Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

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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.

An increasing number of studies have revealed that histone deacetylase (HDAC) mediated histone deacetylation is important for mammalian oocyte development. However, nonselective HDAC inhibitors (HDACi) were applied in most studies; the precise functions of specific HDAC classes during meiosis are poorly defined. In this study, the class IIa-specific HDACi MC1568 was used to reveal a crucial role of class IIa HDACs in the regulation of histone deacetylation during porcine oocyte meiosis. Besides, the functions of HDACs and histone acetyltransferases in regulating the balance of histone acetylation/deacetylation were also confirmed during oocyte maturation. After the validation of nontoxicity of MC1568 in maturation rate, spindle morphology, and chromosome alignment, effects of MC1568 on developmental competence of porcine somatic cell nuclear transfer (SCNT) embryos were evaluated, and data indicated that treatment with 10 μM MC1568 for 12 hours following electrical activation significantly enhanced the blastocyst rate and cell numbers. Moreover, results showed that optimal MC1568 treatment increased the H4K12 acetylation level in SCNT one cells and two cells. In addition, MC1568 treatment stimulated expression of the development-related genes OCT4, CDX2, SOX2, and NANOG in SCNT blastocysts. Collectively, our investigation uncovered a critical role of class IIa HDACs in the regulation of histone deacetylation during oocyte meiosis. Furthermore, for the first time, we showed that MC1568 can improve the in vitro development of porcine SCNT embryos. These findings provide an alternative HDACi for improving animal cloning efficiency and may shed more light on nuclear reprogramming.

ABSTRACT. NR5A2 has been implicated in processes as diverse as steroidogenesis, cellular proliferation, ovarian follicular development, ovulation, and fertility in mammals. However, data about the relationship between NR5A2 and prolificacy in mammals are lacking. In the present study, we identified and characterized NR5A2 of Hu sheep, and investigated the correlation between NR5A2 and reproductive performance. The full-length coding region was 1488 bp, and the gene was conserved in mammals. We found a positive correlation between NR5A2 mRNA levels in the ovary and the ovulation rate and litter size of Hu sheep. We detected two single nucleotide polymorphisms (T40C and T1419C) in the coding sequence of NR5A2. At the third and average parity, litter size of Hu ewes with CC genotype at T40C locus was larger than those of ewes with TT 12766 Y.X. Li et al.©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 12765-12775 (2015) or TC genotypes; at the T1419C locus, Hu ewes with TT genotype was greater than those of ewes with CC genotype at the third parity. Our findings demonstrated that NR5A2 was associated with reproductive performance in Hu sheep, a high prolificacy breed.

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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.