Li Han

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.

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.

Development and evaluation of a novel DNA vaccine expressing inhibin α (1–32) fragment for improving the fertility in rats and sheep

Inhibin is an important protein hormone in regulating folliculogenesis. Immunization against inhibin can improve follicle developments. The objective of present study is to investigate inhibin DNA immunization as a potential tool for improving follicle development and litter sizes of female animals. In our study, the inhibin DNA vaccine was constructed with inhibin alpha (1-32) fragment inserted into the C termination of HBsAg-S. Ninety rats and forty sheep were immunized with inhibin DNA vaccine. In rats, immunization against inhibin resulted in increase of positive sera ratio (P<0.01). The treatment was accompanied by a significant increase in the total number of mature follicles of >0.8mm in diameter on the onset of estrous cycle after twice immunization (31.0+/-3.9 in the test groups versus 27.4+/-5 in control groups) and after third immunization (35.2+/-6.7 in the test groups versus 30.3+/-5.2 in control groups). Litter sizes were significantly (P<0.05) bigger in rats treated with inhibin DNA vaccine (12.7+/-4.5 n=6) than in control (9.8+/-4.5). In sheep, twinning rate in test groups (39.2%) was significantly higher than that in control groups (10%) after immunization (P<0.05). These results indicated that inhibin was an important factor in improvement of fertility in rats and sheep, and demonstrated that DNA immunization against inhibin could induce more mature follicles resulting in increased litter sizes. Our results revealed that inhibin DNA vaccine may be an alternative to the use of exogenous gonadotrophins for increasing ovarian follicular development and improving animal fertility.

The chemopreventive effect of β-cryptoxanthin from mandarin on human stomach cells (BGC-823)

β-Cryptoxanthin, a provitaminic carotenoid, present in many fruits and vegetables, has been associated with decreased risk of chronic diseases, including cancer. The influence of β-cryptoxanthin derived from mandarin on the proliferation of the stomach tumor cell line BGC-823 was tested using MTT and cell count assay at 72 h and dose-response (from 0.01 to 20 μM). β-Cryptoxanthin suppressed the cell migration by the scratch assay. Furthermore, β-cryptoxanthin induced an accumulation of cells in the G1/G0 phase of the cell cycle (as detected by flow cytometry), which was in accordance with an increased expression of p21 and down regulations of cyclin D1 and cyclin E, detected by Western blot analysis, and β-cryptoxanthin increased the mRNA levels of retinoic acid receptor β (RARβ) with the treatment at 10 μM for 24 h. Collectively, the above findings suggest that β-cryptoxanthin could be therapeutic in the treatment of stomach cancer cell in vitro.

Characterization of the Mechanism of Inhibin α-Subunit Gene in Mouse Anterior Pituitary Cells by RNA Interference

Inhibin, a member of the transforming growth factor-β [TGF-β] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary–gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of β-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-β concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-β in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinβ which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.

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.

Knockdown of CEBPβ by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis.

Cultured ovarian granulosa cells (GCs) are essential models to study molecular mechanisms of gene regulation during folliculogenesis. CCAAT enhancer binding proteins β (CEBPβ) has been identified in the ovary and is critical for follicular growth, ovulation and luteinization in mice. In the present study, hormonal treatment indicated that luteinizing hormone (LH) and exogenous human chorionic gonadotropins (hCG) significantly increased the expression of CEBPβ in porcine GCs. By RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors, CEBPβ gene was successfully knocked down in porcine GCs, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respectively. We further found that knockdown of CEBPβ significantly increased the expression of p-ERK1/2. Furthermore, CEBPβ knockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium. To uncover the regulatory mechanism of CEBPβ knockdown on cell cycle and steroids synthesis, we found that the mRNA expression of bcl-2 (anti-apoptosis), StAR and Runx2 (steroid hormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3 and p53), hormonal synthesis (CYP11A1) and cell cycle (cyclinA1, cyclinB1, cyclinD1) were down-regulated, suggesting that knockdown of CEBPβ may inhibit apoptosis, regulate cell cycle and hormone secretions at the transcriptional level in porcine GCs. Furthermore, knockdown of CEBPβ significantly increased the expression of PTGS2 and decreased the expression of IGFBP4, Has2 and PTGFR which are important for folliculogenesis in porcine GCs. In conclusion, this study reveals that CEBPβ is a key regulator of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis.

Effects of parity on uterine involution and resumption of ovarian activities in postpartum Chinese Holstein dairy cows.

Favorable uterine involution and ovarian activity are very important for the next reproductive cycle of postpartum cows. The objective of this study was to evaluate the effect of parity on uterine involution and resumption of ovarian activity in Chinese Holstein dairy cows after calving under similar postpartum nutritional conditions. Traits of the status of uterus and ovaries detected by ultrasonography, dry matter intake (DMI), milk yield, body condition score (BCS), and estradiol concentration in milk samples were analyzed for 46 Chinese Holstein dairy cows in various parities (primiparous=18; biparous=13; multiparous=15). The results showed that there was no significant difference for DMI, BCS, and milk yield among different parities; all cows were considered to be under similar nutritional conditions. Days of the previous gravid uterine horn involution were significantly greater in primiparous dairy cows than in biparous and multiparous dairy cows. Days from calving to ovulation (first and second) and the number of follicular waves to first ovulation were significantly greater in primiparous cows than in multiparous cows. In summary, there was a significant negative relationship between parity and postpartum uterine involution and resumption of ovarian activity in Chinese Holstein cows under similar body conditions.

Oral vaccination with inhibin DNA delivered using attenuated Salmonella choleraesuis for improving reproductive traits in mice

The objective of this study was to examine the efficacy and safety of a novel inhibin vaccine containing inhibin α (1–32) fragments in mice. A recombinant plasmid pVAX‐asd‐IS was constructed by inserting recombinant inhibin α (1–32) and the hepatitis B surface antigen S into the plasmid in which the asd gene, rather than the kanamycin gene, was a selection marker. Ninety Kuming mice were divided into six groups consisting of 15 mice each. First group was (C1) injected with 200 µl of PBS, second (C2) received 1 × 1010 CFU of crp−/asd− C500/pVAX‐asd and served as vector control, third did not receive any treatment (C3), while fourth, fifth, and sixth group received 1 × 1010, 1 × 109, 1 × 108 CFU of the recombinant inhibin vaccine crp−/asd− C500/pVAX‐asd‐IS (group T1, T2, T3), respectively. Western blotting demonstrated that recombinant expressed inhibin protein possessed immune function and that this plasmid could replicate for up to 40 generations stably. Vaccination with this strain at a dose of 1 × 1010 CFU/200 µl per mouse induced high anti‐inhibin antibody levels, significantly increased large‐follicle production in T1 group (p < 0.05) and average litter size (p > 0.05) compared with control groups. Integration studies showed no evidence of inhibin fusion gene integrated into mices genome 2‐month after immunization. These results suggest that the vaccine described in the present study may provide a safe method to improve reproductive traits in animals. A trend towards increased litter size and significant increase in large follicle population depict that this vaccine may have direct application in large animal industry.

Evaluation of efficacy, biodistribution and safety of antibiotic-free plasmid encoding somatostatin genes delivered by attenuated Salmonella enterica serovar Choleraesuis

We describe here a balanced-lethal system using an Asd(+) expression plasmid pVGS/2SS-asd encoding two copies of somatostatin (SS) genes carried by Δasd/Δcrp double mutant Salmonella enterica serovar Choleraesuis (named C501). The advantage of this novel system is the use of asd (aspartate-semialdehyde dehydrogenase) gene as selection marker to replace the antibiotic resistance markers, thus eliminating the industrial cultivation and environmental problems. We then evaluated the efficacy, biodistribution and safety of antibiotic-free plasmid delivered by strains C501. Mice orally immunized with C501 (pVGS/2SS-asd) had significantly higher levels of anti-SS total IgG and IgA antibodies than control mice and demonstrated a bias toward Th2-associated responses (IgG1/IgG2a ratio>1). Safety evaluation indicated that vaccinated mice displayed no abnormal clinical signs and histological changes. Biodistribution result revealed that the GS/2SS message was detected in several examined tissues with the exception of ovary and brain, but was rapidly cleared from the body (approximately 10 days). Furthermore, the risk of integration of plasmid pVGS/2SS-asd into the host cellular genome was considered to be negligible. These results may have important implications for the use of vaccine strain C501 (pVGS/2SS-asd) in domestic animals and prompt new perspectives on the safety of DNA vaccines delivered by attenuated bacteria.