Activin receptor type IIB in rohu (Labeo rohita): molecular characterization, tissue distribution and immunohistochemical localization during different stages of gonadal maturation

Abstract

Activin receptor type IIB (ActRIIB) is a transmembrane serine/threonine kinase receptor which plays a pivotal role in regulating the reproduction in vertebrates including teleost. Earlier studies have documented its importance in governing gonadal maturation in higher vertebrates. However, reports on the regulation of fish reproductive system by ActRIIB gene are still limited. Here, we report the identification and characterization of ActRIIB cDNA of Labeo rohita, a commercially important fish species of the Indian subcontinent. The full-length gene encoding rohu ActRIIB was cloned and found to be of 1674 bp in length. Functional similarities were evident from evolutionary analysis across vertebrates. Real-time PCR to measure the expression of ActRIIB transcript in rohu revealed significant mRNA levels in gonads followed by non-reproductive tissues, including the brain, pituitary and muscle. With respect to different gonadal maturation stages, predominant expression of ActRIIB mRNA was observed during the pre-spawning phase of both sexes. To further delineate its role in rohu reproduction, a recombinant protein of the extracellular domain of ActRIIB (rECD-ActRIIB) was produced, and polyclonal antibody is raised against the protein for its immuno-localization studies during different gonadal maturation stages. Strong immunoreactivity was noticed in the pre-vitellogenic oocytes which decreased dramatically in the fully mature oocytes. Similarly, the strong and intense immunoreactivity was found in the spermatids and spermatocytes of the immature testis, and eventually the intensity reduced with the progression of the maturation stage. These results provide the first evidence of the presence of ActRIIB in rohu gonadal tissues. Taken together, our observations lay the groundwork for further understanding and investigating on the potential role of ActRIIB in fish reproduction system in the event of gonadal maturation.

Keywords: Activin receptor type IIB; Immunohistochemistry; Labeo rohita; Polyclonal Ab; Real-time expression; Recombinant extracellular domain protein; Serine/threonine kinase.