Cathepsin D (CAT-D) is a well-known aspartic protease that serves a function as house-keeping lysosomal enzyme in all somatic cells. Its existence in reproductive tissues is highly variable, even in the somatic derived epithelial cells of reproductive tract. InMacrobrachium rosenbergii, existence of MrCAT-D and its translational product was detected in both somatic cells (Sertoli-like supporting cells) and developing spermatogenic cells as well as along accessory spermatic ducts. Specifically, MrCAT-D was localized onto the sperm surface rather than within the acrosomal matrix, as evident by similar staining pattern of anti-CAT-D on live and aldehyde fixed sperm. MrCAT-D in testicular extracts and sperm isolates showed active enzyme activities towards its specific fluorogenic substrate (MCA-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys (Dnp)-D-Arg-NH₂). MrCAT-D also exerted its function towards hydrolyzing filamentous actin, the meshwork of which is shown to be localized at the junction between germ cells and supporting cells and spermatogonia inM. rosenbergiitesticular epithelium. Together, we have localized MrCAT-D transcript and its translational product in both supporting and germ cells of testis and claimed its enzymatic function towards actin degradation, which may be related to sperm release from the epithelial cell interaction.

Coelomocytes are the first line of immune defense in marine animals. Their distributions are greatly variable even in the close animal species. In this study, we used lectin staining to aid in the classification and purification of these cells for further investigation of SOD distribution among coelomocytes of H. scraba. We classified coelomocytes into four types: type 1, lymphocytes; type 2, phagocytes; type 3, spherulocytes; and type 4, giant cells. Among four lectins used, Con A appeared to give a broad reactivity against most coelomocytes, except for giant cells. In addition, phagocytes usually engaged the highest fluorescent intensity with most lectins, with the exception of PNA, for which spherulocytes possessed the highest fluorescent intensity. Using FACS for fraction collection, it was found that F1 fraction contained the purest phagocyte population (> 95%), which was highly reactive with anti- superoxide dismutase (SOD) as revealed by immunoblotting and immunofluorescence staining, although some...

Sperm activation occurring in both male and female reproductive tract involves a highly complex series of biomolecular dynamics, particularly on membrane lipids and proteins. In mammals, the universal anticipation in cholesterol (CHO) sequestration plays a role in mammalian sperm maturation/capacitation, subsequently enhancing sperm fertilizing ability. In shrimp, we have previously shown that the level of cholesterol (CHO) is significantly reduced in vas deferens sperm when compared with sperm in the testes, presumably due to the sequestering action of the lipid-binding protein, one of which is Niemann-Pick Type C-2 (NPC2). Here, we used recombinant MrNPC2 protein to treat testicular sperm (Tsp) and quantitatively compared the amount of CHO with non-treated or 2-OH-β-CD treated sperm. HPTLC of the extracted lipids from Macrobrachium rosenbergii sperm revealed the presence of major phospholipids and CHO. Tsp treated with 2-OH-β-CD or MrNPC2 showed lower CHO levels, which was comparable to that of the vas deferens sperm as verified by the Amplex Red assay. Finally, the enhanced levels of protein tyrosine phosphorylation and ionophore-induced AR levels in the MrNPC2 treated Tsp significantly increased in a concentration-dependent manner, similar to that of the Vsp sperm. Altogether, our results indicated the importance of MrNPC2 as CHO moderator in sperm membrane, leading to an enhancement in sperm fertilizing ability.