Search

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3β-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P 4) and estradiol (E 2). PGR knockdown significantly downregulated the levels of StAR and 3β-HSD mRNA and decreased the production of P 4 , whereas no effects on CYP19A1 mRNA expression and E 2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3ʹ-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy. • PGR is a direct target of unconservative_15_2570409. • Unconservative_15_2570409 mediates PGR transcriptional target genes by targeting PGR. • PGR affects steroidogenesis in sheep GCs through steroid hormone-related genes. [ABSTRACT FROM AUTHOR]

l -arginine (L-Arg) is a semiessential amino acid that plays crucial roles in the reproductive performance of animals. This research aimed to investigate the effect of supplementing L-Arg on endometrial epithelial cells (EECs) of Hu sheep. In vivo , female Hu sheep were randomly divided into three groups: control group (n = 5), nutrient-restricted group (n = 5), and L-Arg supplemented nutrient-restricted group (n = 5). Then, the effect of L-Arg on ovine endometrial growth and antioxidant capacity was assessed. We found that L-Arg supplementation promoted the growth of endometrial ductal gland invaginations (DGI), and alleviated oxidative stress in nutrient-restricted sheep. In order to investigate its mechanism, a H 2 O 2 -induced EECs oxidative stress model was established, and roles of L-Arg in EECs oxidation resistance, proliferation, apoptosis and endocrine activity were studied in vitro. Our results showed that L-Arg markedly decreased the release of reactive oxygen species (ROS) and malonaldehyde (MDA), and enhanced the expression and activity of certain antioxidant enzymes in EECs challenged by the H 2 O 2 (p < 0.05). Supplementation of L-Arg significantly reduced the effect of 200 μM H 2 O 2 on the viability of EECs (p < 0.05). In addition, EECs treated with L-Arg significantly alleviated the G0/G1-phase cell cycle arrest, apoptosis, and the inhibition of endometrial growth factors expression caused by H 2 O 2 (p < 0.05). Overall, the results demonstrate that L-Arg performs crucial roles in maintaining the proliferation of ovine EECs, endocrine activity and inhibiting apoptosis through reducing oxidative stress. This study offers a theoretical basis for using L-Arg to improve sheep the uterine function. • L-Arg enhances the growth of endometrial DGI in nutrient-restricted Hu sheep. • L-Arg alleviates endometrial oxidative stress induced by nutrient-restriction. • L-Arg inhibits the apoptosis, enhances proliferation and endocrine activity of EECs. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Activin/inhibin is an important factor for the fecundity of Hu sheep, and it is involved in follicular development in ovaries. Inhibin subunit beta A (INHBA) participates in the synthesis of activin A and inhibin A. In this study, we also noted a positive correlation between INHBA level and the secretion of both activin A and inhibin A in culture medium. Nevertheless, both knockdown and overexpression of INHBA downregulated the expression of Inhibin Subunit Alpha (INHA). Based on RNA-Sequencing, we further examined the effect and molecular mechanism of INHBA knockdown in GCs on mRNA expression. A total of 1,687 differentially expressed genes (DEGs) were identified (Fold change ≥ 2; False-discovory-rates (FDR) ≤ 0.01), of which 602 genes were upregulated and 1,087 genes were downregulated in the INHBA interference group compared with the control groups. Gene Ontology (GO) enrichment indicated that these DEGs were mainly involved in the regulation of cell cycle, protein serine/threonine kinase activity, and actin cytoskeleton reorganization. Moreover, DEGs were significantly enriched in 40 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including P53, progesterone-mediated oocyte maturation, and PI3K-AKT signaling pathways. We also noted a positive correlation between INHBA level and many PI3K/Akt/mTOR pathway-related genes at the gene or/and protein expression. Overall, this study may contribute to a better understanding of the roles of INHBA on GCs of prolific sheep, as well as the molecular effect of low INHBA expression on GCs, clarifying some reproductive failures. • INHBA knockdown inhibits cell cycle progression on granulosa cells in vitro. • INHBA silence promotes apoptosis through P53 pathway. • INHBA silence reduces the concentration of activin A and inhibin A. • INHBA participates in PI3K/AKT signal pathway. [ABSTRACT FROM AUTHOR]

Pituitary gonadotropins play a pivotal role in reproduction. Long noncoding RNAs (lncRNAs) have been identified as important regulators in the hypothalamic–pituitary–ovarian (HPO) axis associated with reproduction. However, the contributions of lncRNAs to pituitary gonadotropin secretion remain largely unknown. Therefore, this work was performed to uncover the functional mechanisms of the novel lncRNA TCONS_00083279 (lncRNA SM2) and its potential targeting pathway oar-miR-16b/TGF-beta/SMAD2, which is associated with gonadotropin secretion in sheep pituitary cells. In the present study, the lncRNA SM2 showed high expression levels in the sheep pituitary gland, and it was located in both the nucleus and the cytoplasm of pituitary cells. lncRNA SM2 knockdown inhibited pituitary cell proliferation and FSH and LH secretion. The function of the lncRNA SM2 was sponged by oar-miR-16b, and this regulated the growth and gonadotropin secretion of pituitary cells by modulating SMAD2, as shown by the dual-luciferase reporter assay. FSH and LH levels were both upregulated by SMAD2 overexpression. Moreover, the levels of the lncRNA SM2, SMAD2 and TGFR1, as well as FSH and LH, in sheep pituitary cells increased significantly under gonadotropin-releasing hormone (GnRH) stimulation (p < 0.05). This work illustrates that the lncRNA SM2 regulates gonadotropin secretion in the Hu sheep anterior pituitary by targeting the oar-miR-16b/TGF-β/SMAD2 signaling pathway, providing a valuable resource for understanding the molecular mechanisms underlying sheep reproduction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The current study aimed to examine the effects of dietary spirulina supplementation in high-energy (HE) diets on fatty acid metabolism in sheep, and preliminarily explored the potential mechanisms underlying the associated autophagy-mediated regulation of lipid metabolism. In a 2 × 3 factorial design, including six treatment combinations of two metabolisable energy diets (10 and 11 MJ/kg DM), three spirulina supplementation levels (0, 1%, and 3%) were used. Serum alanineaminotransferase (ALT) (p = 0.003) and aspartatetransaminase (AST) (p = 0.002) activities increased, whereas total PUFA content (p < 0.001) decreased in the liver of lambs fed a HE diet. With the addition of spirulina, serum ALT (p = 0.037) and AST (p = 0.014) activities decreased, whereas EPA (p = 0.004), GLA (p = 0.019), n-6 PUFA (p = 0.005), and total PUFA contents (p = 0.019) increased. Moreover, the crude protein content in the Longissimus thoracis et lumborum (LTL) increased (p = 0.013), the expression of PPARα and PPARγ was up-regulated, while ELOVL2 was down-regulated in liver and LTL (p < 0.05). Spirulina supplementation increased mRNA expression levels of autophagy-associated genes, including that of Beclin-1, AMPK, and ULK1 (p < 0.05). In conclusion, spirulina supplementation in a HE diet exerted a protective effect on the liver, increased PUFA content, and modulated expression levels of autophagy-related genes in growing lambs. [ABSTRACT FROM AUTHOR]

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. • The secretion of T hormone and testosterone synthesis-related enzymes in LCs were inhibited by siRNA-Nanos3. • The BAX/Bcl-2 mRNA and protein increased by inhibiting Nanos3. • Nanos3 regulates the apoptosis of the LCs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of spirulina supplementation in a high-energy (HE) diet on lipid metabolism, oxidative status and immunity in Hu lambs. The lambs were assigned to two groups receiving either a standard diet (ST) or a HE diet. Each group was divided into three subgroups: no spirulina supplementation (control), 1% spirulina supplementation, or 3% spirulina supplementation. The body fat, serum cholesterol, triacylglycerol and oxidative stress increased in lambs fed the HE diet. However, 3% spirulina supplementation in the HE diet reduced above parameters and enhanced antioxidant capacity, including increased SOD activity and T-AOC content in serum and Longissimus thoracis et lumborum (LTL). Additionally, lambs receiving 3% spirulina supplementation showed an improvement in immunity-related parameters, including increased IgG concentration in serum and red and white blood cell counts. In conclusion, 3% spirulina supplementation in HE diet ameliorated lipid metabolic disorder and oxidative stress caused by a HE diet. • HE diets significantly increased serum cholesterol, triacylglycerol and GR value. • HE diets significantly decreased shear force of LTL. • Spirulina supplementation relieved lipid metabolic disorder caused by HE diet. • Spirulina supplementation improved antioxidant status in Hu lambs. • 3% spirulina has the best effect on Hu lambs. [ABSTRACT FROM AUTHOR]