Guohua Hua

Polymorphism of the growth hormone gene and its association with growth traits in Boer goat bucks

In the present study, the polymorphism of growth hormone (GH) gene was analyzed as a genetic marker candidate for growth traits in Boer goat bucks. Two single nucleotide polymorphisms (SNPs) - A781G (Ser/Gly35) and A1575G (Leu147), were identified by GH gene sequencing and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis. AA genotype resulted in a significant decrease in birth chest girth (P=0.03) and weaning weight (P=0.014) comparing to AB genotype, while CC genotype contributed to weaning height (P=0.04) greater than CD genotype. When in combination, AACD genotype was undesired for lower scores in a series of growth traits including body weight, length, height, and chest girth at birth and weaning, as well as the pre-weaning daily gain and body weight at age of 11 months. These results indicate that new molecular markers associated with caprine growth traits can be used in MAS (marker-assisted selection) in Boer goat bucks.

Polymorphisms in toll-like receptor 1 and 9 genes and their association with tuberculosis susceptibility in Chinese Holstein cattle.

Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a pivotal role in the innate and adaptive immune systems. Studies have shown that TLR variants play roles in various human infectious diseases. The aim of the present study was to investigate whether the functional genetic variations at positions C632T, G1409A, A1475C, G1550A and G1596A in TLR1 and at A2700G and A3156G in TLR9 confer susceptibility or resistance to bovine tuberculosis (bTB). Genotyping of the TLR1 and TLR9 gene polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-strand conformation polymorphism (PCR-SSCP) in 586 Chinese Holstein cows (154 infected with bTB, 432 non-infected). The frequencies of the GH and HH genotypes at TLR1-G1596A differed significantly between bTB-infected and non-infected animals [p=0.001 for both GH and HH; GH odds ratio (OR)=2.43 95% confidence interval (CI) (1.47-4.03); and HH OR=1.49 95% CI (0.85-2.62)]. There was a trend toward an increased relative risk of tuberculosis (TB) incidence in the CD genotype at the TLR1-A1475C locus [p=0.056, OR=1.59 95% CI (0.98-2.58)]. The present study suggests that variants in the TLR1 gene are associated with susceptibility to bTB, whereas no significant association can be inferred from the polymorphisms in the TLR9 gene.

Action mechanism of inhibin α-subunit on the development of Sertoli cells and first wave of spermatogenesis in mice.

Inhibin is an important marker of Sertoli cell (SC) activity in animals with impaired spermatogenesis. However, the precise relationship between inhibin and SC activity is unknown. To investigate this relationship, we partially silenced both the transcription and translation of the gene for the α-subunit of inhibin, Inha, using recombinant pshRNA vectors developed with RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Clontech Laboratories, Mountain View, Calif). We found that Inha silencing suppresses the cell-cycle regulators Cyclin D1 and Cyclin E and up-regulates the cell-cycle inhibitor P21 (as detected by Western blot analysis), thereby increasing the number of SCs in the G1 phase of the cell cycle and decreasing the amount in the S-phase of the cell cycle (as detected by flow cytometry). Inha silencing also suppressed Pdgfa, Igf1, and Kitl mRNA levels and up-regulated Tgfbrs, Inhba, Inhbb, Cyp11a1, Dhh, and Tjp1 mRNA levels (as indicated by real-time polymerase chain reaction [PCR] analysis). These findings indicate that Inha has the potential to influence the availability of the ligand inhibin and its antagonist activin in the SC in an autocrine manner and inhibit the progression of SC from G1 to S. It may also participate in the development of the blood–testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa. Real-time PCR and Western blot analyses of Inha, Inhba, and Inhbb mRNA and Inha levels over time show that Inha plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice.

YAP forms autocrine loops with the ERBB pathway to regulate ovarian cancer initiation and progression

Mechanisms underlying ovarian cancer initiation and progression are unclear. Herein, we report that the Yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, interacts with ERBB signaling pathways to regulate the initiation and progression of ovarian cancer. Immunohistochemistry studies indicate that YAP expression is associated with poor clinical outcomes in patients. Overexpression or constitutive activation of YAP leads to transformation and tumorigenesis in human ovarian surface epithelial cells, and promotes growth of cancer cells in vivo and in vitro. YAP induces the expression of epidermal growth factor (EGF) receptors (EGFR, ERBB3) and production of EGF-like ligands (HBEGF, NRG1 and NRG2). HBEGF or NRG1, in turn, activates YAP and stimulates cancer cell growth. Knockdown of ERBB3 or HBEGF eliminates YAP effects on cell growth and transformation, whereas knockdown of YAP abrogates NRG1- and HBEGF-stimulated cell proliferation. Collectively, our study demonstrates the existence of HBEGF & NRGs/ERBBs/YAP/HBEGF & NRGs autocrine loop that controls ovarian cell tumorigenesis and cancer progression.

A review of research progress of FecB gene in Chinese breeds of sheep

FecB gene is a major gene responsible for high prolificacy firstly identified in Booroola Merino sheep. Subsequently, many other aspects of the FecB including endocrinology, fetal and postnatal growth were studied. A forced PCR-RFLP method was performed to screen some Chinese breeds or strains of sheep to determine if FecB gene is responsible for their high prolificacies. The FecB gene was present in some Chinese prolific breeds of sheep, such as Huyang, Small Tail Han (STH), Cele, Duolang sheep and Chinese Merino prolific strains, but absent in the low prolific sheep breeds such as Mongolia, Chinese Merino, Tan, Xinjiang, Hulunbeier, Inner Mongolia Fine Wool and Northeastern Half-fuzz Sheep. It has been confirmed that FecB gene was associated with high prolificacy in some Chinese breeds or strains of sheep. Moreover, introducing FecB gene to some low prolific breeds of sheep by crossbreeding system can improve the reproductive traits.

YAP induces high-grade serous carcinoma in fallopian tube secretory epithelial cells.

Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from fallopian tube secretory epithelial cells (FTSECs). However, the molecular mechanisms underlying the initiation and progression of HGSC derived from FTSECs remains unclear. In this study, we found that the Hippo/Yes-associated protein (YAP) signaling pathway has a critical role in the initiation and progression of fallopian tube and ovarian HGSC. Importantly, YAP was overexpressed in inflammatory and cancerous fallopian tube tissues. Further, overexpression of wild-type YAP, or constitutively active YAP in immortalized FTSECs, induced cell proliferation, migration, colony formation and tumorigenesis. Moreover, the Hippo/YAP and the fibroblast growth factor (FGF) signaling pathways formed an autocrine/paracrine-positive feedback loop to drive the progression of the FTSEC-derived HGSC. Evidence in this study strongly suggests that combined therapy with inhibitors of YAP (such as verteporfin) and FGF receptors (such as BGJ398) can provide a novel therapeutic strategy to treat fallopian tube and ovarian HGSC.

An immunological method to screen sex-specific proteins of bovine sperm

This study was designed to identify sex-specific antibodies (SSAb) in rabbit antisera against bovine sex-sorted sperm, and capture sex-specific proteins of bovine X- or Y- proteins by SSAb. The rabbit antisera against bovine X- or Y-sperm were first produced by a series of immunological approaches, and further purified through immuno-neutralization with excess sex-sorted Y- or X-sperm, respectively, to remove non-sex specific antibodies and enrich sex-specific antibodies. After removal of non-sex specific antibodies, the purified rabbit sera with enriched sex-specific antibodies were screened for sex-specific antibodies by immunofluorescence staining and flow cytometry. The results showed that 3.0, 2.2, and 4.2% of unsorted sperm, sex-sorted X-sperm, and sex-sorted Y-sperm were recognized by the purified rabbit antisera against Y-sperm, respectively, whereas 29.2, 19.7, and 3.9% of unsorted sperm, sex-sorted X-sperm, and sex-sorted Y-sperm were recognized by the purified rabbit antisera against X-sperm. These results suggested that the purified rabbit antisera against X-sperm contained SSAb that preferentially bound to sex-sorted X-sperm. Subsequently, the purified rabbit antisera against X- or Y-sperm were used to immunoprecipitate sex-specific proteins in bovine sperm proteins, and a 30-kDa protein was specifically captured by the rabbit antisera against X-sperm. In conclusion, our results implied that this 30-kDa protein might be a sex-specific protein in bovine X-sperm, which has the potential to be used in immunological procedures for sexing sperm.

The four and a half LIM domains 2 (FHL2) regulates ovarian granulosa cell tumor progression via controlling AKT1 transcription.

The four and a half LIM domains 2 (FHL2) has been shown to play important roles in the regulation of cell proliferation, survival, adhesion, motility and signal transduction in a cell type and tissue-dependent manner. However, the function of FHL2 in ovarian physiology and pathology is unclear. The aim of this study was to determine the role and functional mechanism of FHL2 in the progression of ovarian granulosa cell tumors (GCTs). Immunohistochemical analysis indicated that FHL2 was overexpressed in GCT tissues. Cellular localization of FHL2 in GCT cells was cell cycle dependent. Knockdown of FHL2 suppressed GCT cell growth, reduced cell viability and inhibited cell migration. Consistently, ectopic expression of FHL2 in GCT cells with very low endogenous FHL2 promoted cell growth, improved cell viability and enhance cell migration. Importantly, overexpression of FHL2 promoted GCT progression in vivo. Mechanistic studies indicated that FHL2 regulates AKT1 gene expression in vitro and in vivo. Knockdown of FHL2 or AKT1 in GCT cell lines induced very similar phenotypes. Ectopic expression of constitutively active AKT1 rescued FHL2 knockdown-induced arrest of GCT cell growth and reduction of GCT cell viability, suggesting that FHL2 regulates GCT cell growth and viability through controlling AKT1 expression. Finally, co-immunoprecipitation and chromatin immunoprecipitation analyses indicated that FHL2 functions as a co-activator of NFκB and AP-1 to regulate AKT1 gene transcription. In conclusion, results from the present study indicate that FHL2 exerts its oncogenic action in GCT cells via controlling AKT1 gene expression. FHL2 is a promising target for the development of novel drugs against ovarian granulosa cell tumor.

Somatostatin and its receptors: functional regulation in the development of mice Sertoli cells.

Recently, Sertoli cells have been ascertained as the target for the regulatory peptide somatostatin (SST). Therefore, the present study investigated the expression of somatostatin receptors, their age-related alterations and homologous regulation by in vitro treatment with SRIF14 on mice Sertoli cells; furthermore, it dealt with SRIF14 action on growth progression, apoptotic activity and related gene expressions in these cells. We found that mice Sertoli cells expressed all SST1-5 receptors with differential intensities. Age-related real-time PCR of all somatostatin receptors identified abundance of SSTR2 and SSTR5 mRNA level during Sertoli cell developmental period. Furthermore, higher level of these two receptors was independent of SRIF14, as treatment with SRIF14 failed to induce both receptor expressions when compared with control. Somatostatin treatment elicited a dose-dependent decrease in forskolin stimulated cAMP production. Low (100pM and 10nM) and high dosage (1μM) groups of SRIF14 significantly promoted apoptosis, while all treatment groups led to dose dependent cessation (P<0.05) of G1 phase of cell cycle as further validated by increase in casp3, decrease in bcl2, elevation of P21 (all by western blot) and decrease in Igf1 expressions, similarly, SST treatment caused a dose dependent suppression in the mRNA level of kitl gene, which is important in the regulation of spermatogenesis. These findings suggest that somatostatin and its receptors (SSTR2 and SSTR5) are important markers in the regulation and development of Sertoli cell; furthermore, it portrays physiological inhibitory role in Sertoli cell development by inducing apoptosis and cell cycle arrest.

Integrating RNA-seq and GWAS reveals novel genetic mutations for buffalo reproductive traits

Genome-wide association study (GWAS) has been applied in buffalo breeding programs and been used to identify a number of candidate genes associated with buffalo reproductive traits. The genetic code of specific genes underlying buffalo reproductive traits remains unclear. Association study that measures both genetic and transcriptional variation has been applied for the investigation of complex traits. To investigate genes involved in buffalo reproductive traits, integrated RNA-seq results were investigated of buffalo granulosa cells and candidate genes which were reported to be associated with buffalo reproductive traits in a previous GWAS. A large number of variants were detected by RNA-seq, and 214 variants were located within the buffalo reproductive candidate genes identified by GWAS. A further association study in 462 Italian Mediterranean buffalo indicated that 25 SNPs distributed in 13 genes were associated with reproductive traits. Of the 13 genes, 11 were expressed in granulosa cells of all antral follicle development stages, and significant difference was found in the expression of NDUFS2 between follicles of diameter <8 mm and > 8 mm. These findings extend the results of GWAS by expanding the knowledge about new and potentially functional single-nucleotide polymorphisms and provide useful information about regulatory genes affecting buffalo reproductive traits.