Es-BMP2 Mediates Spermatogenesis In Chinese Mitten Crab Eriocheir Sinensis By Regulating Kinesin-14 KIFC1 Expression

Eriocheir sinensis is one of the model species for studying spermatogenesis of decapod crustaceans.Studying the spermatogenesis kinetics and signal pathway regulation of E.sinensis provides an important theoretical basis for elucidating its reproductive regulation mechanism.Bone morphogenetic proteins(BMPs)are an important branch of the transforming growth factor ?(TGF?)superfamily and are widely involved in physiological activities such as embryonic development,cell proliferation,and cell differentiation.This article mainly explores the function and possible molecular mechanism of BMP2 in the TGF? superfamily during spermatogenesis of E.sinensis.We cloned the bmp2 homologous gene from the E.sinensis testis tissue.The Chinese mitten crab es-bmp2 gene CDS is 1215 bp and encodes 404 amino acids.Es-BMP2 mainly contains a transmembrane domain and a TGF? domain.Compared with homologous sequences of other species,it was found that the amino acid sequence of the TGF? domain is relatively conserved.Phylogenetic tree analysis found that it was a branch with Portunus trituberculatus BMP2 protein.Subsequently,Es-BMP2 fusion protein was expressed prokaryotically and mice were injected intraperitoneally with antigen to prepare Es-BMP2 mouse polyclonal antibody,which can be used in immunoblotting and immunofluorescence experiments.To investigate the expression level of Es-BMP2 in various tissues of E.sinensis,RT-PCR and Western blot experiments were carried out.The results showed that the expression levels of es-bmp2 mRNA and protein in testis were relatively high.Es-BMP2 is mainly expressed in spermatogonia,spermatocytes and spermatids.The expression level in mature spermatozoa is significantly reduced,and the signal is mainly concentrated on the side of acrosome.Injecting es-bmp2 specific dsRNA successfully in vivo RNAi of E.sinensis and histological observation of the testis revealed that a large number of sperm nucleus developmental deformities appeared in the spermatozoa in the RNAi group,indicating that it may be involved in the sperm formation process of E.sinensis.Therefore,we tested the expression level of the important motor protein Es-KIFC1 in the formation of the sperm nucleus and acrosome in the Chinese mitten crab.The results showed that the expression level of Es-KIFC1 in the testis of the es-bmp2 knockdown group was significantly reduced compared with the control group.In primary cultured cells of testis tissue,the expression of Es-KIFC1 was also suppressed after transfection with es-bmp2 dsRNA.The above results indicate that Es-BMP2 is involved in the E.sinensis spermatogenesis and regulates the expression of kinesin-14 motor Es-KIFC1.