Tissue distribution of activin βA and βB expression in grass carp.
(A) RT-PCR of activin βA and βB expression in selected tissues and brain areas. Total RNA was isolated from the tissues/brain areas as indicated and subjected to RT-PCR with primers specific for carp activin βA (Act βA) and βB (Act βB), respectively. The authenticity of PCR products was confirmed by PCR Southern with DIG-labelled probes for respective gene targets with parallel PCR for β actin as internal control. (B) LCM capture of immuno-identified lactotrophs (PRL cells), gonadotorphs (LH cells) and somatotrophs (GH cells) from grass carp pituitary cells. The three cell types (marked by black arrows) were identified by immunostaining using antisera for PRL, LH and GH, respectively. After pulsed with infrared laser, the target cells with signals for respective hormones were captured on LCM HS cap and subjected to RT-PCR with primers for different targets. (C) Expression of activin βA and βB as well as their receptors ActRIB and ActRIIB in immuno-identified pituitary cells. Pure populations of PRL, LH and GH cells captured on LCM HS cap (×25 or ×250 cells/cap) were used for RT-PCR with primers for carp activin βA and βB, respectively (upper panel). For detection of ActRIB and ActRIIB, RNA samples prepared from the respective cell types (×250 cells/cap) with reverse transcription (+RT) were used for RT-PCR using primers for carp ActRIB and ActRIIB, respectively (lower panel). Parallel PCR in RNA samples without reverse transcription (-RT) was used as negative control and PCR with the RT sample prepared from mixed populations of pituitary cells (Pit cells) was used as positive control. In these experiments, RT-PCR for β actin was routinely performed to serve as the internal control.