Study Of Contraception Effects Of FSH-Occludin Fusion Protein By Targeting Sertoli Cells

Background:Sertoli cell provides mechanical and nutritional support to about 30~50 developing germ cells at different stages of the seminiferous epithelial cycle during spermatogenesis. Furthermore,adjacent Sertoli cells in the seminiferous epithelium form an almost impermeable barrier near the basement membrane known as the blood–testis barrier (BTB).This BTB creates a unique microenvironment that sequestered virtually all the events associated with postmeiotic germ cell maturation from the systemic circulation. It also restricts the access of nutrients, hormones, electrolytes, and other biological substances from the interstitium to the developing germ cells behind the BTB.Tight junctions (TJs) between Sertoli cells at the basal compartment form the BTB.At the late stage VIII through early stage IX, preleptotene and leptotene spermatocytes must traverse the BTB, where junctions are rapidly broken and subsequently reassemble to allow the spematocytes to pass through.The translocation of germ cells across the seminiferous epithelium involves not only the dynamic modulation of TJs at the BTB, but also the extensive restructuring of actin-based adherens junctions (AJs) between Sertoli cells as well as between Sertoli and germ cells. thereafter, so that differentiating germ cells could move towards the adluminal compartment for further development.Several studies have demonstrated the unique function of occludin in TJs. Occludin, a 65 kDa protein,was the first TJ-integral membrane protein identified in multiple epithelia,Each occludin molecule consists of four transmembrane domains with a long carboxyl-terminus and a short amino-terminus in the cytoplasm,one intracellular loop,and two extracellular loops.The first extracellular domain is involved in cell-cell adhesion function. The second extracellular loop apparently is needed for the assembly and sealing of TJs, any change of occludin can destroy TJs then AJs,and lead to germ cell fall off from epithelial,TJs plays a key role in spermatogenesis by causing mature sperm to be released from the surrounding Sertoli cells. The integrity of the epithelial cell layer(s) is maintained by intercellular junctional complexes composed of tight junctions (TJs), adherens junctions, and desmosomes,if non-target interfere the function of Occludin would induce TJs functional lesion in other tissues. So specific interfering the function of TJs should be a new choice in developing male contraceptive.Follicle-stimulating hormone (FSH) is the central hormone of mammalian reproduction and FSH action is necessary for gonadal stimulation at puberty and gamete production during the fertile phase of life.FSH effects on gametogenesis by binding to a specific receptor that is located only on the surface of Sertoli cells in the testis and granulosa cells in the ovary. FSH is consists of two subunits, the a- and theβ-subunits, the a-subunit is non covalently joined to theβ-subunit to form a biologically active hormone, but it is theβ-subunit, that confers hormone specificity.Object:In this study,we choose receptor binding region of FSH-βand the secondary extracellular domain of Occludin as objective fragment and recombination. To obtain fusion protein through Pichia Pastoris expression technology and expect to target Sertoli cell then interfere its TJs . It should provide an attractive candidate for development of a contraceptive vaccine.Methods:There are three parts in our study:①The construction of pPIC9KFSH-Occludin yeast expression vector. Firstly, the full length cDNA encoding FSH and Occludin was amplified respectively from a mouse brain and testicle by RT-PCR method .Then mutated the aim amino acids by Site-Direct Mutation and partial overlapping double PCR get recombination gene .Finally, the recombination gene was subcloned into Not I and EcoR I sites of pPIC9K vector. The recombinant expression vectors were verified by DNA sequence test.②The Pichia yeast expression,purification of FSH-Occludin protein and the primary test of its biological activity. The verified recombinant vector was transformed into Pichia strain GS115 by electroporation. The positive clones grown on the HIS drop out medium were selected and confirmed by PCR analysis. The transformants with multiple inserts were screened in vivo by their resistance to G418, and Mut+ phenotype by Mut screening. A time course study of expression was performed to optimize the protein yield.Large baffled flasks were used to scale up the expression, and the culture supernatant were harvested on the time point obtained from previous study.The culture supernatant containing the recombinant protein were demineralized and dialysis, purified by Sephadex G-50 column, and froze in -70℃.The purified protein was confirmed by SDS-PAGE and Western blot analysis. Its biological activity was tested by immunohistochemical assay.③Study immune contraception effects of the FSH-occludin fusion protein . The adult male Balb/c mice were immunized with the fusion protein, detected the sepcific antibody,testosterone(T)and FSH concentrations in mice serum, to observe histopathologic damage,pregnant rates and number of offsprings.Results:1.The amplified full length cDNA encoding FSH and Occludin were exactly the same sequence as Genbank data(NM 008045 and NM 008756). Two amino acid of FSH sequence (36aa and 39aa)were mutated by Site-Direct Mutagenesis technology in order to decrease biology activity.The mutants were confirmed by sequence analysis that there are mutated as design.Two different pPIC9K FSH-Occludin [FSH-β(33aa-100aa) and the second extracellular loop of occludin (207-228aa),(197-241aa) respectively] yeast expression vectors were obtained. These vectors were also confirmed by sequence analysis.2.Two different FSH-Occludin fusion proteins were obtained from the culture supernatant of yeast expression system. After purified,they were used to test their biological activity.3.The adult male Balb/c mice were immunized with FSH-occludin fusion protein, Two fusion protein can effectively inducing specific humoral antibody response and effected on experiment group mice fertilization without disturbing the hormone balance. The fusion proteins can targeting interfere with Sertoli cell, the sperm cell density in seminiferous tubules and the tail of epididymis of experiment group was obviously reduced than control group.There were big interspace between the Sertoli cells of the experimental group, TJs between Sertoli cells had been broken,Sertoli cells did not support germ cell and basal lamina of seminiferous tubules dissolved partly.The pregnant rate and the number of offspring in experimental group were obviously fewer than control group and contraception effect is reversible. Conclusin:1.In summary, occludin is a useful marker to monitor inter-Sertoli TJs assembly .The second external loop of occludin in particular the outermost region of the loop, apparently is important to confer to the TJs functionality and the inter-Sertoli TJs permeability barrier.2.the use of an occluding peptide or other peptide-based reagents homologous to selected TJs or AJs proteins to impair spermatogenesis may provide a potential approach to arrest spermatogenesis.The contraceptive effect of the two fusion proteins is reversible, though disrupt the BTB. Therefore, our research may pave the way for a safty,efficacious and reversible contraceptive method.