Seasonal dynamics in gonadotropin secretion and E2-binding in the ...

Fish Physiol Biochem (2005) 31:183–188 DOI 10.1007/s10695-006-0022-5

RESEARCH ARTICLE

Seasonal dynamics in gonadotropin secretion and E2-binding in the catfish Heteropneustes fossilis R. Kirubagaran Æ B. Senthilkumaran Æ C. C. Sudhakumari Æ K. P. Joy

Ó Springer Science+Business Media B.V. 2006 Abstract In the present investigation, significant annual/seasonal variations were noticed in plasma and pituitary gonadotropin (GTH) which were correlated with gonado-somatic index, plasma estradiol17b, and nuclear E2 receptor (NE2R) in the pituitary, hypothalamus and telencephalon. The NE2R concentrations and dissociation constant (kd) values showed significant seasonal variations with high values in the late preparatory phase and low values in the postspawning phase. The NE2R levels were the highest in the pituitary, followed by the hypothalamus and telencephalon in all the seasons. In the prespawning phase, ovariectomy (OVX) elicited a strong negative feedback on GTH secretion with a bimodal pattern of release and elevated the NE2R levels and kd values, without producing any significant change in the resting phase suggesting that E2

R. Kirubagaran (&) Æ B. Senthilkumaran Æ C. C. Sudhakumari Æ K. P. Joy National Institute of Ocean Technology, Pallikaranai, Chennai 601302, India e-mail: [email protected] B. Senthilkumaran Æ C. C. Sudhakumari Department of Animal Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Gachibowli, Hyderabad 500 046, India K. P. Joy Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

appears to exert differential feedbacks on GTH secretion. Keywords Catfish Æ Estradiol-17b Æ E2-binding Æ Gonadotropin Æ Ovariectomy

Introduction In teleosts, gonadal steroids exert feedback actions on the brain-pituitary axis to modulate gonadotropin (GTH) secretion (Goos 1987). The degree and nature of the steroid feedback show variations during different phases of the reproductive cycle (Van Oordt 1987). The steroid-binding cells are located largely in the ventral telencephalon and hypothalamus of the brain, and in gonadotrops of the pituitary. Surprisingly, studies on steroid receptor binding dynamics in the brain and pituitary of fishes are meager. Although changes in gonadal steroids levels in relation to the annual reproductive cycle, gametogenesis and spawning were monitored in many species, studies correlating such changes with gonadotropin (GTH) content are confined largely to salmonids and cyprinids (Fostier et al. 1983; Kobayashi et al. 1986). Therefore, in the present investigation, a detailed study was conducted in a siluroid catfish, Heteropneustes fossilis, on the following aspects of the brainpituitary-gonadal axis: (1) annual/seasonal changes in the plasma/pituitary GTH and plasma E2 levels, (2)

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effects of ovariectomy (OVX) on GTH, E2 and testosterone levels to elucidate E2 feedback relations, and (3) dynamics of nuclear E2 receptor (NE2) binding sites in the telencephalon, hypothalamus and pituitary in relation to season and OVX.

Materials and methods

Fish Physiol Biochem (2005) 31:183–188

fractions as described by Wisel et al. (1991). The protein concentration of each aliquot was estimated by the method of Lowry et al. (1951). The assay was performed according to the method of Korenman (1975). The NE2R levels were quantified by Scatchard analysis. Data were analyzed using a one-way analysis of variance (ANOVA) followed by Newman–Keul’s multiple test and Student’s t-test for seasonal variation and kd values, respectively.

Annual/seasonal study Adult female catfish H. fossilis were collected in different phases of annual reproductive cycle. After 48 h of arrival of animals to the laboratory, the fish were weighed and blood was collected for the estimation of plasma levels of GTH and E2. The pituitaries and ovaries were dissected out to quantitate GTH content and gonado-somatic index (GSI). The telencephalon, hypothalamus and pituitary were removed quickly on ice for the NE2R binding assay. OVX About 180 fish were ovariectomized in the prespawning phase following standard protocols and were maintained in aquaria with circulating well water under natural photoperiod and ambient temperature. For sham OVX (180 fish), all the steps were followed except that the ovaries were not removed. After 4–6 weeks of OVX/sham OVX, blood was collected and plasma was separated for analysis of GTH, E2 and testosterone levels. The telencephalon, hypothalamus and pituitary were removed quickly on ice for the NE2R binding assay. Analysis of samples The RIA of steroid hormones were carried out following the method developed and validated by Lamba et al. (1983) in H. fossilis. The lower detection sensitivity of RIA for steroids was 10 pg ml)1. Plasma GTH levels were measured using a heterologous RIA system, following the method of Goos et al. (1986). The lower detection sensitivity of RIA for GtH-II was 800 pg ml)1. For NE2R binding assay, the telecephalon, hypothalamus and pituitary collected in the seasonal study and OVX experiment were pooled separately for the preparation of nuclear

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Results The GSI of female catfish showed a significant annual variation (Fig. 1). The plasma E2 levels varied during the reproductive cycle with a major peak in the early prespawning phase and a minor peak in July. The plasma GTH were undetectable in the resting phase, appeared in the early preparatory phase and increased through March to give a minor peak in the late preparatory phase and a major peak in July, and thereafter it decreased sharply. The GTH levels in the pituitary were monitored in different phases of the reproductive cycle (Fig. 2). According to the Newman–Keul’s analysis, all the values were significantly different from each other except that in April and June. The NE2R levels (Fig. 2) showed significant seasonal variations in the telencephalon, hypothalamus and pituitary with peak values in the late preparatory phase (April). The titre of the NE2R decreased significantly in May when the major E2 peak was evident and the GTH content in the plasma decreased with a simultaneous increase in the pituitary. It increased in June and July when changes in the E2 and GTH levels were the opposite. The pituitary recorded the highest titre of NE2R followed by the hypothalamus and telencephalon in all the seasons. The kd values showed a positive correlation with the NE2R levels (Table 1). The plasma levels of E2 decreased significantly while the testosterone levels were not detectable at fourth and sixth week of OVX (Fig. 3). The plasma GTH levels increased significantly after OVX with the peak rise at week 4. The levels of NE2R increased in the telencephalon and hypothalamus significantly at both the durations of OVX compared to that of the respective sham control groups. But the magnitude of the increase was significantly low in the sixth week compared to


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Fig. 1 Annual variations of plasma gonadotropin (GTH) and E2 levels, and gonadosomatic index (GSI) in the female catfish, H. fossilis during ovarian cycle (means–SEM; group size n=5; ND, not detectable; one-way ANOVA—Newman–Keul’s test; P < 0.05)

Fig. 2 Seasonal variations in NE2R levels in telencephalon, hypothalamus, and pituitary and plasma E2 and GTH levels in the female catfish, H. fossilis (means–SEM; group size n=5; one-way ANOVA—Newman–Keul’s test)

that of the fourth week. Similarly, the pituitary NE2R levels were elevated significantly at both the durations but without any significant difference between the durations. The magnitude of the increase was in the order pituitary < hypothalamus < telen-

cephalon. The kd values of all the tissues increased significantly at the fourth week of OVX (Table 2), the increase was 3-fold in the hypothalamus. At the sixth week, the kd value was significantly high only in the pituitary.

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Table 1 Equilibrium dissociation constant (kd) of E2 receptors in nuclear extracts of hypothalamus, telencephalon, and pituitary of female catfish, H. fossilis during the ovarian cycle (means – SEM; group size n = 5; one-way ANOVA—Newman–Keuls’ test) kd values in the order of nM

Reproductive phases

Hypothalamus Late preparatory (April) Early prespawning (May) Late prespawning (June) Spawning (July) Postspawning (September)

0.98 0.30 0.46 0.34 0.28

– – – – –

Telencephalon

0.010 0.017 0.019 0.005 0.011

0.270 0.130 0.160 0.225 0.090

– – – – –

0.017 0.023 0.018 0.014 0.004

Pituitary 0.630 0.375 0.410 0.560 0.200

– – – – –

0.020 0.014 0.024 0.011 0.016

Fig. 3 Effects of different durations of ovariectomy (OVX, 4 weeks and 6 weeks) on NE2R levels in telencephalon, hypothalamus, and pituitary and plasma E2, testosterone (T),

and GTH levels in female catfish, H. fossilis (means–SEM; group size n = 5; one-way ANOVA—Newman–Keul’s test)

Table 2 Equilibrium dissociation constant (kd) of E2 receptors in nuclear extracts of hypothalamus, telencephalon, and pituitary in 4 and 6 week ovariectomized catfish, H. fossilis

(means – SEM; group size n = 5; *P < 0.001; control vs. experimental groups; Student’s ‘t’ test)

Groups

kd values in the order of nM Hypothalamus

4 4 6 6

WK WK WK WK

SHAM OVX SHAM OVX

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0.36 0.92 0.38 0.39

– – – –

0.010 0.015* 0.009 0.011

Telencephalon 0.19 0.27 0.22 0.23

– – – –

0.005 0.008* 0.007 0.005

Pituitary 0.380 0.550 0.375 0.530

– – – –

0.012 0.014* 0.010 0.013*


Discussion As has been previously reported in this species (Sundararaj and Vasal 1976), the present study also shows that gonadal recrudescence measured in terms of the GSI is directly related to the seasonal changes attributed by environmental parameters. The annual changes in the GSI were also correlated with plasma E2 levels in this species by Lamba et al. (1983). The sharp rise in the GSI during this period correlates negatively with the plasma E2 levels but positively with the plasma GTH levels. There is also a strong negative correlation between plasma E2 and GTH levels. In the resting phase, plasma E2 level was not detectable as was also observed by Lamba et al. (1983) and indicated cessation of ovarian steroidogenesis. The heterologous RIA data of GTH show that both the plasma and pituitary contents showed significant variations during the reproductive cycle of the catfish. Experimental evidences in support of the existence of a strong negative feedback are provided further by the OVX data. The OVX data show that there is no sign of feedback in the resting phase as both GTH and E2 levels are undetectable. Thus, during the annual reproductive cycle of the catfish, the E2-induced feedback varies according to the season as has been reported earlier (Van Oordt 1987). Data on E2 receptor dynamics during the reproductive cycle or after OVX are lacking for teleosts. The present study shows significant seasonal variations of NE2R levels in the pituitary, hypothalamus and telencephalon during the reproductive cycle with the concentrations greatest in the late preparatory phase and lowest in the postspawning phase. The greatest concentration of NE2R in the pituitary is in agreement with similar observations in mammals (Ginsburg et al. 1975). The low titre of NE2R observed in the catfish tissues in comparison with mammalian tissues may be due to their low volume or low number of E2-concentrating cells. Low titres of cytosolic E2 receptors were also reported in the brain and pituitary of dogfish (Jenkins et al. 1980). Variations in E2 receptor titres have also been reported in the brain and pituitary of rat during the estrous cycle (Ginsburg et al. 1975) and in the liver of teleosts (Smith and Thomas 1991; Campbell et al. 1994). In the ovareictomized catfish with low E2 titre, the NE2R levels increased significantly in the order:

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pituitary < hypothalamus < telencephalon. The kd values reported in the catfish tissues are higher than those reported in rat (Anderson et al. 1973) but lower than that reported for E2 cytosolic receptors in the dogfish (Jenkins et al. 1980). As the kd value is directly related to receptor abundance and inversely to binding affinity of the receptors, the data show an inverse relationship between receptor abundance and affinity was the highest in the postspawning and early spawning (May) phases compared to other seasons with lower affinity. The affinity also decreased in all the tissues after 4 weeks of OVX and remained low in the pituitary even at the sixth week. Recently, three different forms of estrogen receptors (ERs) have been reported in Atlantic croaker Micropogonias undulatus influencing ligand specificity and receptor function (Hawkins and Thomas 2004). Real-time polymerase chain reaction conducted by Filby and Tyler (2005) also revealed some clear distinctions in the ontogenic and tissue expression of ERs, suggesting different functions for each ER subtype. Hence, the changes in the kd value observed in the present study would be the reflection of the presence of different ERs forms and the change in their ratio in this species. In conclusion, GTH and E2 profiles showed significant annual/seasonal variations during the reproductive cycle. The E2-induced feedback modulation of GTH secretion (synthesis, storage and release) is mediated through differential interactions with NE2R in the pituitary, hypothalamus and telencephalon. Acknowledgements Professor G.D. Niswender, Colorado State University, Fort Collins, CO, USA, Prof. Henk Goos, University of Utrecht, The Netherlands and Dr. G.L. Kumari, NIHFW, New Delhi, India are gratefully acknowledged for providing us antisera for estradiol-17b, catfish GTH & antiGTH and testosterone, respectively, as a gift.

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