Immunization with oral KISS1 DNA vaccine inhibits testicular Leydig cell proliferation mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes in goats.

This study aimed to analyze the effect and mechanism of immunization of oral KISS1 DNA vaccine on the proliferation of goat testicular Leydig cells. Ten 8-week-old male goats were randomly divided into KISS1 DNA vaccine and control groups for immunization (five goats each group). These goats were sacrificed at 8 weeks after primary immunization, and the tissue samples of hypothalamus, pituitary, and testis and Leydig cell samples were collected for RT-PCR and CCK8 assay. Immunization with the oral KISS1 DNA vaccine effectively inhibited the proliferation of Leydig cells, the expression of hypothalamus KISS1, GPR54, and GnRH mRNA, pituitary GnRHR and LH mRNA, testicular LHR mRNA, and apoptosis-inhibitory gene Bcl-2 mRNA in Leydig cells. By contrast, the immunization enhanced the mRNA expression of apoptosis-promoting gene Bax and Clusterin in Leydig cells. These findings indicate that immunization with the oral KISS1 DNA vaccine can inhibit the proliferation of goat testicular Leydig cells mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes.

Temporal expression of the KISS1/GPR54 system in goats' testes and epididymides and its spatial expression in pubertal goats.

Kisspeptin, encoded by the KISS1 gene, and its receptor GPR54 are essential in puberty onset and male fertility due to their central regulatory roles. However, the roles of KISS1/GPR54 in peripheral tissues remain unclear. This study aimed to investigate the temporal expression patterns of KISS1/GPR54 in goat testes and epididymides and its spatial expression patterns in pubertal goats. Immunohistochemical analysis revealed that kisspeptin/GPR54 were localized in Leydig, Sertoli, and germ cells of pubertal goats' testis, as well as in principal and basal cells of the epididymis. RT-PCR revealed a marked variation in the KISS1/GPR54 expressions in the testes and epididymides from the age of first week to adulthood. KISS1 and GPR54 mRNA levels in testes decreased from the age of first week to two months and then increased from two months to puberty and adulthood. The KISS1 and GPR54 mRNA levels in Leydig cells decreased from the age of one week to two months and increased from two months to puberty, and then decreased from puberty to adulthood. Only GPR54 mRNA levels in the epididymides increased from the age of one week to two months and puberty, and then decreased from puberty to adulthood. RT-PCR analysis showed the different spatial expression patterns of KISS1/GPR54 in pubertal goat tissues. The KISS1 mRNA level was high in the hypothalamus, moderate in pancreas, liver, epididymis and testis; and low in the other tissues. The GPR54 expression was high in the pancreas and testis; moderate in pituitary, hypothalamus and mesenteric lymph node; and low in the other tissues. In conclusion, the KISS1/GPR54 system possessed distinct temporal expression profiles in goats' testes and epididymides, as well as different spatial expression patterns in pubertal goat tissues, which implied the possible local role of this system in goats' testes, epididymides, and other peripheral tissues.

Local expressions and function of Kiss1/GPR54 in goats' testes.

Kisspeptin, encoded by the Kiss1 gene, and its receptor GPR54 have a central regulatory role in the male reproduction. However, their functions in peripheral tissues, such as testes, remain unclear. This study investigated the local expressions and function of Kiss1/GPR54 in goats' testes. The mRNA expression of Kiss1/GPR54 in pubertal goat Leydig cells was detected through reverse transcriptase PCR (RT-PCR), and its protein expression in Leydig cells or the testis was examined by immunohistochemistry and Western blot analysis. Isolated and cultured Leydig cells were treated with different concentration of kisspeptin (0, 1, 10 and 100 µM) and kisspeptin antagonist for 4, 24 or 72 h. The direct effect of kisspeptin on testosterone secretion and Kiss1/GPR54 mRNA expression was evaluated by ELISA and RT-PCR. Kiss1/GPR54 mRNA and protein were expressed in Leydig cells and spermatids, and GPR54 were expressed in Sertoli cells. Kisspeptin treatment significantly stimulated testosterone secretion in Leydig cells, with the highest levels found under 24 h of treatment with 10 µM kisspeptin. Treatment with kisspeptin + kisspeptin antagonist significantly reduced the kisspeptin-stimulated testosterone secretion in Leydig cells. Kisspeptin treatment significantly enhanced the expression of Kiss1/GPR54 mRNA in Leydig cells. These data suggest the local expressions of Kiss1/GPR54 in goats' testes and its autocrine role in Leydig cells, which is helpful in understanding the regulation role of kisspeptin/GPR54 system in other peripheral tissues.

Potent effect of KISS1-54 DNA vaccine compared with KISS1-10 DNA vaccine in inhibiting the fertility of female rats.

BACKGROUND: Most studies on immunocastration currently focused on male animals. However, immunization of male animals does not completely inhibit sexual behavior and fertility. This study aimed to compare the immunocastration effect of KISS1 DNA vaccines encoding different lengths of kisspeptins in female rats for effective castration effects on both male and female rats. METHODS: Fifteen female rats were randomly divided into three groups. The rats in T1 group or T2 group was orally given respectively KISS1-54 or KISS1-10 DNA vaccines with fused tPA signal peptide, and the control group (Group C) was orally administered with empty vector vaccine, at a dose of 5 × 109 CFU/rat at weeks 0, 3 and 6 of the study. Blood samples were collected by retroorbital bleeding before primary immunization and at weeks 3 and 9 after primary immunization. RESULTS: Both KISS1-54 and KISS1-10 DNA vaccines induced the body's humoral immune response, and the anti-kisspeptin antibody titres in the T1 group were significantly higher than that in T2 and C groups (p < 0.05). The rats in T1 group has lower serum kisspeptin and estradiol levels than those in T2 and C groups and smaller litter size of rats than those in the control group after mating (p < 0.05). No significant difference was observed between T2 and C groups. The levels of KISS1 and GPR54 mRNA in the hypothalamus and ovaries of the T1 group were significantly lower than that in control group. However, the levels of KISS1 mRNA in the T2 group were significantly lower than that in the control group only in ovaries (p < 0.05). CONCLUSION: The oral KISS1-54 DNA vaccine with fused tPA signal peptide was more effective than that KISS1-10 DNA vaccine in suppressing fertility of female rats.