H2AFZ, RNF4 and NR4A1 loci are associated with boar semen quality by population association studies.

Spermatogenesis is a complex process regulated by many genes. In this study, H2AFZ, RNF4 and NR4A1 genes were selected as candidate genes for boar semen quality traits based on their functions during spermatogenesis, and the associations of three loci (H2AFZ c.192 + 210-192 + 213delCGAT, RNF4 c.374 + 358 T > C and NR4A1 c.956 + 796 A > G) with sperm quality traits were analyzed in Duroc (n = 185), Large White (n = 87) and Landrace (n = 49) pig populations. The results showed H2AFZ c.192 + 210-192 + 213delCGAT AA boars produced 1.52% lower abnormal sperm rate (ASR) than AB boars in Landrace pigs (p < 0.05); RNF4 c.374 + 358 TC boars produced 0.31 × 108/ml higher sperm concentration (SCON) than CC boars (p < 0.05) in Large White pigs; NR4A1 c.956 + 796 A > G was associated with ASR in Duroc and Large White pigs and was associated with sperm motility (MOT) in Large White and Landrace pigs. This study indicated the H2AFZ, RNF4 and NR4A1 loci were the potential molecular markers for improving the semen quality traits in boars.

miR-638 Inhibits immature Sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene.

Numerous studies have demonstrated that microRNAs (miRNAs) play important roles in cell growth, apoptosis and spermatogenesis. Our previous study showed that miR-638 was differentially expressed in sexually immature and mature testes of Large White boars. Here we reported that sperm-associated antigen 1 (SPAG1) was a direct target gene of miR-638. Moreover, miR-638 inhibited cell proliferation and cell cycle, and promoted apoptosis of porcine immature Sertoli cells. Key genes including phosphorylated phosphatidylinositide 3-kinases (p-PI3K) and phosphorylated serine/ threonine kinase (p-AKT) in PI3K/AKT pathway as well as cell cycle factors including c-MYC, cyclin-D1 (CCND1), cyclin-E1 (CCNE1) and cyclin-dependent kinase 4 (CDK4) were all significantly down-regulated after overexpression of miR-638 or RNAi of SPAG1. Notably, mRNA levels of SRY-related HMG-box 2 (SOX2) and POU domain, class 5, transcription factor 1 (POU5F1) essential for spermatogonia proliferation were significantly suppressed in SPAG1 siRNA- transfected ST cells. This study suggests that miR-638 regulates immature Sertoli cell growth and apoptosis by targeting SPAG1 gene which can indirectly inactivate PI3K/AKT pathway, and plays roles in pig spermatogenesis.

miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4 (RNF4) gene.

A growing number of reports have revealed that microRNAs (miRNAs) play critical roles in spermatogenesis. Our previous study showed that miR-762 is differentially expressed in immature and mature testes of Large White boars. Our present data shows that miR-762 directly binds the 3' untranslated region (3'UTR) of ring finger protein 4 (RNF4) and down-regulates RNF4 expression. A single nucleotide polymorphism (SNP) in the RNF4 3'UTR that is significantly associated with porcine sperm quality traits leads to a change in the miR-762 binding ability. Moreover, miR-762 promotes the proliferation of and inhibits apoptosis in porcine immature Sertoli cells, partly by accelerating DNA damage repair and by reducing androgen receptor (AR) expression. Taken together, these findings suggest that miR-762 may play a role in pig spermatogenesis by regulating immature Sertoli cell growth.

Characterization of swine testicular cell line as immature porcine Sertoli cell line.

Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions.

Exploiting RNA-sequencing data from the porcine testes to identify the key genes involved in spermatogenesis in Large White pigs.

Mammalian testis development and spermatogenesis play critical roles in male fertility. However, little genomic information is available for porcine sexually mature and immature testis. Presently, we detected approximately 76% of previously annotated genes that were expressed in the porcine testes by RNA sequencing. Taking an FDR of 0.001 and a |log2Ratio| of 1 as cutoffs, 10,095 genes were significantly differentially expressed between two stages, including 242 spermatogenesis-associated genes. These genes were significantly enriched to GO BP terms concerning spermatogenesis, male gamete generation, developmental process and sexual reproduction; to the KEEG pathways, including focal adhesion, ECM-receptor interaction, and phagosome. 186 extended transcripts, 1273 alternative splicing events and 2846 SNPs were detected in spermatogenesis-associated DEGs. Two PIWIL4 SNPs were successfully validated and suggested to be the potential molecular markers for semen quality. This study will help identify the specific genes and isoforms that are active in porcine spermatogenesis and sexual maturity.

Study on TCM Syndrome Differentiation of Primary Liver Cancer Based on the Analysis of Latent Structural Model.

Primary liver cancer (PLC) is one of the most common malignant tumors because of its high incidence and high mortality. Traditional Chinese medicine (TCM) plays an active role in the treatment of PLC. As the most important part in the TCM system, syndrome differentiation based on the clinical manifestations from traditional four diagnostic methods has met great challenges and questions with the lack of statistical validation support. In this study, we provided evidences for TCM syndrome differentiation of PLC using the method of analysis of latent structural model from clinic data, thus providing basis for establishing TCM syndrome criteria. And also we obtain the common syndromes of PLC as well as their typical clinical manifestations, respectively.

Discovery of two potential DAZL gene markers for sperm quality in boars by population association studies.

The Deleted in AZoospermia Like (DAZL) gene is a member of the DAZ (deleted in azoospermia) gene family which are critical for successful germ cell development in diverse animals. In previous research, we discovered two SNPs (DAZL c.570+385 A>G, DAZL c.735+150 C>A) associated with litter size traits in sows. Here we selected DAZL gene as a candidate gene for boar sperm quality traits based on its function on the formation of germ cells during spermatogenesis, and then analyzed the associations of the two SNPs with sperm quality traits in Duroc (n=185), Large White (n=87) and Landrace (n=49) pig populations. The results showed DAZL c.570+385 AG boars had significantly higher motility (MOT) than GG boars (P<0.05) in Large White and Landrace pigs; Duroc DAZL c.735+150 CA boars had significantly lower MOT and lower sperm concentration (SCON) than CC and AA boars (P<0.05), respectively; Large White DAZL c.735+150 CC boars produced lower abnormal sperm rate (ASR) than CA and AA boars (P<0.05), with favorable allelic C substitution effect -0.94% (P<0.05). Our research indicated that DAZL c.570+385 A>G and DAZL c.735+150 C>A locus were the potential molecular markers for improving the sperm quality traits in pigs.

Associations of TCF12, CTNNAL1 and WNT10B gene polymorphisms with litter size in pigs.

In previous research, several WNT signaling pathway genes including transcription factor 12 (TCF12), catenin alpha-like protein 1 (CTNNAL1) and wingless-type MMTV integration site family, member 10B (WNT10B) were differentially expressed in PMSG-hCG stimulated preovulatory ovarian follicles of Large White and Chinese Taihu sows. In the present research, these three genes were selected as the candidate genes for litter size traits in pigs. Four mutations (TCF12 c.-201+65 G>A, TCF12 c.-200-300 G>A, CTNNAL1 c.1878 G>C and WNT10B c.*12 C>T) were detected in eleven pig populations, and results indicated CTNNAL1 c.1878 G and WNT10B c.*12 C were the major alleles in all tested pig populations, while TCF12 c.-201+65 A and TCF12 c.-200-300 A were the major alleles in several Chinese native pig breeds. Association analysis of four mutations with litter size in Large White and DIV pigs showed that both the signficant differences of total number born (TNB) and number born alive (NBA) among three genotypes and the significance of additive effects appeared at TCF12 c.-200-300 G>A and CTNNAL1 c.1878 G>C loci, suggesting these two mutations might be reliable markers for pig selection and breeding.