Cloning and expression analysis of Shvasa and the molecular regulatory pathways implicated in Cd-induced reproductive toxicity in the freshwater crab Sinopotamon henanense.

Cadmium (Cd), a widespread, severely toxic heavy metal, can cause serious reproductive toxicity in animals. However, the molecular pathways associated with Cd-induced effects remain unknown. In this study, we first cloned the vasa gene (Shvasa) and characterized the VASA protein (ShVASA) in Sinopotamon henanense. We then investigated the molecular mechanisms of Cd-induced reproductive toxicity. Shvasa was specifically expressed in the ovary and testis. ShVASA was abundant in early ovarian development and significantly less abundant in mature ovaries. During oogenesis, ShVASA was abundant and evenly distributed in the cytoplasm of the oogonium and previtellogenic oocytes, but gradually accumulated in the nuclear periphery of vitellogenic and mature oocytes. As Cd concentration increased, ShVASA abundance decreased gradually in proliferation-stage ovaries, and increased gradually in mature ovaries. Notably, at the small and large growth stages, ShVASA was upregulated following exposure to 14.5 mg/L Cd and downregulated following exposure to 29 mg/L Cd. In contrast to the unexposed control, ShVASA accumulated around the nuclear periphery in Cd-exposed previtellogenic oocytes and scattered gradually into the cytoplasm in Cd-exposed vitellogenic and mature oocytes. Shvasa RNA interference (RNAi) downregulated Shnanos and Shpiwi, but simultaneous Cd exposure and Shvasa RNAi significantly upregulated Shnanos and downregulated Shpiwi. These data suggested that Cd disrupted Shvasa expression and function, as well as the functions of Shnanos and Shpiwi, leading to severe reproductive toxicity in S. henanense.

Histological analysis of oogenesis and ovarian development of the freshwater crab Sinopotamon henanense.

The morphological and cytological changes of oogenesis and ovarian development were described in the freshwater crab Sinopotamon henanense through macroscopic and microscopic examinations. Serial histological dissections of the ovaries demonstrated that oocyte development was asynchronous. Oogenesis was divided into four distinct stages including six phases: oogonium stage, the first phase (OI) and the second phase (OII) comprising the previtellogenic stage, the third phase (OIII), the fourth phase (OIV) and the fifth phase (OV), comprising the vitellogenic stage and the sixth phase representing the mature stage. Furthermore, examining and analyzing the gonadosomatic indices showed that the developmental cycle of the ovary was closely related to season, and indicated that the breeding season of S. henanense was between May and June. Ovarian development was classified into six stages: proliferation stage, small growth stage, large growth stage, pre-maturation stage, mature stage and spawning stage. Ovaries varied in size and color during each developmental stage, which were closely related to the status and proportions of oogonia and primary oocytes. Although there were cases that oocytes at two or more phases were present at each stage, ovary developmental stages were substantially different. These results provide an important base for studies of the regulatory mechanisms of oogenesis in this compared to other brachyuran species, and will be useful for the aquaculture of S. henanense and related species.