could promote the proliferation, suppress apoptosis, and decrease the global nucleic acid methylation (5 mC and m 6 A) levels of iSCs. After we profiled mRNA and long non-coding RNA (lncRNA) expression by transcriptome sequencing on iSCs (treated by 250 μM AA for 36 h), 1232 mRNAs and 937 lncRNAs were identified to be differentially expressed (DE). Gene enrichment analysis found multiple significantly enriched biological pathways, including oxidoreductase activity, cell proliferation and apoptosis, regulation of hormone level, regulation of catalytic activity, developmental process, ATP metabolism and reproductive process. Specifically, for the reproductive process, 49 up- and 36 down-regulated DE mRNAs (including highly expressed genes, such as Tfcp2l1, Hmgcs1, Mmp7, Fndc3a, and Zfp36l1) are involved. Moreover, AA supplementation could promote the secretion of anti-müllerian hormone, inhibin B and lactate, and enhance the activity of lactate dehydrogenase as well. Taken together, AA could promote the reproductive function of pig iSCs, potentially through reprogramming the global transcriptome, and elevating hormone secretion and metabolite production., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)