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Published in final edited form as: Gen Comp Endocrinol. 2008 Sep 10;159(2-3):158–169. doi: 10.1016/j.ygcen.2008.08.016

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

Padmasana Singh a, Amitabh Krishna a,*, Rajagopala Sridaran b, Kazuyoshi Tsutsui c
PMCID: PMC7927428  NIHMSID: NIHMS1672080  PMID: 18809405

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 B2 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.

Keywords: GnRH I, GnRH I receptor, Bradykinin, Bradykinin B2 receptor, GnIH, Ovary, Calotes versicolor, Reproductive cycle

1. Introduction

We have demonstrated the presence of GnRH I and high-affinity GnRH I binding sites in the ovary of many vertebrate species from fish to bird (Singh et al., 2007). Since the half-life of GnRH I is very short and GnRH I is found in undetectable amounts in peripheral circulation, it is proposed that ovarian GnRH I receptors are activated by locally produced GnRH I (Hsueh and Jones, 1981; Singh et al., 2007). A number of studies have shown that GnRH and its receptor are also expressed in the ovaries of various vertebrate groups, suggesting that the peptide hormone may have intra-ovarian functions in addition to its hypophysiotrophic activity. However, so far the role of ovarian GnRH I in the regulation of reproductive processes has received little attention. The various forms of GnRH found in the ovary of fish and rat to have roles in steroidogenesis (Habibi et al., 1988, 1989), apoptosis (Billig et al., 1994; Parborell et al., 2002), and meiotic maturation of the oocyte (Hillensjo and LeMaire, 1980; Pati and Habibi, 1998; Nabissi et al., 1997). Another possible function of GnRH is related to its role in gonadogenesis. In rainbow trout, the expression of GnRH I might be required for growth and differentiation of a new wave of germ cells during the reproductive cycle. In Calotes versicolor it has been reported that not high temperature but GnRH can bring recrudescence and androgen production in the quiescent testis (Shanbhag et al., 2000). Thus, the actions of GnRH I within the ovary are diverse and depend on the state of reproductive phase. Previous studies showed significant variation of ovarian GnRH I and its receptor during the rat reproductive cycle (Kogo et al., 1995). Synthesis of ovarian GnRH I and the presence of its receptor across the reproductive cycle have been investigated only in a non-mammalian vertebrate rainbow trout (Uzbekova et al., 2000, 2001).

The factor(s) regulating GnRH I and its receptor in the ovary is (are) not known. Activity of GnRH I in hypothalamus is regulated by hypothalamic neurotransmitters. In general catecholamine, norepinephrine and epinephrine increased GnRH I release; where endogenous opioides such as β endorphin inhibits GnRH I release (Wierman, 1996; Kalra, 1993; Levine, 1997). Bradykinin has been demonstrated to stimulate GnRH I release from the hypothalamus (Shi et al., 1998). Induction of GnRH I release through bradykinin has been reported in brain (Shi et al., 1999). Bradykinin acts directly on the GnRH I neurons mediated through bradykinin B2 receptor. Kihara et al. (2000) have shown a bradykinin producing system in the porcine ovarian follicle and suggest the importance of this peptide in follicular development and ovulation.

In recent years, a novel hypothalamic neuropeptide inhibiting gonadotrophin release was discovered in quail and was designated gonadotrophin inhibitory hormone (GnIH) (Tsutsui et al., 2000). GnIH is also effective in inhibiting gonadotrophin release in vitro and in vivo in several avian species (Tsutsui et al., 2000; Osugi et al., 2004; Ciccone et al., 2004; Ubuka et al., 2006). In birds, cell bodies and terminals of GnIH neurons are localized in the paraventricular nucleus (PVN) and median eminence (ME), respectively (Tsutsui et al., 2000; Bentley et al., 2003; Ubuka et al., 2003; Ukena et al., 2003). GnIH acts directly on the pituitary via a novel G protein-coupled receptor for GnIH to inhibit not only gonadotrophin release but also gonadotrophin synthesis (Ciccone et al., 2004; Yin et al., 2005; Ubuka et al., 2006). Furthermore, GnIH inhibits gonadal development and maintenance by inhibiting gonadotrophin release and synthesis in birds (Ubuka et al., 2006; Tsutsui and Ukena, 2006; Tsutsui et al., 2006, 2007). Recent studies have further shown that GnIH neurons project not only to the ME but also to GnRH neurons in birds (Bentley et al., 2003; Ubuka et al., 2008). GnIH receptor is also expressed in GnRH neurons (Bentley et al., 2006; Ubuka et al., 2008). Therefore, GnIH may act on GnRH I neurons at the level of the hypothalamus to inhibit gonadotrophin release and synthesis as well as acting at the level of the pituitary in birds. Furthermore, GnIH and its receptor have been detected in gonads and accessory reproductive organs in sexually mature birds (Bentley et al., 2008). The distribution of GnIH and its receptor in the reproductive system suggests a potential for autocrine/paracrine regulation of gonadal steroid production and germ cell maturation in birds.

The present study was undertaken to investigate the changes in the concentration of GnRH I and its receptor in the ovary of C. versicolor during the reproductive cycle in order to understand their significance in ovarian functions. To understand the regulation of ovarian GnRH I and its receptor, seasonal variation in ovarian concentration of bradykinin, bradykinin B2 receptor and GnIH will be correlated with the changes in the concentration of GnRH I and its receptor in the ovary of C. versicolor during the reproductive cycle.

2. Materials and methods

2.1. Sample collection

All experiments were conducted in accordance with the guide and use of wild animal approved by the Department Research Committee, Banaras Hindu University, Varanasi, India. Adult female C. versicolor (10–15 g) four to six in number were procured locally from Varanasi (28″18′N; 83°E) during their different reproductive phases of annual reproductive cycle: resting phase, recrudescence phase, previtellogenic phase, vitellogenic phase, ovulatory phase and regression phase. Animals were killed by decapitation within 6 h as they were brought to laboratory. Ovaries were dissected out. Ovaries of one side from each animal were kept frozen at −40 °C for immunoblot and of the other side were fixed in bouin’s fixative at room temperature for immunohistochemistry. The fixed tissues were dehydrated in ethanol and embedded in paraffin wax. Sections (5 μm) were cut and mounted on the gelatin coated slides.

2.2. Classification of ovary based on the reproductive status in the reptile C. versicolor

Calotes versicolor, a seasonal breeding lizard is widely distributed in India. It has a single annual cycle of reproductive activity in Varanasi. C. versicolor has a long breeding season that breeds from June to September (Varma, 1970). Based on the size and type of follicles in the ovary, the reproductive cycle of C. versicolor are divided into following phases:

  1. Resting phase: ovary small in size containing only primordial and primary follicles and some early previtellogenic secondary follicles (November–February).

  2. Recrudescence/progressive phase: ovary increases in size due to the presence of many growing previtellogenic secondary follicles along with several primordial and primary follicles (March–April).

  3. Previtellogenic phase: ovary containing several large previtellogenic as well as a few vitellogenic secondary follicles in different stage of growth (May).

  4. Preovulatory or vitellogenic phase: ovary containing many vitellogenic mature tertiary follicles. Developing follicles of all size occur in the ovary (June–July).

  5. Ovulatory phase: ovary containing several mature yolk-filled tertiary follicles. Developing follicles of all size occur in the ovary (June–September).

  6. Regression phase: ovary containing many corpus luteum and atretic follicles. Some primary and early secondary follicles are also present (September–October).

2.3. Classification of ovarian follicles in C. versicolor

Ovarian follicles in C. versicolor are classified into following types on the basis of size, presence of vacuoles and amount and type of yolk deposition (Devaraj and Shivanandappa, 1989): (a) Primary follicles: primary follicle having single layer of granulosa cells. Developing follicles of all sizes occur in the ovary during the breeding season. It contains three types of granulosa cells, small, intermediate and large pyriform cells. Small and intermediate cells are round while large pyriform cells has narrow protoplasmic prolongation which appears to traverse the zona pellucida. Several nuclei are distributed irregularly in the periphery of the ooplasm of the developing follicles. Number of the nuclei varies. These nuclei degenerate before the yolk deposition starts. (b) Secondary/previtellogenic follicles: pyriform cells become smaller and granulosa becomes single layered and monomorphic. Theca is differentiated into more cellular theca interne and fibrous theca externa. (c) Secondary/vitellogenic follicles: it is characterized by the deposition of yolk in the ooplasm. The cells of the follicular epithelium are stretched, reduced in size and separated from each other. The thecal layers have increased in thickness and are loosely constructed. (d) Preovulatory follicles: ovary appears large and heavy due to yolk deposition. Size of the follicles increases measuring 6–8 mm in diameter in case of C. versicolor.

2.4. In vitro study

To determine the effects of GnIH and estradiol on the expression of ovarian GnRH I receptor protein, the in vitro study was performed. The two doses of GnIH, low and high and two doses of 17β estradiol, were adopted for the study. The two doses for estradiol were selected based on the study of Khosravi and Leung (2003), whereas doses for GnIH was selected based on our preliminary study. Female C. versicolor was collected during late March and early April and sacrificed by decapitation as soon as they were brought to the laboratory. Their ovaries were quickly taken out and cleaned for any adhered fat tissue and oviduct in medium Dulbecco Modified Eagle’s Medium (DMEM) (Himedia, Mumbai, India) containing 250 U/ml penicillin and 250 μg/ml streptomycin sulfate. Culture medium was a mixture of DMEM (with sodium pyruvate and l-glutamine) and Ham’s F-12 (1:1; v:v) (Himedia, Mumbai, India) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 0.1% BSA (Sigma) and pH 7.2 was adjusted. Each ovary was cut into two equal halves. After initial incubation for 2 h at 28 °C, culture medium was discarded and ovaries were finally cultured in 1 ml of medium in a humidified atmosphere with 95% air and 5% CO2 for 24 h at 28 °C with either GnIH (1 and 100 ng/ml) or 17β estradiol (1 and 10 ng/ml). Control tubes did not receive any treatment. Ovaries were cultured by the method as described previously with some modifications (Khan and Rai, 2008). Each treatment group was run in triplicate. Ovaries cultured under these conditions appear healthy and do not show any sign of necrosis. Ovaries were collected at the end of culture, washed several times with PBS and kept frozen at −40 °C for immunoblot study.

2.5. Immunohistochemistry

Ovarian sections were processed through standard protocols of immunohistochemistry (Singh et al., 2007). Sections were deparaffinized, rehydrated in graded ethanol and endogenous peroxide was quenched with 0.3% H2O2 in methanol. The sections were soaked in 0.05 mol/l Tris–cl—0.15 mol/l NaCl (TBS pH 7.4). Sections were preincubated in 10% normal horse serum in PBS for 1 h to reduce background staining. The tissue sections were then incubated with the primary antibody (see Table 1 for detail) in PBS for 1 h at room temperature. Following a rinse in TBS for 15 min slides were incubated with the secondary antibody. Detection system used was ABC universal staining kit (sc-PK-6200; Vector Laboratories, Inc., Burlingame, CA, USA). Peroxidase activity was revealed with 0.03% 3,3′-diaminobenzidine tetra hydrochloride (DAB; Sigma Chemicals Co., St. Louis, USA) in 0.05 M Tris pH 7.6 and 0.1% H2O2. Sections were rinsed in buffer. Nucleus was counterstained with Ehrlich’s hematoxylin, dehydrated and mounted with DPX. To test the specificity of the immunoreaction in the control section the primary antiserum was replaced by: (1) 1% normal horse serum; (2) after preabsorption of GnRH I antiserum with GnRH I antigen (200 ng of GnRH I per ml), GnIH antiserum with GnIH antigen (10 μg of GnIH per ml) and bradykinin antiserum with bradykinin antigen (200 ng of bradykinin per ml). For preabsorption, the antigens were added to diluted antisera, incubated overnight at 4 °C, centrifuged and then the supernatant was used.

Table 1.

Details of antibodies used for immunohistochemistry and slot/Western blot

Antibody Type Target species Source Concentration used for IHC Concentration used for slot/Western Blot Control
GnRH GnRH I Human Peninsula Lab Inc, San Carlos, CA, USA (Cat No. IHC-7201) 1:1500 1:2000 Preabsorbed control
GnRH receptor GnRH I receptor Human Santa Cruz Biotechnology Inc. (Cat No. sc 8028) 1:25 1:250 Negative control
GnIH GnIH Quail Kazuyoshi Tsutsui, Tokyo, Japan 1:5000 1:2000 Preadsorbed control
Bradykinin Bradykinin Human Peninsula Lab Inc, San Carlos, CA, USA (Cat No. IHC 7051) 1:2000 1:2000 Negative control
Bradykinin receptor Bradykinin B2 receptor Human BD Transduction Lab, CAS, USA (Cat No. 610451) 1:25 1:250 Negative control
Actin β-Actin Chicken Santa Cruz Biotechnology Inc. (Cat No. sc 47778) 1:500
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2.6. Slot blot

For each reproductive phase from ovarian pool of at least three animals, protein was extracted, as described elsewhere (Chanda et al., 2004). An equal amount of protein, as determined by Folin’s method, was equated to equal volume with PBS. Ten microliters of this sample was loaded on NC membrane using Millipore slot blot apparatus. Non-specific sites were blocked with 5% non-fat dried milk in PBS, 0.02% Tween 20. Membrane was then incubated with the primary antibody (see Table 1 for detail) in PBS for 1 h at room temperature. Immunodetection was performed with anti-rabbit IgG conjugated horseradish peroxidase (1:500 v/v). Finally membrane was developed with Enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA). Experiments were repeated three times with the same result. Equal loading was confirmed with Ponceau S staining. Validation of slot blot assay was performed using serially diluted ovarian protein samples ranging between 0.01 and 2 μg/μl. The intensity of the protein bands of the slot blot was quantified using densitometry. The graph plotted between the amounts of protein loaded and the intensity of the protein bands showed strong correlation (Fig. 2A for GnRH, r2 = 0.96; B for GnIH, r2 = 0.98 and C for bradykinin, r2 = 0.87). No specific band was found in the blot processed with antibody preabsorbed with their respective antigen. The intra-assay coefficient of variation was <7.5% and the sensitivity of the blot was 0.1 μg.

Fig. 2.

Fig. 2.

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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).

2.7. Western blot

An equal amount of protein, as determined by Folin’s method, was loaded on 10% SDS–PAGE for electrophoresis. Separated proteins were then transferred on nitrocellulose membrane (Sigma). Non-specific sites were blocked with 5% non-fat dried milk in TBS, 0.02% Tween 20. Membrane was then incubated with primary antibody (see Table 1 for detail) in PBS for 1 h at room temperature. Immunodetection was performed with anti-mouse IgG conjugated horseradish peroxidase (1:500 v/v). Finally membrane was developed with DAB or detected with Enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA). Experiments were repeated three times using four different ovarian samples. Equal loading was confirmed with Ponceau S or with β actin.

2.8. Data analysis

The relative levels of different signals were quantified using a computer-assisted image-analysis system (Image J versus 1.36, NIH, USA). The densitometric data were expressed as the means ± SEM. Seasonal changes were analyzed by one-way ANOVA followed by Duncan’s multiple range post hoc tests. Student t-test and correlation studies were performed by using SPSS software 12 for windows (Apache software foundation) to compare the data from different groups.

3. Result

3.1. Relative concentration of GnRH I and GnRH I receptor in the ovary (Table 2, Figs. 1 and 3)

Table 2.

Intensity and distribution of gonadotrophin releasing hormone I, bradykinin and their receptors and gonadotrophin inhibiting hormone immunoreactivity in the ovary of C. versicolor during its reproductive cycle.

Type of follicle Intensity
GnRH I GnRH I receptor GnIH Bradykinin Bradykinin B2 receptor
Resting phase
Primary follicle
Oocyte + + + + +
Granulosa + + ++ + +
Theca * * * * *
Secondary previtellogenic follicle
Oocyte + + + + ++
Granulosa ++ ++ ++ ++ +++
Theca + + + + ++
Stroma +++ ++ +++ +++ ++
Recrudescence phase
Primary follicle
Oocyte + + + + +
Granulosa ++ + ++ + +
Theca * * * * *
Secondary previtellogenic follicle
Oocyte ++ ++ + ++ +
Granulosa +++ +++ ++ +++ +++
Theca ++ ++ + ++ ++
Stroma ++ ++ +++ + ++
Previtellogenic phase
Primary follicle
Oocyte + + + + +
Granulosa + + ++ + +
Theca * * * * *
Secondary previtellogenic follicle
Oocyte + ++ + + ++
Granulosa ++ +++ + ++ +++
Theca + ++ + + +
Stroma + + + + +
Vitellogenic phase
Primary follicle
Oocyte + + + + +
Granulosa + + ++ + +
Theca * * * * *
Secondary previtellogenic follicle
Oocyte + ++ + + +
Granulosa ++ +++ ++ ++ +
Theca + ++ + + +
Secondary vitellogenic follicle
Oocyte +++ +++ +++ ++ ++
Granulosa + ++ ++ + ++
Theca + + + + +
Stroma * * * * *
Ovulatory phase
Primary follicle
Oocyte + + + + +
Granulosa + + ++ + +
Theca * * * * *
Secondary previtellogenic follicle
Oocyte + ++ + ++ ++
Granulosa ++ +++ ++ +++ ++
Theca + ++ + + +
Secondary vitellogenic follicle
Oocyte + ++ ++ ++ ++
Granulosa ++ +++ + +++ +++
Theca + + + ++ +
Tertiary/preovulatory follicle
Oocyte +++ +++ +++ +++ +++
Granulosa ++ ++ ++ ++ +
Theca + + + ++ +
Stroma * * * * *
Regression phase
Primary follicle
Oocyte + + + + +
Granulosa ++ ++ ++ + +
Theca * * * * *
Secondary vitellogenic follicle
Oocyte ++ + + + +
Granulosa +++ ++ ++ ++ ++
Theca + + + + +
Stroma +++ +++ + + +
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Scores for intensity of immunoreactivity are as follows: −, absence of immunoreactivity; +, mild; ++, moderate; +++, intense; *, absence of the cell in the section.

Fig. 1.

Fig. 1.

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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. 3.

Fig. 3.

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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).

Presences of GnRH I and GnRH I receptor were demonstrated immunohistochemically in the ovary of C. versicolor. The immunoreactivity of both GnRH I and GnRH I receptor were mainly localized in the granulosa cells of the primary follicles and stroma or interstitial cells of the ovary during the early stage of follicular development from resting to previtellogenic phases. As the follicles grow, GnRH I immunostaining changes from moderate to mild in the primary and secondary follicles. During recrudescence phase, immunostaining of GnRH I increases in the granulosa cells showed moderate to intense staining. Atretic follicles also showed moderate staining in the granulosa cells. GnRH I immunoreactivity declines in the follicles during previtellogenic phase. During vitellogenic phase, GnRH I immunoreactivity shifted from the granulosa cells of the follicles to the periphery of the cytoplasm of oocytes. During ovulatory phase, the largest follicle showed strong immunoreactivity along the periphery of the oocytes. Although the other smaller follicles showed only moderate immunoreactivity in the oocyte, granulosa cells showed mild immunoreactivity. Theca cells generally showed mild to no immunoreactivity. Finally, during post-ovulatory regression phase, GnRH I immunoreactivity was found mainly in the stroma cells.

The pattern of GnRH I receptor immunoreactivity was nearly the same as described for GnRH I. Maximum GnRH I receptor immunoreactivity was observed in previtellogenic follicles and large ovulatory follicles. In general, in the previtellogenic follicles, immunoreactivity was generally observed in granulosa cells, whereas in the vitellogenic follicles immunoreactivity was mainly in the periphery of the oocytes. A transiently strong immunoreactivity was noticed in the ovulatory follicles. Thereafter, GnRH I receptor immunoreactivity declines and remains low during the regression phase.

The densitometric analysis of GnRH I and GnRH I receptor immunoblots showed significant variation in the intensity of immunoreactivity in the ovary during different phases of reproductive cycles. The ovary shows significantly higher intensity of GnRH I during resting and progressive phases as compared with the other reproductive phases. It gradually declines to reach to its lowest level during ovulatory phase. Immunoreactivity rises again during the regression phase. The Western blot analysis of GnRH I receptor in C. versicolor showed a single immunoreactive band at 62 kDa. Densitometric analysis of Western blot for GnRH I receptor during different reproductive phases showed a marked variation. The intensity of GnRH I receptor immunoblot increased during the ovarian recrudescence (progressive phase) to attained a peak during the previtellogenic phase and then declines sharply during vitellogenic and ovulatory phases. The intensity of immunoblot of GnRH I receptor remains substantially low during regression and resting phases.

3.2. Relative concentration of GnIH in the ovary of Calotes versicolor in their reproductive cycle (Table 2, Figs. 1 and 3)

During the resting phase a strong GnIH immunoreactivity was observed in stroma cells and moderate immunoreactivity in granulosa cells of the primary follicles. As the follicles grow, immunoreactivity decreased in the secondary follicles and finally showed a low immunoreactivity in granulosa cells of the previtellogenic follicles. The GnIH immunoreactivity shifted from granulosa cells to the periphery of the oocytes in previtellogenic follicles to the vitellogenic follicles. A moderate to strong GnIH immunoreactivity was observed in the periphery of the oocytes of the vitellogenic and ovulatory follicles. The primary follicles during the resting phase showed higher intensity of immunoreactivity than the primary follicles during regression.

Densitometric analysis of slot blot showed a high GnIH concentration during resting phase as compared to the brain which decreases sharply during preparatory and previtellogenic phases. It again increases at a moderate level during the vitellogenic phase when GnRH I remained low.

3.3. Relative concentration of bradykinin and bradykinin B2 receptor in the ovary (Table 2, Figs. 1 and 4)

Fig. 4.

Fig. 4.

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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).

High bradykinin immunoreactivity was observed in granulosa cells of primary and secondary follicles during resting and recrudescence phases. It decreased during previtellogenic phase. A mild immunoreactivity for bradykinin was observed in granulosa cells and theca cells of the secondary follicles. Bradykinin immunoreactivity was mainly observed at the periphery of the oocytes in vitellogenic and ovulatory follicles. However, secondary follicles still showed immunoreactivity in granulosa cells.

Pattern of bradykinin B2 receptor immunoreactivity was nearly the same as that of bradykinin. An intense immunoreactivity for bradykinin B2 receptor was observed in the primary and secondary follicles during resting, recrudescence and previtellogenic phases. A mild immunoreactivity for bradykinin B2 receptor was observed at the periphery of the oocytes in vitellogenic follicles. During the preovulatory phase, bradykinin B2 receptor immunoreactivity was intense. It remains moderate in primary follicles during regression phase.

Densitometric analysis of slot blot for bradykinin showed a significantly higher intensity of immunoreactivity during resting and recrudescence phases, decreases during previtellogenic and vitellogenic phases. It again increases during ovulatory phase and remain low during regression.

Densitometric analysis of Western blot for bradykinin B2 receptor showed two immunoreactive bands at 42 and 44 kDa. Bradykinin B2 receptor protein showed high immunoreactivity during resting, progressive and previtellogenic phases but no detectable amount of bradykinin B2 receptor immunoreactivity was found during vitellogenic and ovulatory phases.

3.4. Correlation between ovarian GnRH I and GnRH I receptor with bradykinin and bradykinin B2 receptor and GnIH (Fig. 5)

Fig. 5.

Fig. 5.

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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.

Relative density value of the immunoblots for GnRH I, GnRH I receptor, GnIH, bradykinin and bradykinin B2 receptor are based on analysis of total ovarian extract with specific antibodies (shown in Figs. 3 and 4). Changes in ovarian concentration of GnRH I and GnRH I receptor was correlated with ovarian concentration of bradykinin, bradykinin B2 receptor and GnIH during reproductive phases using computer based SPSS program (Table 3). GnRH I and GnRH 1 receptor showed a strong positive correlation with Bradykinin B2 receptor (p < 0.01) during the reproductive cycle. Bradykinin also showed positive correlation only with upper band bradykinin B2 receptor (p < 0.05) during the reproductive cycle (Table 3). GnRH I receptor showed negative correlation (p < 0.01) with GnIH only during the period of follicular development from resting to vitellogenic phases (Fig. 5).

Table 3.

Multiple correlations between GnRH I, GnIH, bradykinin, GnRH I R and bradykinin B2 receptor

GnRH I GnIH Bradykinin GnRH I R Bradykinin B2 R (U) Bradykinin B2 R (L)
GnRH I 1 .354 .302 .210 .702** .822**
GnIH .354 1 .274 −.403 .065 .122
Bradykinin .302 .274 1 .027 .506* .459
GnRH I R .210 −.403 .027 1 .745** .642**
Bradykinin B2 R (U) .702** .065 .506* .745** 1 .928**
Bradykinin B2 R (L) .822** .122 .459 .642** .928** 1
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*

Correlation is significant at the 0.05 level (two-tailed).

**

Correlation is significant at the 0.01 level (two-tailed).

3.5. Effect of GnIH and 17 β estradiol on ovarian GnRH I receptor expression in vitro (Fig. 6)

Fig. 6.

Fig. 6.

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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.

The effects of two doses of GnIH (low and high doses) and two doses of 17β estradiol on in vitro changes in expression of GnRH I receptor are shown in Fig. 6. Both low and high doses of GnIH (1 or 100 ng/ml) and two doses of 17β estradiol (1 or 10 ng/ml) suppress (p < 0.05) GnRH I receptor expression in the ovary of C. versicolor. However, the two doses of GnIH and 17β estradiol showed no significant changes (p > 0.05) in the relative intensity of GnRH I receptor.

4. Discussion

In this study, we present evidence for the presence of neuropeptides, GnRH I, bradykinin and their receptors and GnIH in the ovary of C. versicolor during different reproductive stages. The presence of GnRH I and GnRH I receptor together with GnIH in the ovary provides further support the notion that there is a long-standing evolutionary relationship between the GnRH I and GnIH system in the gonads as suggested recently in the brain of the vertebrate classes (Bentley et al., 2006; Ubuka et al., 2008). This neuroendocrine system may be playing an important role in the gonadal function. This is the first report to our knowledge of the expression of bradykinin, bradykinin B2 receptor and GnIH in the ovary of any reptile. The occurrence of GnRH I and its receptor in gonads has earlier been demonstrated in at least two reptiles, the leopard gecko (Ikamoto and Parks, 2007) and Calotes versicolor (Singh et al., 2007).

The present study demonstrates the significant seasonal variation in the relative abundance of GnRH I and GnRH I receptor in the ovary of C. versicolor during the reproductive cycle. Reproductive stage dependent expression of GnRH I and GnRH I receptor were demonstrated previously in the rat and rainbow trout (Schirman-Hildesheim et al., 2005, 2006; Kogo et al., 1995; Uzbekova et al., 2000, 2001). In C. versicolor, the peak abundance of GnRH I immunostaining was observed in secondary follicles during recrudescence phase; subsequently, the abundance of immunostaining gradually declines with the increase in the follicular development and finally attained the lowest concentration during vitellogenic and ovulatory follicles. Whereas, the GnRH I receptor immunostaining gradually increases from the recrudescence phase to previtellogenic phase with increasing follicular growth. This suggests that the increase in GnRH I receptor abundance in the C. versicolor coincides with the period of active follicular development and is an agreement with the earlier reports on rainbow trout (Uzbekova et al., 2001) and rat (Kogo et al., 1995). Whether locally produced GnRH I regulates the concentration of GnRH I receptor in the ovary is not known. In the present study, the highest abundance of GnRH I was detected much prior to the peak abundance of GnRH I receptor during previtellogenic stage of the ovary in C. versicolor. This suggests that the ovarian GnRH I and GnRH I receptor are not coregulated. This also raises the possibility that GnRH I may not be regulating its own receptor in the ovary of C. versicolor. This is in agreement with an earlier in vitro study which failed to demonstrate up regulation of GnRH I receptor by GnRH I in the granulosa cells of the small antral follicles (Schirman-Hildesheim et al., 2005).

The changes in the pattern of GnRH I immunostaining in the ovary of C. versicolor in general corroborates with the finding on rainbow trout (Uzbekova et al., 2002). The abundance of GnRH I immunostaining were relatively higher in the ovary of C. versicolor during reproductive inactive resting phase. During the regression and the resting phases, intense GnRH I immunostaining was observed in the granulosa cells of primary follicles and stroma cells. Observation of GnRH I immunostaining in the stroma cells suggests role of GnRH I in ovarian steroidogenesis. It has earlier been suggested that GnRH I may affect steroidogenesis possibly as a local autocrine and paracrine regulator by modulation of gonadotrophin stimulated steroidogenesis in the ovary (Peng et al., 1994; Vaananen et al., 1997). It has earlier been shown that in the rat ovary local action of GnRH I is predominantly inhibitory (Nathwani et al., 2000). The low circulating estradiol level described for C versicolor during reproductively inactive period (Raddar et al., 2001) may be correlated with the high ovarian GnRH I level found during the resting phase. A mild GnRH I immunostaining in the ovary during vitellogenic and ovulatory phases may be responsible for increased estradiol synthesis during these phases, thus ovarian GnRH I and circulating estradiol level is inversely related (Raddar et al., 2001). A number of studies are available suggesting the regulation of GnRH I gene expression by estradiol (Khosravi and Leung, 2003). It has also been shown that the estradiol decreases GnRH I mRNA level in time and dose-dependent manner (Dong et al., 1996). GnRH I mRNA levels in human granulosa-luteal cells were reduced by treatment with estradiol for 24 h, whereas GnRH I receptor mRNA levels were increased by short term treatment (6 h) but diminished by long term estrogen treatment indicating that estradiol regulates the expression of both GnRH I and its receptor in the ovary (Nathwani et al., 2000).

The presence of GnIH peptide was also detected in the ovary of C. versicolor by immunostaining and further confirmed by immunoblotting. GnIH was mainly localized in the granulosa cells, theca cells, oocytes and stromal or interstitial cells in the ovary of C. versicolor. Immunoblotting analysis suggests relatively highest concentration of GnIH in the ovary during recrudescence phase, subsequently its concentration in the ovary decline gradually to reached lowest concentration during previtellogenic phase. The GnIH concentration then rises again during vitellogenic and ovulatory phases in the ovary. Whether the gonadal GnIH interacts with the gonadal GnRH I and/or GnRH I receptor is not known. In the present study, there appear to be a temporal positive correlation between the expression patterns of GnRH I and GnIH peptides, but showed a negative correlation of GnIH with the GnRH I receptor in the ovary of C. versicolor during its reproductive cycle. This observation was further supported by our in vitro study showing suppressive effect of GnIH on ovarian expression of GnRH I receptor protein. Distribution pattern of GnIH, GnRH I and GnRH I receptor are nearly similar in the ovary of C. versicolor as also demonstrated in the avian brain (Ubuka et al., 2008). Thus, the spatial association of GnIH, GnRH I and GnRH I receptor in the ovary appears to be conserved irrespective to the site of their synthesis. In birds and hamster, confocal microscopy indicates that the GnRH I and GnIH peptides were on the same optical plane suggesting functional interactions between these peptides (Bentley et al., 2006; Kriegsfeld et al., 2006; Ubuka et al., 2008). The functional significance of these interactions is still unclear, but provides potential for GnIH regulation of GnRH I actions.

In this study, the distribution of GnIH in the ovary further suggests a potential for autocrine and/or paracrine regulation of GnIH on gonadal steroid production and germ cell differentiation and maturation in the lizard. A similar result has been obtained in birds (Bentley et al., 2008). The present finding also suggests the interaction of GnIH with GnRH I and bradykinin because of similar pattern of immunolocalization of these neuropeptides in the ovary of C. versicolor. These data suggest a close interaction between neuropeptides, GnRH I, bradykinin and GnIH in the ovary of C versicolor during the reproductive cycle. To the best of our knowledge, this is the first report showing the colocalization of GnIH, GnRH I and bradykinin in the ovary in any vertebrate.

GnRH I has been involved in a variety of both inhibitory and stimulatory responses in the mammalian ovary (Leung et al., 2003) including a role in follicular atresia or selection (Whitelaw et al., 1995), oocyte maturation (Dekel and Shalgi, 1987) and corpus luteum (Tsafriri and Adashi, 1994). Localization of GnRH I and GnRH I receptor in the granulosa cells of the atretic follicle in ovary of C. versicolor suggests that GnRH I may be regulating follicular selection by promoting atresia. The present study, is thus, in agreement with earlier studies showing greater GnRH I receptor expression in granulosa cells of the atretic follicles in rat ovary (Bauer-Dantoin and Jameson, 1995). It has also been demonstrated that GnRH I inhibited DNA synthesis in vitro (Saragüeta et al., 1997) or induced apoptosis in granulosa cells in rat (Billig et al., 1994). Interestingly, we noticed a shift in GnRH I, GnRH I receptor and GnIH immunostaining from the granulosa and theca cells to the periphery of oocytes during vitellogenic and ovulatory phases. An increase in the immunostaining of GnIH and GnRH I-system in oocyte prior to ovulation suggests their involvement in oocyte maturation in C. versicolor. Similarly in rainbow trout fish, GnRH I peptide transiently appeared in oocytes coincides with the time of oocyte maturation and decreased thereafter (Von Schalburg et al., 1999). Evidence exists for the direct action of GnRH I on in vitro oocyte maturation (Bogerd et al., 2002; Pati and Habibi, 1992). It has been demonstrated that GnRH I induced transcription of several genes associated with the follicular rapture and oocyte maturation during preovulatory period in rat, such as plasminogen activator (Ny et al., 1987), prostaglandin endoperoxidase synthase type 2 (Wong and Richards, 1992) and progesterone receptor (Natraj and Richards, 1993). Thus the actions of GnRH I within the vertebrate ovary are diverse and depend on the stage of ovarian reproductive cycle in vertebrates.

Despite of the fact that the role of the bradykinin in the mammalian ovary is well recognized (Hellberg et al., 1991; Kihara et al., 2000), little is known about the presence of this neuropeptide in the ovary of non-mammalian vertebrates. The present study demonstrates, for the first time, the presence of bradykinin and its receptor using immunohistochemistry and immunoblotting in the ovary of C. versicolor during different reproductive stages. In the present study, bradykinin and bradykinin B2 receptor were localized in the ovarian cells, where GnRH I and GnRH I receptor immunostaining were also localized. This suggests a possibility that ovarian bradykinin may be regulating GnRH I release in the ovary as suggested in the rat hypothalamus (Shi et al., 1999). A significant positive correlation between changes in relative abundance of GnRH I and bradykinin immunostaining in the ovary of C. versicolor during the reproductive cycle further support this possibility, although more detailed studies are needed to confirm this hypothesis.

This study demonstrates the presence of bradykinin and bradykinin B2 receptor in the ovarian follicles and showed a significant variation with intra-ovarian concentration during follicular growth and development. Bradykinin B2 receptor immunostaining showed a gradual decrease beginning from resting phase and continue to decrease until vitellogenic phase. Bradykinin concentration increase transiently during the ovulatory period suggests its role in ovulation in C. versicolor. Previous studies have demonstrated that bradykinin can induce ovulation or potentiate the effect of LH surge in the rat ovary (Yoshimura et al., 1988; Brännström and Hellberg, 1989). Besides bradykinin may induce the release of prostaglandins and cause inflammatory reaction, vasodilation, etc. (Hellberg et al., 1991). Bradykinin may partially stimulate oocyte maturation through its effects on GnRH I (Ekholm et al., 1981). Both bradykinin and bradykinin B2 receptor were mainly localized in the granulosa cells and theca cells of growing follicles and in oocyte during preovulatory maturation. This suggests the physiological importance of bradykinin in the early stage of the follicular development as demonstrated in the mammalian ovary (Kihara et al., 2000). The results from the present study in the C. versicolor showed an increase in the abundance immunostaining for bradykinin during the recrudescence phase. Further bradykinin immunostaining has been predominantly noticed in the secondary follicles. The immunostaining subsequently shifted from follicular cells to the periphery of oocytes during preovulatory period. Similar pattern of immunostaining was also noticed in GnRH I, GnRH I receptor and GnIH. These data suggest a close interaction between neuropeptides, GnRH I, bradykinin and GnIH in the ovary of C. versicolor during the reproductive cycle.

In brief this study showed significant variation in the localization and abundance of immunostaining of the neuropeptides GnRH I, bradykinin, GnRH I receptor, bradykinin B2 receptor and GnIH during different reproductive stages. GnRH I, bradykinin and their receptors and GnIH were mainly localized in the granulosa cells of early growing follicles and in the periphery of cytoplasm of oocyte prior to preovulatory maturation period. This study showed significant positive correlation between the expression pattern of GnRH I and GnIH peptides, but showed a negative correlation of GnIH with GnRH I receptors in the ovary of C. versicolor. The functional significance of these interactions is still unclear, but provides potential for GnIH regulation of GnRH I receptor concentration. This study further suggests a possibility for bradykinin regulating GnRH I synthesis in the ovary. An increase in the immunoreactivity of GnRH I-GnIH system together with bradykinin–bradykinin B2 receptor in the oocyte prior to ovulation suggests their involvement in oocyte maturation. It is thus concluded that the Calotes ovary possess GnRH I–GnIH–bradykinin system and interaction between these neuropeptides may be involved in regulation of follicular development and oocyte maturation. Further studies are needed to confirm this hypothesis.

Acknowledgments

This work was supported in part by Grant-in aid from UGC, New Delhi to A.K.; Scientific research from the Ministry of Education, Science and Culture, Japan (15207007, 16086206 and 18107002) to K.T. and grants from NIH, USA (HD41749, HD52155 and RR03034) to R.S.

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