Changes in GnRH I, bradykinin and their receptors and GnIH in the ovary of Calotes versicolor during reproductive cycle

Abstract

The aim of this study was to investigate changes in the abundance of gonadotrophin releasing hormone I (GnRH I) and GnRH I receptor in the ovary of Calotes versicolor during the reproductive cycle and correlate them with the changes in gonadotrophin inhibitory hormone (GnIH), bradykinin and bradykinin B(2) receptor in order to understand their interaction during ovarian cycle. GnRH I, bradykinin and their receptors and GnIH, were localized immunohistochemically in the ovary. Relative intensity of these peptides was estimated from the contralateral ovary using slot/Western blot followed by densitometry. The immunostaining of GnRH I, bradykinin and their receptors and GnIH were localized in the granulosa cells of previtellogenic follicles and stroma cells, whereas in the peripheral part of the cytoplasm in oocytes of vitellogenic and ovulatory follicles. The GnRH I immunostaining was relatively higher in inactive phase, but was low during active preovulatory phase suggesting inverse correlation with circulating estradiol level. The study showed a positive correlation between the expression pattern of GnRH I and GnIH, but showed a negative correlation between GnIH with GnRH I receptor in the ovary. This study further suggests a possibility for bradykinin regulating GnRH I synthesis in the ovary. An increase in the immunostaining of both GnRH I and GnIH in the oocyte prior to ovulation suggests their involvement in the oocyte maturation. It is thus concluded that the ovary of C. versicolor possesses GnRH I-GnIH-bradykinin system and interaction between these neuropeptides may be involved in the regulation of follicular development and oocyte maturation.

Fig. 1.

All figures are transverse sections of the ovary of the reptile, Calotes versicolor. I: GnRH I, (A) ovary showing immunoreactivity mainly in the granulosa cells (GC) of the growing follicles during resting phase. (B) Recrudescence phase: immunoreactivity mainly in granulosa and theca cells (TC). (C) A mild immunoreactivity was observed in the granulosa and theca cell of the secondary previtellogenic follicles during previtellogenic phase. (D) Vitellogenic phase: immunoreactivity mainly in the periphery of the oocytes (O) during vitellogenic phase. (E) Ovulatory phase follicle: immunoreactivity mainly in the periphery of the oocytes (shown by arrow). (F) Decline in the immunoreactivity in the follicle but stroma showing strong immunoreactivity during regression phase. (G) Preadsorbed control for GnRH showing no immunostaining. II: GnRH I receptor, (A) moderate immunoreactivity for GnRH I receptor in the granulosa cells. (B) Increase in the immunoreactivity in the granulosa and theca cells in the growing follicles during recrudescence phase. (C) A strong immunoreactivity was observed in the granulosa (shown by arrow) and moderate in the oocytes during previtellogenic phase. (D) Immunostaining shifted from granulosa to the periphery of the oocytes (shown by arrow) during vitellogenic follicle. (E) Ovulatory phase: strong immunoreactivity in the periphery of the oocytes of ovulatory follicles while immunoreactivity remains in the granulosa and theca cells of the previtellogenic follicles. (F) Marked decline in the immunoreactivity during regression phase. (G) Negative control showing no immunostaining. III: GnIH, (A) strong immunoreactivity for GnIH was observed in the follicle in the granulosa cells (shown by white arrow) and in the stroma (shown by black arrow) during resting phase. (B) Immunoreactivity declined to a moderate level during recrudescence. (C) Mild immunoreactivity in granulosa and theca cells during previtellogenic follicles. (D) Immunoreactivity confined only at the periphery of the oocytes (shown by arrow) in vitellogenic follicles. (E) Strong immunoreactivity only at the periphery of the oocytes during ovulatory phase. (F) Moderate immunoreactivity was observed during regression phase. (G) Preadsorbed control for GnIH showing no immunostaining. IV: Bradykinin, (A) moderate immunoreactivity in granulosa cells and theca cells during resting phase. (B) Moderate immunoreactivity in granulosa cells (GC) and theca cells during recrudescence phase. (C) Decline in the immunoreactivity during previtellogenic phase. (D) Immunoreactivity only at the periphery of the oocyte during vitellogenic phase. (E) Strong immunoreactivity at the periphery of the ovulatory follicle (shown by arrow) and strong immunoreactivity in granulose cells of secondary previtellogenic follicle. (F) Decreased immunoreactivity during regression phase. V: Bradykinin B₂ receptor, (A) strong immunoreactivity for bradykinin B₂ receptor in granulosa, theca cells (TC) and oocytes (O) during resting phase. (B) Strong immunoreactivity during recrudescence phase. (C) Strong immunoreactivity during previtellogenic phase. (D) Decrease in immunoreactivity and confined only to periphery of the oocytes (shown by arrow) during vitellogenesis. (E) Strong immunoreactivity at the periphery of the oocytes of ovulatory follicle (shown by arrow) and strong immunoreactivity in the granulosa, theca and oocytes of the previtellogenic follicle. (F) Moderate immunoreactivity observed in the granulosa cells and mild in the oocytes during regression phase.

Fig. 2.

Validation of slot blot assay. The amount of protein loaded and intensity of protein band in the slot blot showing strong correlation for: (A) GnRH I (r² = 0.96), (B) GnIH (r² = 0.98) and (C) bradykinin (r² = 0.87).

Fig. 3.

Slot blot for GnRH I (Fig. 3A) and GnIH (Fig. 3B) and Western blot for GnRH I receptor (Fig. 3C). Densitometric analyses of the blot are given in bar graph. Values are represented as means ± SEM. Bar bearing different superscripts (a–e) indicate significant difference between the specific values (p < 0.05).

Fig. 4.

Immunoblot analysis of bradykinin and bradykinin B₂ receptor. Densitometric analyses of the blot are represented as means ± SEM. Values are represented as means ± SEM. Bar bearing different superscripts (a–e) indicate significant difference between the specific values (p < 0.05).

Fig. 5.

The correlation between GnRH I receptor and GnIH during follicular development from resting to vitellogenic phase. Correlation analysis was performed between the mean densitometric values of each band (relative to the band density of resting phase). Values were obtained from four different set of experiments. r² = coefficient of determination.

Fig. 6.

Effect of GnIH (1 and 100 ng) and 17β estradiol (1 and 10 ng) on expression level of GnRH 1 receptor by the ovary of C. versicolor. Values are represented as means ± SEM. *Values are significantly (p < 0.05) different, control versus treatment groups. No significant difference was observed between the two doses of same treatment.