Testosterone Triggers the Brain–Pituitary–Gonad Axis of Juvenile Female Catfish (Heteropneustes fossilis Bloch) for Precocious Ovarian Maturation

General and Comparative Endocrinology 126, 23–29 (2002) doi:10.1006/gcen.2001.7751, available online at http://www.idealibrary.com on

Testosterone Triggers the Brain–Pituitary–Gonad Axis of Juvenile Female Catfish (Heteropneustes fossilis Bloch) for Precocious Ovarian Maturation Basant K. Tiwary,* ,1 R. Kirubagaran,† and Arun K. Ray* ,2 *Department of Animal Physiology, Bose Institute, P-1/12, CIT Scheme VII M, Calcutta-700 054, India; and †National Institute of Ocean Technology, NIOT Campus, Chennai-601 302, India Accepted November 2, 2001

in brain of juvenile rainbow trout and induced precocious maturation (Crim and Evans, 1982). The release of GnRH from neurons in the hypothalamus controls pituitary gonadotropin (GtH) levels, which in turn regulate reproductive activity (Sherwood, 1987). The GnRH neurons of the preoptic area (POA) directly innervate the pituitary in teleosts (Yamamoto et al., 1998). Amano et al. (1995) demonstrated an increase in the number of neurons expressing salmon GnRH (sGnRH) mRNA in the POA and the ventral telencephalon during maturation and concluded that sGnRH neurons in these areas are involved in the regulation of gonadal maturation of masu salmon (Oncorhynchus masou) through GtH secretion. Sex steroids exert positive feedback effects at the brain and pituitary levels in immature animals. Steroid administration has increased brain GnRH contents in European eels, Anguilla anguilla (Dufour et al., 1985, 1989, 1993), and rainbow trout, Oncorhynchus mykiss (Breton and Sambroni, 1996). Activated sGnRH gene expression was observed in the preoptic area in precocious male masu salmon (O. masou) after treatment with another androgen, 17␣-methyl testosterone (Amano et al., 1994). Testosterone (T) also showed a positive effect on the accumulation of GtH in the pituitary gland of juvenile male and female rainbow trout (Crim and Evans, 1979; Gielen et al., 1982). Crim et al. (1981) observed similar findings with estrogens and aromatizable androgen. Crim and Evans (1982) noticed in-

The brain–pituitary– gonad axis of precociously matured females (PMFs) of Indian catfish (Heteropneustes fossilis), produced by testosterone treatment during juvenile stages, was analyzed by studies on immunoreactive gonadotropin-releasing hormone (ir-GnRH) secreting cells of the preoptic area of brain, plasma levels of gonadotropin (GtH-II), testosterone (T), and estradiol-17␤ (E 2). GnRH cells of PMFs were large and strongly immunoreactive in comparison to control females. PMFs showed higher plasma levels of GtHII, T, and E 2 than did control females. The ovaries of PMFs contained ripe ova, whereas control females had ova at maturing stages. This study suggests testosterone-mediated activation of the brain–pituitary– ovarian axis for precocious maturation in juvenile catfish. © 2002 Elsevier Science (USA) Key Words: precociously matured female (PMF); GnRH; GtH-II; steroids; ovary.

INTRODUCTION Gonadal steroids have both negative and positive feedback effects on the brain–pituitary axis (Trudeau, 1997). It exerted positive feedback on the gonadotropin-releasing hormone (GnRH) producing system 1

Present address: Department of Zoology, Chandernagore Government College, Chandernagore, Hooghly, West Bengal, India. 2 To whom correspondence and reprint requests should be addressed. 0016-6480/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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creased plasma GtH levels and precocious maturity in juvenile trout after prolonged treatment with testosterone. Testosterone accelerates the development of the catfish GnRH system in the brain of immature African catfish, Clarias gariepinus (Dubois et al., 1998). Testosterone stimulates the secretion of maturational gonadotropin (GtH-II) from pituitary cells both in vivo and in vitro in rainbow trout and eel, respectively, but this action was not mimicked by estradiol-17␤ (Breton et al., 1997; Huang et al., 1997). A long-term treatment with T positively influenced GnRH-stimulated GtH-II release in juvenile striped bass, Morone saxatilis and cultured goldfish pituitary cells (Holland et al., 1998; Lo and Chang, 1998). Goos et al. (1986) explained this phenomenon by showing influence of testosterone on the synthesis of GnRH and proposed that GnRH neurons can be stimulated by exogenous testosterone for synthesis as well as for release. Testosterone has a predominant role in control of GtH-II secretion in goldfish and trout (Kobayashi et al., 1989; Breton and Sambroni, 1996). Nonaromatizable androgen like 5␣-dehydrotestosterone (5␣-DHT) was also found to increase GtH content in teleost pituitaries, but to a lesser extent than an aromatizable one, suggesting its direct action on GnRH cells (Borg, 1994). It appeared that testosterone also had some physiological role in precocious maturation of Indian catfish (Tiwary et al., 1998). Therefore, immunocytochemical localization of GnRH cells in the preoptic area of precociously matured females (PMFs) was performed for assessing the effect of testosterone on reproductive brain of the fish. Simultaneously, plasma levels of GtH-II, estradiol-17␤ (E 2), and testosterone were measured during the preparatory phase of the reproductive cycle (April) in this catfish using radioimmunoassay (RIA). Histological study on the ovaries of PMFs was conducted to assess the ultimate effect of testosterone-activated brain–pituitary– gonad axis on the extent of reproductive maturity in PMFs.

MATERIALS AND METHODS Production of PMFs Precociously matured females were produced in our laboratory by subjecting fresh hatchlings of catfish to immersion treatment in testosterone propionate at

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Tiwary, Kirubagaran, and Ray

the dose of 300 ␮g/L for 1 month and subsequently were reared in controlled laboratory conditions for 7 months (Tiwary et al., 1998).

Sampling A total of 10 PMFs and 10 control females were sampled for brain and plasma. Blood was collected in heparinized syringes and centrifuged in cold (4°) at 450xg for plasma separation. All females were decapitated, and their brains were dissected out and fixed in freshly prepared Bouin’s fluid.

Immunocytochemistry of GnRH Cells The brain and pituitary were dissected out in all fishes and fixed in freshly prepared Bouin’s fluid for 24 h. The tissues were embedded in paraffin and cut at 30-␮m thickness in sagittal planes, which ensured that the majority of preoptic cells were not split between subsequent sections and thus not counted more than once. All sections were mounted on gelatin-coated slides and air-dried for 30 min. The sections were rehydrated in 0.01 M phosphatebuffered saline (PBS, pH 7.6) and treated with normal goat serum (1:20 dilution). Primary antiserum against salmon GnRH (a gift from N. M. Sherwood, University of Victoria, Canada) at a 1:1000 dilution was applied to each section, and then slides were incubated in a closed moist chamber for 48 h at 4°. Sections were washed for 10 min in two changes. The sections were then incubated in goat anti-rabbit IgG (Sigma, St. Louis, MO) at 1:20 dilution for 60 min. Sections were again rinsed in PBS for 20 min and incubated in peroxidase–antiperoxidase (PAP, Sigma) at a dilution of 1:200 for 60 min and again rinsed in PBS for 10 min. Then, sections were washed in 0.05 M Tris–HCl (pH 7.6) for 5 min in two changes and were treated with 0.05% 3,3⬘-diaminobenzidine tetrahydrochloride (DAB, Sigma) with 0.01% H2O 2 in 0.05 M Tris–HCl for 3 to 10 min, rinsed in distilled water, dehydrated through graded alcohol, cleared in xylene, and mounted with DPX. The POA is a continuum of cells in the basal telencephalon and the mediobasal hypothalamus. The GnRH staining was quantified by counting numbers and measuring the size of immunoreactive cell bodies in these brain areas. The relative staining intensity of immunoreactive cell bodies in the POA and ir material

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Brain–Pituitary–Gonad Axis in Catfish

in the pituitary was also estimated. The number of GnRH-ir preoptic cells was counted in each immunostained section, and their major and minor diameters were measured in catfish following the method of Yamamoto et al. (1995). Only GnRH-ir cells containing nuclei with clearly identifiable boundaries were measured, which eliminated the possibility of recounting of the same cell in subsequent sections. Immunostained sections were examined under 400⫻ magnification, and all of the cells stained more densely than background were counted visually in the POA of PMFs and control female fish (n ⫽ 10) without knowing the origin of the sample. Because GnRH-ir neurons of the POA were ovoid in shape, major and minor diameters of these ir cells were measured with a micrometer selected randomly from the sections.

Radioimmunoassay of Hormones Sampling. Blood samples were collected in tubes by heparinized syringes separately from 10 female catfish, in both the PMF and control groups, by caudal vein puncture under anesthesia during April. Samples were centrifuged at 450xg at 4° for 15 min to separate plasma. Plasma was stored at ⫺20° until it was assayed for GtH-II, E 2, and T. Radioimmunoassay of GtH-II. The RIA for GtH-II was carried out by a heterologous RIA system following the method of Goos et al. (1986). This assay was characterized and validated by Senthilkumaran and Joy (1994) in Indian catfish (H. fossilis). Displacement of the labeled gonadotropin was studied by diluting standard gonadotropin in the concentration range of 10 pg to 10 ng/50 ␮l. Standard and samples were incubated in duplicate for 24 h with 250 ␮l antiserum (anti-catfish gonadotropin) at a dilution of 1:5000, which gave 40% binding in the titer assay (determined by serial dilution of the antiserum). Then, labeled GtH-II (10,000 cpm in 200 ␮l of the diluent veronal buffer) was added. Goat anti-rabbit serum was added on day 3. On day 4, the assay was terminated with 2 ml of the diluent and centrifuged at 1000xg. The precipitate was counted after decanting the supernatant in a Beckman gamma counter for 1 min. All incubations and centrifugation were carried out at 4°. Calculation was performed after logit–log transformation. Extraction of serum samples. Then, 50-␮l aliquots of serum samples were taken in duplicate, and the vol-

ume was made up to 200 ␮l with gelatin–phosphate buffer (GPB). After addition of 1 ml of diethyl ether, each tube was vortexed for 1 min. The ether phase decanted into assay tubes and evaporated to dryness in vacuo at 37°. Ether blank was prepared by drying 1 ml of diethyl ether under identical conditions. Radioimmunoassay of steroids. Serum levels of estradiol-17␤ and testosterone were measured by radioimmunoassay following the method developed and validated by Lamba et al. (1981) in this species (H. fossilis). The dried serum extracts, as well as the extracted internal standards, were reconstituted with 100 ␮l of GPB. Various concentrations of the authentic nonradioactive steroids in 100 ␮l of GPB were pipetted in duplicate in another set of tubes. These were used for obtaining an external standard curve. For determining nonspecific binding and total counts, 100 ␮l of the tracer solutions of various steroids was added to tubes containing 200 ␮l of GPB only. Into all of the above tubes, except those kept for nonspecific binding and total counts, were added 100 ␮l of the appropriate [ 3H] steroid solution and 100 ␮l of the corresponding antiserum. Thus, the total volume in every tube was maintained at 300 ␮l. All tubes were then incubated overnight at 4° in a refrigerator, after which they were kept in an ice-water bath at 4°, and 500 ␮l of ice-cold charcoal suspension was added within 3 min to all of the tubes except those meant for estimation of total counts. The tubes were shaken in the ice bath for 10 min from the time of addition of charcoal to the first tube and then centrifuged at 4° for 20 min in a refrigerated centrifuge. The supernatant was decanted into scintillation vials containing 1 ml of ethanol and 10 ml of scintillation fluid. Then, the vials were counted for 1 min in a liquid scintillation spectrometer having 50% counting efficiency for tritium.

Histology of Ovary After fixation in Bouin’s fluid, gonadal morphology was studied using histological preparations of 7 ␮m cross section per fish, stained with hematoxylin and counterstained with eosin. A total of 10 fish in each of the PMF and control groups were examined. Histological studies were performed on five sections taken from five different locations along the cranial– caudal axis. The diameter of oocytes was measured with the help of a micrometer.

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Tiwary, Kirubagaran, and Ray

262 ⫾ 14 in controls. The major diameter of ir cells increased from 7.5 ⫾ 0.33 to 11.3 ⫾ 1.30 ␮m, whereas the minor diameter increased from 4.5 ⫾ 0.92 to 6.0 ⫾ 0.78 ␮m.

Maturational Gonadotropin PMFs showed significantly high levels (8.2 ⫾ 0.69 ng/ml) of GtH-II, in comparison to control females (6.9 ⫾ 0.37 ng/ml) (Fig. 3).

Sex Steroids E 2 and T levels in plasma of PMFs were compared to those of control females during the preparatory phase of the gonadal cycle (Fig. 3). The plasma level of E 2 showed about a 2.5-fold higher value (10.21 ⫾ 0.34 ng/ml) in PMFs, in comparison to that of control females (4.04 ⫾ 0.23 ng/ml) (P ⬍ 0.001). Similarly, the T level in plasma of PMFs (7.23 ⫾ 0.25 ng/ml) was significantly higher than that of controls (3.04 ⫾ 0.18 ng/ml). FIG. 1. Microphotographs showing ir-GnRH cells in the preoptic area of (A) control female and (B) PMF during the preparatory phase of the ovarian cycle (⫻750).

Statistical Analysis All values were represented as means ⫾ standard deviations. The analyses of the differences in PMFs compared to control values were performed using a t test. Significance was based on the probability of occurrence being ⬍ 0.001 in all statistical procedures.

RESULTS Immunoreactive GnRH Cells GnRH cells in the POA of PMFs showed strong immunoreactivity in comparison to control females during the preparatory phase (April) of the ovarian cycle (Fig. 1). In addition, PMFs showed a significant increase in number and size of GnRH cells in the POA during this period (Fig. 2). The number of cells, on average, in PMFs was 345 ⫾ 23, in comparison to

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FIG. 2. Differences in major and minor diameters of ir-GnRH cells in the POA of PMFs and control females during the preparatory phase. Error bar represents ⫾ SD.

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A major function of steroids is also to synchronize the different components of the brain–pituitary– gonad axis over the reproductive process. In teleosts, it is well established that steroids exert positive and negative feedback effects notably on the maturation of the

FIG. 3. Plasma levels of gonadotropin, estradiol-17␤ and testosterone in PMFs and control females during the preparatory phase. Plotted as means ⫾ SDs.

Ovarian Histology The average diameter of oocytes in ovaries of PMFs was significantly greater (576.28 ␮m) than that of control females (132.48 ␮m) (P ⬍ 0.001). The ovaries in PMFs were packed with large ova at a ripe or mature stage, and smaller oocytes were few in number and seen only toward the periphery (Fig. 4). Ripe ova were classified based on features such as migration of nucleus to periphery, dissolution of nuclear membrane, and coalescence of yolk granules. By contrast, control females had predominantly cortical alveoli or yolk vesicle stages. These stages were identified based on the presence of numerous small nucleoli in oval or round nuclei or small yolk vesicles surrounding the nuclei, respectively.

DISCUSSION It is well known that precocious sexual maturation can be induced in vertebrates by administering gonadal-, pituitary-, or hypothalamic-releasing factors.

FIG. 4. Transverse section through ovaries of (A) control and (B, C) PMF showing fully matured ova in PMF during the preparatory phase (⫻500). PN, perinucleolar stage; YG, yolk granules; OW, oocyte wall; RO, ripe ova.

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neuroendocrine circuits controlling the release of the maturational gonadotropin, but the details of these mechanisms are still largely unknown. The dramatic increase in the number and intensity of ir-sGnRH cells in the preoptic area of PMFs appears to be due to the positive action of testosterone or aromatized estradiol17␤ on GnRH cells in brain, as the highest level of aromatase activity in the brain of the African catfish was found in the POA (Timmers et al., 1987). Steroid induction of aromatase may not be direct but requires the mediation of one or more neurohormones or neurotransmitters. Sex steroids showed a positive stimulatory effect on GnRH synthesis in the preoptic area of fish brain (Schreibman et al., 1986; Goos et al., 1986; Grober et al., 1991; Amano et al., 1994). Exogenous androgens initiated activity in specific LHRH-producing nuclei in the brain for induced sexual maturity in platyfish (Schreibman et al., 1986). The action of steroids is in agreement with the abundance of androgen receptors in goldfish forebrain during sexual maturity (Pasmanik and Callard, 1988) and estrogen receptors in the preoptic area of rainbow trout (Anglade et al., 1994). However, neither androgen nor estrogen receptors were ever co-localized with GnRH neurons in fish (Linard et al., 1996). Because the sGnRH promoter does not present perfect estrogen responsive element (ERE) sequences (Kugland et al., 1993), steroids in fish may not directly regulate GnRH synthesis. It is also possible that the regulation by steroid hormones may not always occur through a palindromic sequence, as suggested by various workers (Mitchell et al., 1987; Crone et al., 1990). It is highly probable that steroid effects are mediated through several neuromediators such as dopamine, GABA, and galanine neuropeptide Y, as suggested in rainbow trout (Breton and Sambroni, 1996). The increase in size and number of irGnRH cells in the preoptic area of PMFs may be due to a steroid-mediated increase in GnRH biosynthesis, cell migration, and/or cell birth (Grober et al., 1991). Both aromatizable and nonaromatizable androgens have been found to increase the content of GtH-II in teleosts. Testosterone has positive feedback action on GnRH-secreting neurons as testosterone stimulates in vivo gonadotropin secretion through increasing sGnRH contents in the brain and pituitary of rainbow trout (Breton and Sambroni, 1996). Testosterone potentiates GnRH-stimulated GtH-II release through probable aromatization and subsequent intracellular

© 2002 Elsevier Science (USA) All rights reserved.

Tiwary, Kirubagaran, and Ray

activation of proteinkinase C (PKC) pathways (Lo and Chang, 1998). Steroid activation of the brain–pituitary– gonad axis during juvenile stages of this catfish might have initiated positive feedback regulation of sex steroids for rapid oocyte maturation 3 months ahead of the normal spawning period. However, further experiments with nonaromatizable androgen and assessment of aromatase activity are necessary to confirm this hypothesis. In addition, this technique bears a high potential for developing a technology in advancing the spawning season of this catfish.

ACKNOWLEDGMENTS The authors thank N. M. Sherwood for her gift of salmon GnRH antiserum. The authors are also grateful to H. J. Th. Goos, University of Utrecht, The Netherlands, and the World Health Organization, Geneva, for providing us with catfish GtH-II along with its antibody and antisera for testosterone and estradiol-17␤, respectively, as gifts. B. K. Tiwary is grateful to the Council of Scientific and Industrial Research, Government of India, New Delhi, for financial support in the form of a research fellowship.

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