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Vitamin C (Ascorbic acid, AA), as vital micro-nutrient, plays an essential role for male animal reproduction. Previously, we showed that vitamin C reprogrammed the transcriptome and proteome to change phenotypes of porcine immature Sertoli cells (iSCs). Here, we used LC-MS-based non-targeted metabolomics to further investigate the metabolic effects of vitamin C on porcine iSCs. The results identified 43 significantly differential metabolites (DMs) (16 up and 27 down) as induced by vitamin C (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) treatment of porcine iSCs, which were mainly enriched in steroid related and protein related metabolic pathways. ELISA (Enzyme-Linked ImmunoSorbent Assay) showed that significantly differential metabolites of Dehydroepiandrosterone (DHEA) (involved in steroid hormone biosynthesis) and Desmosterol (involved in steroid degradation) were significantly increased, which were partially consistent with metabolomic results. Further integrative analysis of metabolomics, transcriptomics and proteomics data identified the strong correlation between the key differential metabolite of Dehydroepiandrosterone and 6 differentially expressed genes (DEGs)/proteins (DEPs) (HMGCS1, P4HA1, STON2, LOXL2, EMILIN2 and CCN3). Further experiments validated that HMGCS1 could positively regulate Dehydroepiandrosterone level. These data indicate that vitamin C could modulate the metabolism profile, and HMGCS1-DHEA could be the pathway to mediate effects exerted by vitamin C on porcine iSCs., Competing Interests: Conflict of interest The authors declare that there are no conflicts of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chloroquine (CQ) is widely used in the therapy against malarial, tumor and recently the COVID-19 pandemic, as a lysosomotropic agent to inhibit the endolysosomal trafficking in the autophagy pathway. We previously reported that CQ (20 μM, 36 h) could reprogram transcriptome, and impair multiple signaling pathways vital to porcine immature Sertoli cells (iSCs). However, whether CQ treatment could affect the metabolomic compositions of porcine iSCs remains unclear. Here, we showed that CQ (20 μM, 36 h) treatment of porcine iSCs induced significant changes of 63 metabolites (11 up and 52 down) by the metabolomics method, which were involved in different metabolic pathways. Caffeic acid and esculetin, the top two up-regulated metabolites, were validated by ELISA. The combined analysis of metabolomics and transcriptome showed caffeic acid and esculetin to be highly correlated with multiple differentially expressed genes (DEGs), including Ndrg1, S100a8, Sqstm1, S100a12, S100a9, Ill1, Lif, Ntn4 and Peg10. Furthermore, esculetin treatment (53 nM, 36 h) significantly decreased the viability and proliferation, suppressed the mitochondrial function, whereas promoted the apoptosis of porcine iSCs, similar to those by CQ treatment (20 μM, 36 h). Collectively, our results showed that CQ treatment induces metabolic changes, and its effect on porcine iSCs could be partially mediated by esculetin., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2024 Elsevier Ltd. 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.)

Identification of stably expressed gene(s) as internal reference(s) for different experimental conditions is key to the accurate normalization and quantification of target transcripts. Previously, our RNA-seq study showed that Hprt1, Actb, and 18S rRNA abundances were all significantly altered in porcine immature Sertoli cells (iSCs) during acute heat stress (HS). In the current study, we aimed to identify stable reference gene(s) to study the gene expression dynamics of quick and delayed responses after acute HS treatment of porcine iSCs. A total of six genes previously used in pig testis or Sertoli cells (Hprt1, Top2b, Actb, Rpl32, Gapdh, and 18S rRNA) were chosen to perform RT-qPCR for the control (before acute HS), HS0.5 (acute HS at 43°C for 0.5 h), and HS0.5-R36 (36 h recovery following acute HS) groups. The stability of candidate reference genes was examined by the GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods, and RefFinder to obtain the final rank. Rpl32 and Actb were the two most stable internal reference genes as found by all methods, whereas Hprt1 and 18S rRNA were the two most unstable as ranked by RefFinder. Moreover, expression of six target mRNAs (Ccn1, Ccnb1, Eif4g1, Hdac6, Plk2, and Ptma) was normalized using Rpl32, Actb, or the combination of Rpl32 and Actb, respectively. Therefore, our findings that the most suitable internal references are Rpl32 and Actb provide useful information for further functional investigation on genes regulating the acute HS of porcine iSCs., (© 2022 Wiley-VCH GmbH.)

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.)

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.)