PLAG1 g.8795C>T Mutation Regulates Early Body Weight in Hu Sheep by Weakening miR-139 Binding.

Sheep birth and weaning weights indicate their growth and survival. Thus, identifying molecular genetic markers for early body weight is important in sheep breeding. Pleomorphic adenoma gene 1 (PLAG1) is important for regulating birth weight and body length in mammals; however, its relationship with sheep body weight remains unknown. Here, the 3'-untranslated region (3'-UTR) of the Hu sheep PLAG1 gene was cloned, single nucleotide polymorphisms (SNPs) were screened, genotype-early body weight relationships were analyzed, and the possible molecular mechanism was explored. PLAG1 3'-UTR sequences with five forms of base sequences plus poly(A) tails were detected in Hu sheep and the g.8795C>T mutation was identified. Luciferase reporter assay indicated that the g.8795C>T mutation influenced PLAG1 post-transcriptional activity. miRBase prediction showed that the g.8795C>T mutation was located in the miR-139 seed sequence binding region, and miR-139 overexpression significantly decreased both PLAG1-CC and PLAG1-TT activities. Moreover, the luciferase activity of PLAG1-CC was significantly lower than that of the PLAG1-TT, but miR-139 inhibition substantially increased both PLAG1-CC and PLAG1-TT luciferase activities, suggesting that PLAG1 is the target gene of miR-139. Thus, the g.8795C>T mutation upregulates PLAG1 expression by weakening its binding with miR-139, promoting PLAG1 expression, and increasing Hu sheep birth and weaning weights.

miR-27a-3p targets NR5A2 to regulate CYP19A1 expression and 17-ß estradiol synthesis in ovine granulosa cells.

Although 17-ß estradiol (E2) synthesis is important in regulating female fertility, we know little regarding the molecular mechanism of miRNA-regulated ovine E2 synthesis. Here, our experiments with granulosa cells (GCs) from Hu sheep revealed miR-27a-3p involvement in E2 synthesis and its association with ovine litter size. First, we showed that miR-27a-3p of sheep and other mammals share a high nucleotide identity. Next, gain- and loss-of-function assays indicated that miR-27a-3p inhibits CYP19A1 expression and E2 synthesis in GCs. Moreover, we demonstrated that NR5A2 is a direct target of miR-27a-3p. Ovine miR-27a-3p suppresses E2 synthesis via the NR5A2 and CYP19A1 axes. We also identified four single nucleotide polymorphisms in the ovine miR-27a gene, and g.-13 G>A and g 0.24 T > G were significantly associated with the first and the second parity litter size, respectively (P < 0.05). In summary, our findings reveal that miR-27a-3p is a novel regulator of E2 synthesis and may predict litter size of Hu sheep, providing insight into mechanisms underlying granulosa cell function and female fertility.

The involvement of translationally controlled tumor protein during lamb rumen epithelium development.

Early weaning is usually applied to improve the reproductive efficiency of sheep in mutton production, while the development of rumen is of vital importance for sheep weaning age. Translationally controlled tumor protein (TCTP) is a highly conserved protein which participates in multiple tissue and organ development. Thus, we hypothesized that TCTP was involved in sheep rumen development. Histological analyses of sheep rumen epithelium showed that the epithelium formed tough shaped papillae without growing from birth to day 15 of age, after which it rapidly developed to functional epithelia on day 45 of age. We then found TCTP expressed in stratum basale, stratum spinosum and stratum granulosum of rumen epithelium. TCTP protein expression remained at a relative low level from day 0 to day 15 of age, it then significantly increased on day 30 (p < 0.05) and gradually decreased until day 60. Furthermore, to explore the role of TCTP in sheep rumen and its regulation, we found the ratio of Ki67 positive cell in stratum basale cells followed the similar pattern as the expression of TCTP. We also found the ratio of acetate:propionate in rumen fluid decreased from day 30 to day 60 of age (p < 0.05). To conclude, our data indicated that TCTP participated in rumen papillae growth by promoting rumen stratum basale cell proliferation.

Different susceptibility to porcine reproductive and respiratory syndrome virus infection among Chinese native pig breeds.

China is rich in native pig breeds, yet information regarding the susceptibility/resistance of local breeds to porcine reproductive and respiratory syndrome virus (PRRSV) infection is lacking. In the present study, an in vitro method based on assessing PRRSV replication in porcine alveolar macrophages (PAMs) was established to evaluate PRRSV susceptibility/resistance in a commercial pig breed (Landrace) and five native pig breeds from Jiangsu and Anhui provinces in China. Expression levels of cytokines (IL-8, IL-10, TNF-α and IFN-γ), Toll-like receptor 3 (TLR3), CD163 (PRRSV receptor), and sialoadhesin (Sn, PRRSV receptor) in infected pigs were determined using real-time PCR, and the association between PRRSV susceptibility/resistance and the abundance of the cytokines and receptors was investigated. The viral replication rate and titer at 0, 6, 12 18, 24 and 36 hours postinfection (hpi) were determined to assess the proliferation dynamics of PRRSV NJGC in PAMs. Based on the PRRSV proliferation dynamics, the results indicated that Dingyuan pigs were the most susceptible to PRRSV infection, whereas Jiangquhai pigs were the least susceptible to PRRSV infection among the six pig breeds tested, as indicated by measuring PRRSV replication and the viral load in PAMs. The different levels of susceptibility to PRRSV infection in PAMs may be associated with differences in the abundance of CD163 (PRRSV receptor), cytokines IL-8, IFN-γ, and TNF-α in Jiangquhai and Dingyuan pig breeds after viral inoculation.

Thyroid hormones alter estrous cyclicity and antioxidative status in the ovaries of rats.

To expand our understanding of the roles of thyroid hormones on female reproduction, we induced hypo- and hyper-T rat models to investigate the roles of thyroid hormones on estrous cyclicity, as well as the antioxidative status in the ovaries of rats. In the current study, our data show that hypothyroidism (hypo-T) and hyperthyroidism (hyper-T) led to significantly reduced body weights and ovarain weights and delayed vaginal opening day. For hyper-T, thyroxine (T4), tri-iodothyronine (T3), progesterone (P4) and follicle-stimulating hormone (FSH) were significantly increased, while estradiol (E2) and luteinizing hormone (LH) were significantly decreased. For hypo-T rats, serum levels of total T4 and T3, E2, P4, FSH and LH were significantly increased, while concentrations of E2 and LH were significantly decreased. For ovary morphology, the numbers of secondary and antral follicles were significantly decreased with more atretic antral follicles and less corpora lutea in both hyper- and hypo-T groups. Both hyper-T and hypo-T treatment significantly decreased the expressions of thyroid hormone receptor α1 in the ovary. Hypo-T significantly reduced nitric oxide (NO), total NO synthase (tNOS), inducible NOS and constitutive NOS activities, but hyper-T increased them. For antioxidative parameters, hypo-T and hyper-T treatment significantly increased malondialdehyde (MDA) contents. The activities of both glutathione peroxidase (GSH-Px) and catalase (CAT) significantly decreased in the hypo-T group but increased in the hyper-T group. Total superoxide dismutase (T-SOD) activity was significantly increased in the hyper-T group. In summary, thyroid hormones alter estrous cyclicity and antioxidative status in the ovary of the rat may act through the NOS signaling pathway.

Cellular Localization of Inhibin alpha-subunit, PKB/Akt and FoxO3a proteins in the ovaries of minipigs.

Experiments were conducted to examine the cellular localization of inhibin alpha-subunit, protein kinase B (PKB/Akt), and FoxO3a proteins in the ovaries of minipigs, Chinese Xiang pigs, by immunohistochemistry. The results indicated that inhibin alpha-subunits were localized in the granulosa cells of follicles at all stages but were not localized in corpora lutea. PKB was localized in the granulosa cells of primordial follicles and in the basal layers of the granulosa cells of preantral and antral follicles, but were not localized in atretic follicles and corpora lutea. FoxO3a was localized in the granulosa cells of follicles at all stages and was extensively localized in the cytoplasma of the luteinized granulosa cells of corpora lutea. Together, the stage- and cell-specific expression patterns of inhibin alpha-subunit, FoxO3a, and PKB suggest that these proteins might play potential roles in follicular development, atresia, and luteinization in the minipig.