Earticle

Retinol-Binding Protein-4 (RBP-4) is a low molecular weight lipocalin, which mainly functions as a carrier for vitamin A. Though liver is the main machinery for synthesis of this protein, it is also detectable in other extrahepatic tissues, for example, ovary, uterus, and placenta. Recent evidences have shown that RBP-4 plays important roles in animal reproduction, for example, promoting the development of uterus and embryo. To the best of our knowledge, our laboratory firstly reported that high level of RBP-4 existed in follicular fluid from follicular cysts in sows. Moreover, we have also found that RBP-4 could be secreted by granulosa cells, and RBP-4 receptor was detected in granulosa cells. However, there is no any evidence on the role of RBP-4 in regulating the follicular development. Therefore, cloning and expression of RBP-4 and preparation of polyclonal antibody could help us to explore the role of RBP-4 in follicular development. The aim of this work was to construct prokaryotic expression system of swine RBP-4 gene. The total RNA was extracted from swine’s normal ovarian tissue. The sequence including the whole length of RBP-4 was amplified by RT-PCR and inserted into pEASY-E1.Then transformed into E. coli BL21(DE3)pLysS after gene sequencing. Three hours later, adding IPTG with the final concentration of 1mmol/L and inducing five hours. After centrifugation, the supernatant was discarded. By adding Glucose to Luria-Bertani broth, the expressions of protein were increased. SDS-PAGE showed that the RBP-4 gene expressed in the form of inclusion body with a molecular weight of 21KD. Western-Blot results showed that the target protein could be specifically recognized by mouse anti-human monoclonal antibody. Prokaryotic expression vector of RBP-4 gene was successfully established, and the gene was successfully expressed n E. coli, which is ready for purification and RBP-4 polyclone antibody. Meanwhile, these results were beneficial to investigate the function of RBP-4 in follicular development.

The present study was aimed to investigate the molecular mechanisms of active immunization against GnRH by focusing on effects of active immunization against GnRH on hypothalamic-pituitary-testicular axis. Adult male rats (n=36) were randomly and equally allocated into three groups: control (no treatment), surgically castrated, or immunized against 50 ug D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 12 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 2-week intervals until rats were killed at 20 weeks of age. At decapitation, testes weight /volume and pituitary weight were recorded. Hypothalamus, pituitary and testis were collected to detect the mRNA expressions of reproduction-related genes with real-time fluorescence quantitative PCR, and the hypothalamus and testes were excised for tissue GnRH detection and morphological observation, respectively. Compared to intact controls, immunocastration reduced (p<0.05) serum concentrations of testosterone, LH and FSH, and the GnRH content in the median eminence, reduced the weight of the hypohysis (p<0.01), and induced testicular atrophy (suppression of spermatogenesis). Furthermore, mRNA expression of GnRH in the hypothalamus, GnRH receptor, LH-β and FSH-β in the pituitary, LH receptor and FSH receptor in the testes, and genes in sex steroid feedback loops (androgen receptor (AR), kisspeptin encoded gene (Kiss-1) and kisspeptin receptor (GPR54) in the hypothalamus were decreased in immunocastrated rats compared to intact controls (p<0.05). Similarly, surgical castration reduced GnRH in the median eminence as well as mRNA expression of GnRH, AR, Kiss-1 and GPR54 in the hypothalamus (p<0.05). These results suggest that active immunization against GnRH reduces GnRH synthesis in the hypothalamus by decreasing androgen-androgen receptor-Kisspeptin-GPR54 signaling pathways and causes dysfunction of the pituitarytesticular axis. Both the loss of GnRH synthesis and dysfunction of the pituitary- testicular axis seem to be mechanisms underlying immunocastration.