Role and mechanism of AMH in the regulation of Sertoli cells in mice.

Sertoli cells produce anti-Müllerian hormone (AMH), a glycoprotein belonging to the transforming growth factor-beta family. AMH mediates the regression of Müllerian ducts in the developing male fetus. However, the role of AMH in the regulation of primary Sertoli cells remains unclear. The present study was designed to investigate the effect of AMH on the viability and proliferation of Sertoli cells, with an additional focus on stem cell factor (SCF). Treatment of Sertoli cells with increasing concentrations of rh-AMH (0, 10, 50, 100, and 800ng/ml) for two days revealed that AMH, at high concentrations, increased apoptosis. These results were confirmed by a significant increase in Caspase-3 and Bax and a decrease in Bcl-2 protein and mRNA expression (P<0.01). Paradoxically, treatment with a low concentration of rh-AMH (10ng/ml), but not higher concentrations (50-800ng/ml), promoted Sertoli cell proliferation, which was verified by an increase in PCNA mRNA (P<0.05). Furthermore, only low concentrations of rh-AMH activated the non-canonical ERK signaling pathway. Similarly, low concentrations of rh-AMH (10-50ng/ml) significantly increased (P<0.05) SCF mRNA and SCF protein levels. These findings indicate that AMH differentially regulates the fate of Sertoli cells in vitro by promoting proliferation at low concentrations and apoptosis at high concentrations. In addition, AMH increased the expression of SCF, an important regulator of Sertoli cell development. Therefore, AMH may play a role in Sertoli cell development.

Association of MAP4K4 gene single nucleotide polymorphism with mastitis and milk traits in Chinese Holstein cattle.

The objective of the studies presented in this Research Communication was to investigate the association of single nucleotide polymorphisms present in the MAP4K4 gene with different milk traits in dairy cows. Based on previous QTL fine mapping results on bovine chromosome 11, the MAP4K4 gene was selected as a candidate gene to evaluate its effect on somatic cell count and milk traits in ChineseHolstein cows. Milk production traits including milk yield, fat percentage, and protein percentage of each cow were collected using 305 d lactation records. Association between MAP4K4 genotype and different traits and Somatic Cell Score (SCS) was performed using General Linear Regression Model of R. Two SNPs at exon 18 (c.2061T > G and c.2196T > C) with genotype TT in both SNPs were found significantly higher for somatic SCS. We found the significant effect of exon 18 (c.2061T > G) on protein percentage, milk yield and SCS. We identified SNPs at different location of MAP4K4 gene of the cattle and several of them were significantly associated with the somatic cell score and other different milk traits. Thus, MAP4K4 gene could be a useful candidate gene for selection of dairy cattle against mastitis and the identified polymorphisms might potentially be strong genetic markers.

RNAi-mediated knockdown of inhibin α subunit increased apoptosis in granulosa cells and decreased fertility in mice.

Inhibin α (INHα), a member of TGFß superfamily, is an important modulator of reproductive function that plays a vital role in follicular changes, cell differentiation, oocyte development, and ultimately in mammalian reproduction. However, the role of inhibin α in female fertility and ovarian function remains largely unknown. To define its role in reproduction, transgenic mice of RNAi-INHα that knock down the INHα expression by shRNAi were used. Inhibin α subunit gene was knocked down successfully at both transcriptional and translational levels by RNAi PiggyBac transposon (Pbi) mediated recombinant pshRNA vectors and purified DNA fragments were microinjected into mouse zygotes. Results showed that transgenic female mice were sub-fertile and exhibited 35.28% reduction in litter size in F1 generation relative to wild type. The decreased litter size associated with the reduction in the number of oocytes ovulated after puberty. Serum INHα level was significantly decreased in both 3 and 6 weeks; whereas, FSH was significantly increased in 3 weeks but not in 6 weeks. Furthermore, suppression of INHα expression significantly promoted apoptosis by up-regulating Caspase-3, bcl2, INHßB and GDF9 and down regulated Kitl and TGFßRIII genes both at transcriptional and translational levels. Moreover, it also dramatically reduced the progression of G1 phase of cell cycle and the number of cells in S phase as determined by flow cytometer. These results indicate that suppression of INHα expression in RNAi-transgenic mice leads to disruption of normal ovarian regulatory mechanism and causes reproductive deficiencies by promoting cellular apoptosis, arresting cellular progression and altering hormonal signaling.

Analysis of the binding sites of porcine sialoadhesin receptor with PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) can infect pigs and cause enormous economic losses to the pig industry worldwide. Porcine sialoadhesin (pSN) and CD163 have been identified as key viral receptors on porcine alveolar macrophages (PAM), a main target cell infected by PRRSV. In this study, the protein structures of amino acids 1-119 from the pSN and cSN (cattle sialoadhesin) N-termini (excluding the 19-amino acid signal peptide) were modeled via homology modeling based on mSN (mouse sialoadhesin) template structures using bioinformatics tools. Subsequently, pSN and cSN homology structures were superposed onto the mSN protein structure to predict the binding sites of pSN. As a validation experiment, the SN N-terminus (including the wild-type and site-directed-mutant-types of pSN and cSN) was cloned and expressed as a SN-GFP chimera protein. The binding activity between SN and PRRSV was confirmed by WB (Western blotting), FAR-WB (far Western blotting), ELISA (enzyme-linked immunosorbent assay) and immunofluorescence assay. We found that the S107 amino acid residue in the pSN N-terminal played a crucial role in forming a special cavity, as well as a hydrogen bond for enhancing PRRSV binding during PRRSV infection. S107 may be glycosylated during PRRSV infection and may also be involved in forming the cavity for binding PRRSV along with other sites, including W2, Y44, S45, R97, R105, W106 and V109. Additionally, S107 might also be important for pSN binding with PRRSV. However, the function of these binding sites must be confirmed by further studies.