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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. • CQ treatment reduced viability of porcine iSCs in a dose-dependent manner. • CQ treatment (20 μM, 36h) elevated oxidative stress, damaged mitochondrial function and promoted apoptosis of porcine iSCs. • Melatonin (MT) (10 nM, 36h) could partially rescue CQ-induced phenotypic defects. • RNA-seq also confirmed that MT could partially rescue CQ-altered gene expression. • RT-qPCR confirmed that the expression of most selected DEGs were consistent with RNA-seq results. [ABSTRACT FROM AUTHOR]

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. • HSP90AA1 over-expression in porcine iSCs promoted cell viability via increasing the PLK2 expression. • HSP90AA1 over-expression in porcine iSCs elevated lactate production, but suppressed the secretion of AMH and estradiol. • Inhibition of HSP90AA1 by 17-AAG treatment of porcine iSCs reduced cell viability and promoted cell apoptosis. • HSP90AA1 inhibition suppressed lactate production, but boosted the secretion of AMH and estradiol. [ABSTRACT FROM AUTHOR]

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 m6A) 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 Zfp36l 1) 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. • AA could promote the proliferation, suppress apoptosis, and decrease the global nucleic acid mthylation levels of iSCs. • AA treatment changed mRNA and lncRNA profiles of iSCs. • AA treatment significantly disturbed the expression of mRNAs (such as Tfcp2l1, Hmgcs1, Mmp7, Fndc3a, and Zfp36l1) involved in reproductive process. • AA supplement could promote the secretion of anti-mullerian hormone, inhibin B and lactate, and the activity of lactate dehydrogenase as well. [ABSTRACT FROM AUTHOR]

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. • Acute heat stress (HS) on porcine iSCs could reduce cell viability, promote apoptosis and increase lactate production. • HS could initiate immediate changes of heat shock-related genes. • Prolonged response to HS involved pathways of cell cycle, apoptosis, reproductive process and Glycolysis/Gluconeogenesis. • Validation experiment confirmed the consistent expression pattern of selected DE mRNAs and HSPs. [ABSTRACT FROM AUTHOR]