Activin A-, Inhibin A-, and FS-Like Molecules Were Present in the Zebrafish Ovary
To examine the expression of activin A, inhibin A and FS in the zebrafish ovary, RT-PCR was first performed using cDNA prepared from zebrafish ovary. A sense primer from a conserved region of (3A cDNA and an antisense primer specific for zebrafish (3A subunit were used to detect pA mRNA expression. As shown in Figure 1A, a 645-base pair (bp) DNA fragment of the expected size was generated upon amplification of ovary cDNA, but not of the control (omitted cDNA sample) or of the corresponding total RNA sample. Sequencing of the PCR product revealed that it was highly similar to the (3A cDNA sequence of other species (data not shown). Similarly, using primers specific for ze-brafish FS cDNA, a DNA fragment with the expected size of 384 bp was obtained from the ovary cDNA sample, but not from the corresponding total RNA sample or the water control (Fig. 1A). The sequence of the PCR product was identical to the published zebrafish FS cDNA sequence.
To further confirm the expression of activin and FS, Western blot analyses were carried out using antibodies against human activin (3A subunit and FS, respectively. Three bands, with molecular sizes of approximately 75, 32, and 26 kDa, were observed when (3A antibody was used (Fig. 1B). The large band may represent a precursor for (3A subunit, while the respective smaller bands are similar in size to mammalian inhibin and activin. The FS antibody also detected several bands, ranging from 30 to 50 kDa (Fig. 1B). These molecular sizes are consistent with the known structure of FS.
FIG. 1. Expression of activin A, inhibin A-like molecule, and FS in the zebrafish ovary. A) PCR using primers specific for activin/inhibin pA subunit and for FS. A cDNA sample (Ovc) prepared from zebrafish ovary was used as the template in PCR to detect the expression of pA subunit mRNA and FS mRNA. Negative controls include the use of water (C) to replace the cDNA sample, and use of the corresponding RNA sample (Ovr). DNA fragments of the expected sizes were obtained when Ovc was used as the template. PCR was performed for 35 cycles. B) Western blot analysis of proteins extracted from the zebrafish ovary using antibodies against human pA subunit and FS under nonreducing conditions. Three bands with approximate sizes of 26, 32, and 75 kDa were observed when antibody against pA subunit was used. FS antibody detected four bands with molecular size ranging from 30 to 50 kDa. MW, Molecular weight marker.