Lipocalin-type Prostagland In D Synthase In Male Reproduction: Basic And Applied Research

Lipocalin-type prostaglandin D synthase (L-PGDS), also known as β -trace protein(β-TP), is a unique member of the Lipocalin superfamily composed of various secretory lipophiilic ligand-carrier proteins with low molecular weight, and it is the first enzyme to be recognized in this superfamily. L-PGDS is localized mainly in the central nervous system and male genital organs of various mammals and is secreted into various body fluids, such as the cerebrospinal fluid, serum, seminal plasma, urine and aminotic fluid. L-PGDS is bifunctional, acting as a PGD₂-producing enzyme and as a potential transporter of hydrophobic molecules. L-PGDS appears to be involved in several functions in vivo including induction of sleep, temperature regulation nocieption and modulation of odour. L-PGDS is also closely associated with fertility, maybe playing an important role in the development and maturation of sperm. Studies indicate that monitoring the concentrations of L-PGDS in the body fluids are useful for the diagnosis of several neurological disorders, dysfunction of sperm formation, and cardiovascular and renal diseases.Although the structure and character of L-PGDS have been researched clearly, the distribution, specific function, metabolic process, mechanism of action and the relationship with others diseases are short of sthenic experiment to confirm. This research focuses on the basic and applied research of L-PGDS.The pPIC9/htL-PGDS was linearized with Bg1 â…¡ followed by transformation of Pichia Pastoris GS115 with electroporation. After PCR analysis of Pichia intergrants, methanol was used to induce the expression of the his-tag protein. One recombinant clone was found to express L-PGDS with a molecular mass of 27 000, which was identical to that of nativeL-PGDS. The band at MT 27 000 was about 18% of the total proteins in the culture supernant, and the expression level could be as high as 27mg/L. The recombinant L-PGDS purified with Ni^NTA resin showed considerable heterogeneity with a major protein band at about Mr 27 000 and two minor bands at about 24 000 and 21 000. According to the results of glycosylation analysis, we speculated that the three protein bands were corresponded to the di- (27 kD), mono-(24 kD), and non-glycosilated (21 kD) isoforms respectively. After forming the complex with purified L-PGDS, the UV spectra of all-trans retinoic acid were red-shifed approximately 30nm. In the immunoblotting test with L-PGDS polyclonal antibody, a highly specific 27kDa band was observed.The above purified recombinant L-PGDS was used as an antigen to immunize BALB/c mice, and then cell fusion was performed with 50% PEG. After limiting dilution, the hybridoma cells were inoculated to BALB/c mouse abdominal cavity to generate ascitic fluid-type L-PGDS McAb. One strain of IgGl L-PGDS McAb was harvested, and the titer was 1:1000, the affinity was 2 X 108 L/mol. Western blotting of human seminal plasma samples probed with L-PGDS McAb was immunoreactive at 27 kD.The variable region genes of L-PGDS McAb were amplified by RT-PCR from hybridoma cells, using 5' degenerate primers of leader sequences. After sequenced, one VH and two V k were obtained. But one of the V k was an aberrant V k transcript, derived from myeloma cell line. The other VH and V k were functional genes, for they were highly homologous to the variable region genes of murine immunoglobulin.The variable region genes of L-PGDS McAb were assembled to a whole ScFv gene by SOE PCR, using a Linker [ (Gly),,Ser]3. Then we constucted ScFv to expression vector pET-28a(+), and expressed it in E.Coli BL21. As a result, many soluble ScFv were demonstrated by SDS-PAGE, which was expressed under 25°C or 28°C and BL21 was abducted by 0. 02 mM IPTG. Thenwe used Ni^NTA affinity chromatography to purify these soluble proteins, and the concentration reached to 2 mg/100 ml. The purified ScFv was proved to have L-PGDS antigen binding activity and specificity by ELISA and immunofluorescence competitive test.The expression of L-PGDS in human testis and epididymidis was assessed by RT-PCR and immunoblotting. Using immunohistochemistry to explore the distribution of L-PGDS in testis , epididymidis and on sperm. L-PGDS on sperm was analyzed by flow cytometry. The semen donors were categorized in two groups: normal and oligospermic, then we evaluated their correlation. L-PGDS was strikingly expressed in the testis and caput epididymidis, while moderate to weak expressing was observed in the corpus and cauda epididymidis. The results of immunohistochemistry and immunocytochemistry displayed that L-PGDS was mainly localized within the cytoplasm and the cilia of the epithelial cells in epididymidis and on human sperm. The differences of L-PGDS on sperm were statistically significant between groups. L-PGDS on sperm correlated positively with sperm density(r = 0.700) and progressive motility(r = 0.316). A significant reduction of L-PGDS on sperm was observed in severe oligozoospermic patients compared to normozoospermic subjects, and a significant correlation between L-PGDS on sperm and sperm density was found. The dose of expression was different, which suggested that L-PGDS may play some kind of role during the process of the spermatozoa maturation.In order to examine the clinical value of seminal plasma components in the evaluation of sperm quality and in the differential diagnosis of men with infertility, We analyzed 92 seminal plasmas for acid phosphatase, alpha—glucosidase, fructose and L-PGDS. L-PGDS was analyzed by ELISA procedure. The semen donors were categorized in three clinical groups: normal, oligospermic, azoospermic. We then evaluated whether any of thesebiochemical markers were associated with other parameters of sperm quality, including patient age, sperm density, progressive motility, and ejaculate value. We found that L^PGDS concentration and alpha glucosidase were both significantly associated with sperm quality. L-PGDS concentration correlated positively with sperm density (r = 0.813), progressive motility (r = 0.380), and alpha^glucosidase (r = 0.426). Alpha^glucosidase correlated positively with sperm density (r = 0. 382). Our findings suggest that L-PGDS, nearly just the same as alpha-glucosidase, hints at an obstruction of the seminal ducts, and L-PGDS concentration in seminal plasma is a new marker that may aid in the differential diagnosis of obstructive and nonobstructive azoospermia.In words, we have successfully expressed recombinant L-PGDS with Pichia Pastoris GS115, established a hybridoma cell secreting anti- L-PGDS monoclonal antibodies stably, explored the distribution of L-PGDS in testis , epididymidis and on sperm, established the ELISA method to analysis L-PGDS in seminal, examined the clinical value of four seminal plasma components in the evaluation of sperm quality and in the differential diagnosis of men with infertility.