Gonadotropins are primary hormones that control vertebrate reproduction. In a recent study, we analyzed the impacts of FSH and LH on zebrafish reproduction by disrupting FSH and LH-β genes (fshb and lhb) using transcription activator-like effector nuclease (TALEN) technology. Using the same approach, we successfully deleted FSH and LH receptor genes (fshr and lhcgr) in the present study. In contrast to the deficiency of its cognate ligand FSH, the fshr-deficient females showed a complete failure of follicle activation with all ovarian follicles arrested at the primary growth-previtellogenic transition, which is the marker for puberty onset in females. Interestingly, after blockade at the primary growth stage for varying times, all females reversed to males, and all these males were fertile. In fshr-deficient males, spermatogenesis was normal in adults, but the initiation of spermatogenesis in juveniles was retarded. In contrast to fshr, the deletion of the lhcgr gene alone caused no obvious phenotypes in both males and females; however, double mutation of fshr and lhcgr resulted in infertile males. In summary, our results in the present study showed that Fshr was indispensable to folliculogenesis and the disruption of the fshr gene resulted in a complete failure of follicle activation followed by masculinization into males. In contrast, lhcgr does not seem to be essential to zebrafish reproduction in both males and females. Neither Fshr nor Lhcgr deficiency could phenocopy the deficiency of their cognate ligands FSH and LH, which is likely due to the fact that Fshr can be activated by both FSH and LH in the zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.

The mechanism by which gonadal steroids modulate GH secretion is not known. We have used the reverse hemolytic plaque assay to examine whether gonadal steroid-induced modulation of GH secretion is effected by changes in the population of somatotrophs and/or alterations in their secretory properties. Two groups of Sprague-Dawley rats were studied: group 1 (n = 6) comprised male (M), castrate (Cx), and testosterone-replaced castrate male (Cx + T) rats and group 2 (n = 5) consisted of male (M), female (F), and 17 beta-estradiol-replaced castrate male (Cx + E) rats. The number of plaque-forming cells (expressed as both absolute number and a percentage of all cells) was determined, and secretory status was assessed by measuring plaque areas in response to 0, 0.01, 0.1, 1, 10, and 100 nM GHRH. While mean basal GH plaque areas were similar among the treatment groups of group 1, the maximal GH plaque area was significantly decreased in Cx [16.8 +/- 2.4 vs. 26.4 +/- 3.9 X 10(6) microns2 (mean +/- SEM); P less than 0.05], but not in Cx + T (27.5 +/- 4.1 microns2) rats. The GHRH EC50 was unaffected by castration or T replacement. The percentage and absolute population of somatotrophs were reduced in Cx, but not in Cx + T, rats, while the numbers of lactotrophs remained unchanged in these treatment groups. For group 2, the mean peak GH plaque area was reduced in Cx + E (16.5 +/- 2.9 microns2; P less than 0.001) compared to that in M rats (36.2 +/- 2.3 microns2), but was not significantly different from that in F (13.0 +/- 1.5 microns2) rats. The EC50 was significantly (P less than 0.025) greater in Cx + E (10.9 +/- 2.3 nM) and F (7.9 +/- 1.6 nM) compared to M rats (2.8 +/- 0.7 nM). The absolute somatotroph and lactotroph populations were increased in Cx + E compared to M and F rats, as were the populations of other pituitary cell types. Testosterone enhances GH secretion by increasing the secretory capacity, but not the sensitivity, of somatotrophs to GHRH and by recruiting the function of a subpopulation of somatotrophs. Estradiol reduces the secretory capacity and sensitivity of somatotrophs to GHRH, but increases the population of somatotrophs, lactotrophs, and non-GH- and non-PRL-secreting cells.(ABSTRACT TRUNCATED AT 400 WORDS)