chi-miR-130b-3p regulates the ZEA-induced oxidative stress damage through the KEAP1/NRF2 signaling pathway by targeting SESN2 in goat GCs.

As a dominant mycotoxin, zearalenone (ZEA) has attracted extensive attention due to its estrogen-like effect and oxidative stress damage in cells. In order to find a way to relieve cell oxidative stress damage caused by ZEA, we treated goat granulosa cells (GCs) with ZEA and did a whole transcriptome sequencing. The results showed that the expression level of Sesterin2 (SESN2) was promoted extremely significantly in the ZEA group (p < .01). In addition, our research demonstrated that SESN2 could regulate oxidative stress level in GCs through Recombinant Kelch Like ECH Associated Protein 1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The overexpression of SESN2 could reduce the oxidative damage, whereas knockdown of SESN2 would aggravate the oxidative damage caused by ZEA. What's more, microRNA (miRNA) chi-miR-130b-3p can bind to SESN2 3'-untranslated region (3'UTR) to regulate the expression of SESN2. The mimics/inhibition of chi-miR-130b-3p would have an effect on oxidative damage triggered by ZEA in GCs as well. In summary, these results elucidate a new pathway by which chi-miR-130b-3p affects the KEAP1/NRF2 pathway in GCs by modulating SESN2 expression in response to ZEA-induced oxidative stress damage.

Characterization of sheep spermatogenesis through single-cell RNA sequencing.

Spermatogenesis is an important biological process in male reproduction. The interaction between male germ cells and somatic cells during spermatogenesis, is necessary for male reproductive activities. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. Here, we present the first comprehensive, unbiased single-cell transcriptomic study of sheep spermatogenesis using 10× genomics single cell sequencing (scRNA-seq). We collected scRNA-seq data from 11 772 cells from the adult sheep testis and identified all known germ cells (including early primary spermatocytes, late primary spermatocytes, round spermatids, elongated spermatids, and sperm), and somatic cells (Sertoli cells and Leydig cells), as well as one somatic cell that unexpectedly contained leukocytes. The functional enrichment analysis indicated that several pathways of cell cycle, gamete generation, protein processing, and mRNA surveillance pathways were significantly enriched in testicular germ cell types, and ribosome pathway was significantly enriched in testicular somatic cell types. Further analysis identified several stage-specific marker genes of sheep germ cells, such as EZH2, SOX18, SCP2, PCNA, and PRKCD. Our research explored for the first time of the changes in the transcription level of various cell types during the process of sheep spermatogenesis, providing new insights for sheep spermatogenesis and spermatogenic cell development.

Reinterpreting sheep muscle strand-specific RNA sequencing data showing extensive 3'UTR extensions.

The more complex 3' UTR in higher organisms may have the function of increasing post-transcriptional gene regulation. Recent RNA sequencing technologies have provided us with the possibility to capture the complete 3' UTR landscape of different species and cells. However, no systematic analysis of sheep-related 3' UTR has been performed. Here, we conducted a detailed analysis of the 3' UTR with the primary goal of identifying intact 3' UTR landscapes in the sheep muscles of the three developmental stages. Based on strand-specific RNA sequencing (ssRNA-seq) data, we found that thousands of genes in sheep muscle are continuously transcribed after the UTR of the reference genome (Oar_v4.0). More than 66% of the 3' UTR extensions exhibit similar expression trends to their upstream gene exons. These 3' UTR extensions strongly enrich thousands of conserved microRNA binding sites. The 3' UTR extension-associated RNA of PFKM (PuaRNA) was predicted to be derived from the 3' UTR of PFKM. In sheep myocytes, myotubes and various tissues, the expression pattern of PuaRNA is positively correlated with that of PFKM. Taken together, these new 3' UTR annotations greatly extend the range of mammalian post-transcriptional regulatory networks, which have a particular impact on the regulation of sheep muscle development.

The Expression Pattern of p32 in Sheep Muscle and Its Role in Differentiation, Cell Proliferation, and Apoptosis of Myoblasts.

The complement 1q binding protein C (C1QBP), also known as p32, is highly expressed in rapidly growing tissues and plays a crucial role in cell proliferation and apoptosis. However, there are no data interpreting its mechanisms in muscle development. To investigate the role of p32 in sheep muscle development, an 856 bp cDNA fragment of p32 containing an 837 bp coding sequence that encodes 278 amino acids was analyzed. We then revealed that the expression of p32 in the longissimus and quadricep muscles of fetal sheep was more significantly up-regulated than expression at other developmental stages. Furthermore, we found that the expression of p32 was increased during myoblasts differentiation in vitro. Additionally, the knockdown of p32 in sheep myoblasts effectively inhibited myoblast differentiation, proliferation, and promoted cell apoptosis in vitro. The interference of p32 also changed the energy metabolism from Oxidative Phosphorylation (OXPHOS) to glycolysis and activated AMP-activated protein kinase (AMPK) phosphorylation in sheep myoblasts in vitro. Taken together, our data suggest that p32 plays a vital role in the development of sheep muscle and provides a potential direction for future research on muscle development and some muscle diseases.

Melatonin regulates the antioxidant capacity of sheep granulosa cells through a novel uORF-Nrf2aa mediated Nrf2/KEAP1 pathway.

Ovarian dysfunction stands as a prevalent contributor to female infertility, with its etiology intertwined with genetic, autoimmune, and environmental factors. Within the ovarian follicles, granulosa cells (GCs) represent the predominant cell population. Alterations in GCs, notably oxidative stress (OS) and the consequential surge in reactive oxygen species (ROS), play pivotal roles in the orchestration of ovarian function. Nrf2aa, a newly identified upstream open reading frame (uORF), is situated within the 5' untranslated region (5'UTR) of sheep Nrf2 mRNA and is regulated by melatonin, a crucial intrafollicular antioxidant. In this study, we have noted that Nrf2aa has the capacity to encode a peptide and exerts a negative regulatory effect on the translation efficiency (TE) of the Nrf2 CDs region. Further in vitro experiments, we observed that interfering with Nrf2aa can enhance the cellular functionality of GCs under 3-np-induced oxidative stress, while overexpressing Nrf2aa has the opposite effect. Furthermore, overexpression of Nrf2aa counteracts the rescuing effect of melatonin on the cellular functions of GCs under oxidative stress conditions, including estrogen secretion, proliferation, apoptosis, and many more. Finally, we confirmed that Nrf2aa, by regulating the expression of key proteins in the Nrf2/KEAP1 signaling pathway, further modulates the antioxidant levels in GCs.

Research progress on the development of pennycress (Thlaspi arvense L.) as a new seed oil crop: a review.

Compared with other crops, pennycress (Thlaspi arvense L.) is a niche emerging oil crop. In recent years, research on pennycress has been increasingly reflected in various directions. Pennycress belongs to the Brassicaceae family and was introduced from Eurasia to North America. It has been found worldwide as a cultivated plant and weed. In this paper, we review the advantages of pennycress as a supplementary model plant of Arabidopsis thaliana, oil and protein extraction technology, seed composition analysis based on metabolomics, germplasm resource development, growth, and ecological impact research, abiotic stress, fatty acid extraction optimization strategy, and other aspects of studies over recent years. The main research directions proposed for the future are as follows: (1) assemble the genome of pennycress to complete its entire genome data, (2) optimize the extraction process of pennycress as biodiesel, (3) analyze the molecular mechanism of the fatty acid synthesis pathway in pennycress, and (4) the functions of key genes corresponding to various adversity conditions of pennycress.

Effects of Zearalenone on Apoptosis and Copper Accumulation of Goat Granulosa Cells In Vitro.

Zearalenone (ZEA), also known as F-2 toxin, is a mycotoxin. Despite numerous reports of ZEA impairing livestock production performance and fertility, little information is available, including information about the mechanism underlying damage to cell metal ion transport. Copper, which is essential for cell survival as a metal ion, can consist of a variety of enzymes that facilitate abundant metabolic processes. However, the accumulation of copper in cells can have toxic effects. Here, we intended to determine whether ZEA could impair goat granulosa cells (GCs) and alter the cellular copper concentration. GCs were divided into a negative control (NC) group (cells cultured with 0.1% dimethyl sulfoxide (DMSO) for 8 h) and a ZEA group (cells cultured with 200 µmol/L ZEA diluted in DMSO for 8 h). The results showed that ZEA could inhibit GC proliferation and impair cell viability. GCs showed significant increases in the apoptosis rate and oxidative stress levels, while their ability to synthesize estrogen decreased. In addition, RNA-seq results showed dramatic changes in the expression of copper transport-related genes. The expression levels of ATPase copper transporting alpha (ATP7A) and ATPase copper transporting beta (ATP7B) were significantly downregulated (p < 0.01), while the expression of solute carrier family 31 member 1 (SLC31A1) was not modified in the ZEA group compared with the NC group. In accordance with these trends, the copper concentration increased significantly in the ZEA group (p < 0.01). In summary, our results show that ZEA can negatively affect GCs and cause copper accumulation. This finding may provide a prospective line of research on the relationship between ZEA and the transport of copper ions in GCs.

Melatonin protects Leydig cells from HT-2 toxin-induced ferroptosis and apoptosis via glucose-6-phosphate dehydrogenase/glutathione -dependent pathway.

HT-2 toxin is a mycotoxin commonly found in food and water that can have adverse effects on male reproductive systems, including testosterone secretion. Ferroptosis and apoptosis are two types of programmed cell death that have been implicated in the regulation of cellular functions. Melatonin, a powerful antioxidant with various physiological functions, has been shown to regulate testosterone secretion. However, the mechanisms underlying the protective effects of melatonin against HT-2 toxin-induced damage in testosterone secretion are not fully understood. In this study, we investigated the effects of HT-2 toxin on sheep Leydig cells and the potential protective role of melatonin. We found that HT-2 toxin inhibited cell proliferation and testosterone secretion of Leydig cells in a dose-dependent manner and induced ferroptosis and apoptosis through intracellular reactive oxygen species accumulation, leading to lipid peroxidation. Exposure of Leydig cells to melatonin in vitro reversed the defective phenotypes caused by HT-2 toxin via a glucose-6-phosphate dehydrogenase/glutathione-dependent mechanism. Interference of glucose-6-phosphate dehydrogenase disrupted the beneficial effect of melatonin on ferroptosis and apoptosis in HT-2 toxin-treated Leydig cells. Furthermore, similar results were observed in vivo in the testes of male mice injected with HT-2 toxin with or without melatonin treatment for 30 days. Our findings suggest that melatonin inhibits ferroptosis and apoptosis by elevating the expression of glucose-6-phosphate dehydrogenase to eliminate reactive oxygen species accumulation in HT-2 toxin-treated Leydig cells. These results provide fundamental evidence for eliminating the adverse effects of HT-2 toxin on male reproduction.

Necroptosis-related regulatory pattern and scoring system for predicting therapeutic efficacy and prognosis in ovarian cancer.

BACKGROUND: Ovarian cancer is difficult to treat and is, therefore, associated with a high fatality rate. Although targeted therapy and immunotherapy have been successfully used clinically to improve the diagnosis and treatment of ovarian cancer, most tumors become drug resistant, and patients experience relapse, meaning that the overall survival rate remains low. AIMS: There is currently a lack of effective biomarkers for predicting the prognosis and/or outcomes of patients with ovarian cancer. Therefore, we used published transcriptomic data derived from a large ovarian cancer sample set to establish a molecular subtyping model of the core genes involved in necroptosis in ovarian cancer. METHODS AND RESULTS: Clustering analysis and differential gene expression analyses were performed to establish the genomic subtypes related to necroptosis and to explore the patterns of regulatory gene expression related to necroptosis in ovarian cancer. A necroptosis scoring system (NSS) was established using principal component analysis according to different regulatory patterns of necroptosis. In addition, this study revealed important biological processes with essential roles in the regulation of ovarian tumorigenesis, including external encapsulating structure organization, leukocyte migration, oxidative phosphorylation, and focal adhesion. Patients with high NSS scores had unique immunophenotypes, such as more abundant M2 macrophages, monocytes, CD4+ memory T cells, and regulatory T cells. Immune checkpoint CD274 had a greater expression in patients with high NSS values. CONCLUSION: This NSS could be used as an independent predictor of prognosis to determine the sensitivity of ovarian cancer to various small-molecule inhibitors, immune checkpoint inhibitors, and platinum-based chemotherapy drugs.

Comparative Transcriptomic Analysis of Hu Sheep Pituitary Gland Prolificacy at the Follicular and Luteal Phases.

The pituitary gland directly regulates the reproduction of domestic animals. Research has increasingly focused on the potential regulatory mechanism of non-coding RNA in pituitary development. Little is known about the differential expression pattern of lncRNAs in Hu sheep, a famous sheep breed with high fecundity, and its role in the pituitary gland between the follicular phase and luteal phase. Herein, to identify the transcriptomic differences of the sheep pituitary gland during the estrus cycle, RNA sequencing (RNA-Seq) was performed. The results showed that 3529 lncRNAs and 16,651 mRNAs were identified in the pituitary gland. Among of them, 144 differentially expressed (DE) lncRNA transcripts and 557 DE mRNA transcripts were screened in the follicular and luteal phases. Moreover, GO and KEGG analyses demonstrated that 39 downregulated and 22 upregulated genes interacted with pituitary functions and reproduction. Lastly, the interaction of the candidate lncRNA XR_001039544.4 and its targeted gene LHB were validated in sheep pituitary cells in vitro. LncRNA XR_001039544.4 and LHB showed high expression levels in the luteal phase in Hu sheep. LncRNA XR_001039544.4 is mainly located in the cytoplasm, as determined by FISH analysis, indicating that XR_001039544.4 might act as competing endogenous RNAs for miRNAs to regulate LHB. LncRNA XR_001039544.4 knockdown significantly inhibited LH secretion and cell proliferation. LncRNA XR_001039544.4 may regulate the secretion of LH in the luteal-phase pituitary gland via affecting cell proliferation. Taken together, these findings provided genome-wide lncRNA- and mRNA-expression profiles for the sheep pituitary gland between the follicular and luteal phases, thereby contributing to the elucidation of the molecular mechanisms of pituitary function.

Melatonin alleviated oxidative stress induced by energy restriction on sheep Leydig cells through Sirt1/Sod2 pathway.

Energy balance is essential for normal reproduction of ram. However, the effect of energy restriction (ER) on reactive oxygen species (ROS) of sheep Leydig cells (LCs) and the rescuee methods are still unclear. To investigate the in vitro effect of melatonin on cellular ROS in fER-treated sheep LCs and explore the underlying mechanism, Hu sheep LCs were restricted energy using no serum culture medium and resaved with 10 ng/ml melatonin, respectively. The results showed that ER significantly increased MDA level, while decreased CAT, GHS-px expression and ΔΨm (p < 0.05). Meanwhile, ER decreased testosterone concentration and cell proliferation rate (p < 0.05). And the expression of testosterone synthesis-related enzymes was also down-regulated by ER (p < 0.05). Furthermore, we revealed that melatonin reversed the defective phenotypes in ER-treated LCs via Sirt1/Sod2 pathway. The interference of Sirt1 abolished the melatonin-mediated improvement of cellular ROS and testosterone secretion. Taken together, our study firstly indicated that melatonin could alleviate the excessive ROS accumulation and promote testosterone biosynthesis in ER-treated sheep LCs via the activation of Sirt1/Sod2 pathway.

Expression pattern and potential role of Nanos3 in regulating testosterone biosynthesis in Leydig cells of sheep.

Nanos3 is from a highly conserved family of genes with putative RNA-binding activity and plays an important role in germ cell development. Yet, its function in Leydig cells (LCs) for supporting germ cell development, is still poorly understood. In this study, we investigated the expression pattern and function of Nanos3 in the sheep testis. The Nanos3 mRNA and protein were highly expressed in the ram testis. The sheep Nanos3 amino acid sequence was 93.6%-88.6% homologous with those of other mammalian species. The expression of Nanos3 in the ram testis was significantly lower in 9 and 24-month-old sheep than in at 3-month-old sheep (P < 0.05). Immunohistochemistry analysis revealed that sheep Nanos3 was not only localized at spermatogenesis cell but also at LCs. Thus the functions of Nanos3 in LCs were further investigated by using siRNA-Nanos3 in vitro. The secretion of T hormone and expression of testosterone synthesis-related enzymes in LCs were significantly inhibited (P < 0.05) by siRNA-Nanos3. Moreover, the BAX/Bcl-2 mRNA and protein levels were significantly increased (P < 0.05) by siRNA-Nanos3. In addition, we examined the expression levels of Nanos3 mRNA and protein in energy-restricted(ER) rams and in vitro ER LCs. Their levels were also significantly decreased (P < 0.05) and were accompanied by an increased BAX/Bcl-2 mRNA expression compared with the control group (P < 0.05). To the best of our knowledge, our study is the first to show that Nanos3 is an important regulator of testosterone biosynthesis in rams for supporting germ cell development.

Genome-Wide Analysis and Function Prediction of Long Noncoding RNAs in Sheep Pituitary Gland Associated with Sexual Maturation.

Long noncoding RNA (lncRNA) plays a crucial role in the hypothalamic-pituitary-testis (HPT) axis associated with sheep reproduction. The pituitary plays a connecting role in the HPT axis. However, little is known of their expression pattern and potential roles in the pituitary gland. To explore the potential lncRNAs that regulate the male sheep pituitary development and sexual maturation, we constructed immature and mature sheep pituitary cDNA libraries (three-month-old, TM, and nine-month-old, NM, respectively, n = 3) for lncRNA and mRNA high-throughput sequencing. Firstly, the expression of lncRNA and mRNA were comparatively analyzed. 2417 known lncRNAs and 1256 new lncRNAs were identified. Then, 193 differentially expressed (DE) lncRNAs and 1407 DE mRNAs were found in the pituitary between the two groups. Moreover, mRNA-lncRNA interaction network was constructed according to the target gene prediction of lncRNA and functional enrichment analysis. Five candidate lncRNAs and their targeted genes HSD17B12, DCBLD2, PDPK1, GPX3 and DLL1 that enriched in growth and reproduction related pathways were further filtered. Lastly, the interaction of candidate lncRNA TCONS_00066406 and its targeted gene HSD17B12 were validated in in vitro of sheep pituitary cells. Our study provided a systematic presentation of lncRNAs and mRNAs in male sheep pituitary, which revealed the potential role of lncRNA in male reproduction.