Transport of Acrosomal Enzymes by KIFC1 via the Acroframosomal Cytoskeleton during Spermatogenesis in Macrobrachium rosenbergii (Crustacea, Decapoda, Malacostracea).

The spermatogenesis of crustaceans includes nuclear deformation and acrosome formation. The mechanism of acrosome formation is one focus of reproductive biology. In this study, Macrobrachium rosenbergii was selected as the research object to explore the mechanism of acrosome formation. The acrosome contains a large number of acrosomal enzymes for the hydrolysis of the egg envelope. How these acrosomal enzymes are transported to the acrosomal site after synthesis is the key scientific question of this study. The acroframosome (AFS) structure of caridean sperm has been reported. We hypothesized that acrosomal enzymes may be transported along the AFS framework to the acrosome by motor proteins. To study this hypothesis, we obtained the full-length cDNA sequences of Mr-kifc1 and Mr-Acrosin from the testis of M. rosenbergii. The Mr-kifc1 and Mr-Acrosin mRNA expression levels were highest in testis. We detected the distribution of Mr-KIFC1 and its colocalization with Mr-Acrosin during spermatogenesis by immunofluorescence. The colocalization of Mr-KIFC1 and microtubule indicated that Mr-KIFC1 may participate in sperm acrosome formation and nucleus maturation. The colocalization of Mr-KIFC1 and Mr-Acrosin indicated that Mr-KIFC1 may be involved in Acrosin transport during spermiogenesis of M. rosenbergii. These results suggest that Mr-KIFC1 may be involved in acrosomal enzymes transport during spermiogenesis of M. rosenbergii.

Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker ( Larimichthys crocea) under environmental hypoxia.

Oxygen is an essential molecule for animal respiration, growth, and survival. Unlike in terrestrial environments, contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments, thus impacting aquatic animal survival. However, the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown. Here, we used large yellow croaker ( Larimichthys crocea) and large yellow croaker fry (LYCF) cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis, and apoptosis. We confirmed that hypoxia induced the expression of Hif-1α, Hsf1, and Hsp70 in vivo and in vitro. Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress. Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential, enhancing Bcl-2 mRNA and protein expression, inhibiting Bax and caspase3 mRNA expression, and suppressing caspase-3 and caspase-9 activation. Hsp70 suppressed caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription. Hsf1 enhanced Hsp70 mRNA transcription in a similar manner. In summary, the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L. crocea under hypoxic stress.

Expression and functional analysis of cytoplasmic dynein during spermatogenesis in Portunus trituberculatus.

The mechanism of acrosome formation in the crab sperm is a hot topic in crustacean reproduction research. Dynein is a motor protein that performs microtubule-dependent retrograde transport and plays an essential role in spermatogenesis. However, whether cytoplasmic dynein participates in acrosome formation in the crab sperm remains poorly understood. In this study, we cloned the cytoplasmic dynein intermediate chain gene (Pt-DIC) from Portunus trituberculatus testis. Pt-DIC is composed of a p150glued-binding domain, a dynein light chain (DLC)-binding domain, and a dynein heavy chain (DHC)-binding domain. The Pt-DIC gene is widely expressed in different tissues, showing the highest expression in the testis, and it is expressed in different stages of spermatid development, indicating important functions in spermatogenesis. We further observed the colocalization of Pt-DIC and Pt-DHC, Pt-DHC and tubulin, and Pt-DHC and GM130, and the results indicated that cytoplasmic dynein may participate in nuclear shaping and acrosome formation via vesicle transport. In addition, we examined the colocalization of Pt-DHC and a mitochondrion (MT) tracker and that of Pt-DHC and prohibitin (PHB). The results indicated that cytoplasmic dynein participated in mitochondrial transport and mitochondrial degradation. Taken together, these results support the hypothesis that cytoplasmic dynein participates in acrosome formation, nuclear shaping, and mitochondrial transport during spermiogenesis in P. trituberculatus. This study will provide valuable guidance for the artificial fertilization and reproduction of P. trituberculatus.