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a Graduate School for Developmental Biology, Research Group for Comparative Endocrinology, Utrecht University, 3584 CH Utrecht, The Netherlands
b Department for Fish Culture and Fisheries, Wageningen Agricultural University, 6700 AH Wageningen, The Netherlands
ABSTRACT
The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Stress has been shown to interfere with reproduction and the functioning of the BPG axis. The response to chronic and severe stress may require much energy and force the organism to make adaptive choices. Energy that is normally available for processes like growth, immune response, or reproduction will be channeled into restoration of the disturbed homeostasis. Cortisol plays a key role in the homeostatic adaptation during or after stress. In the present study, immature common carp were fed with cortisol-containing food pellets covering the pubertal period. We showed that cortisol caused an inhibition of pubertal development, by affecting directly or indirectly all components of the BPG axis. The salmon GnRH content of the brain was decreased. Luteinizing hormone- and FSH-encoding mRNA levels in the pituitary and LH plasma levels were diminished by long-term cortisol treatment, as was the testicular androgen secretion. Testicular development, reflected by gonadosomatic index and the first wave of spermatogenesis, was retarded.
cortisol, hypothalamus, pituitary, puberty, spermatogenesis, stress, testes
INTRODUCTION
Adaptation to changing environmental conditions is essential for maintenance of physiological homeostasis. Stress can be defined as a disturbance of homeostasis by any kind of external or internal factor, referred to as the stressor. The consequence of the action of a stressor is that the homeostatic equilibrium is threatened. This will induce a coordinated set of behavioral and physiological responses that are compensatory and/or adaptive, enabling the organism to restore its homeostatic set points [1]. These adjustment reactions have been identified as the neuroendocrine stress response [2]. However, the response to prolonged stress may exceed the adaptive capacity. Energy, normally available for processes like growth, immune response, or reproduction, may then be channeled into restoration of the disturbed homeostasis. This may result in maladaptation with adverse effects on reproduction, immune competence, or growth.
Indeed, in several vertebrates, stress has been shown to interfere with reproduction and the functioning of the brain-pituitary-gonad (BPG) axis. In mammals, all levels of the BPG axis are affected [3]. For example, a decrease in plasma LH and hypothalamic LHRH in male rats after chronic restraint stress has been shown [4], and similar results were found in rams and ewes [5]. Furthermore, adult rats submitted to immobilization stress from prepuberty showed decreased plasma LH and plasma testosterone (T) levels [6], whereas Charpenet et al. [7] demonstrated that chronic intermittent immobilization stress induced a strong decrease of plasma T levels and testicular T content in rats, without, however, detectable changes in plasma LH values. The precise mechanisms via which the stress response has its adverse effects on reproduction are still unknown.
Reports on the effects of stress on the reproductive capacity in fish are inconsistent because stimulatory as well as inhibitory effects of stress, or no effects at all, have been described. (Reviewed by Wendelaar Bonga [1]). In male brown trout, Salmo trutta L., acute and chronic stress suppressed the plasma levels of 11-ketotestosterone (11KT). However, plasma gonadotropin levels were elevated following 1 h of handling stress [8]. The variability of these results may depend on the nature and duration of the stressor and the animal model that was used.
In all vertebrates, including fish, cortisol plays a key role in the restoration of homeostasis during or after stress. Furthermore, cortisol has frequently been indicated as the major factor mediating the suppressive effect of stress on reproduction. Carragher et al. [9] showed that implantation of cortisol-releasing pellets in the brown trout, S. trutta L. and in the rainbow trout, Salmo gairdneri Richardson, chronically elevated the plasma cortisol levels and affected a wide range of reproductive parameters. Cortisol-implanted maturing male brown trout had smaller gonads, lower plasma T levels, and their pituitaries had lower gonadotropin content.
The onset and regulation of puberty depends on the functional development of the BPG axis. Several definitions for puberty exist, but for the purpose of this study, puberty will be considered as the period that spans the onset of spermatogonial multiplication until the appearance of the first flagellated spermatozoa. Previous results have shown that in common carp (Cyprinus carpio L.) repeated temperature stress caused elevated plasma cortisol levels [10] and a retardation of the first waves of spermatogenesis (unpublished results).
In the present experiments, stress-induced cortisol levels were mimicked in order to investigate 1) whether the effects of the temperature stress are due to elevated cortisol levels and, if so, 2) on which level the BPG axis is affected by cortisol. Temperature stress-induced cortisol levels [10] were mimicked by feeding the experimental animals with cortisol-containing food pellets. As experimental animals we used isogenic male common carp, with highly uniform and predictable testicular development, with meiosis of spermatogonia starting around 90 days posthatching (dph) [11].
MATERIALS AND METHODS
Animals
Isogenic male common carp (C. carpio L., designated as strain E4xR3R8) were produced by crossing a homozygous gynogenetic E4 female [12] with a YY male of an unrelated homozygous androgenetic male R3R8 [13]. Fry were produced and raised in the facilities of the Department for Fish Culture and Fisheries (Agricultural University, Wageningen, The Netherlands) and transported at 21 dph to our department at the Utrecht University.
During the experiment, the fish were kept at 25°C in a flow-through system, exposed to a 12L:12D regime and fed pelleted dry food daily (91 series; Provimi, Rotterdam, The Netherlands) at a daily ration of 20 g/kg-0.8. Fish were allowed to acclimatize till 63 dph, after which the experiment started.
Experiment 1: Short-Term Steroid Treatment
Cortisol (Steraloids Inc., Wilton, CT)-containing food (100 mg/kg food) was prepared as described by Pickering et al. [14]. In order to determine how to mimic the cortisol profile induced by temperature stress [10], 320 animals were equally divided over four groups.
The first two groups received during 1 wk, once daily, either control food or cortisol-treated food. In the same period the other two groups received either control food or cortisol-treated food daily over a 6-h period, starting at 1000 h (four times, with intervals of 1.5 h). Feeding the fish with cortisol-containing pellets according to the latter regime mimicked the cortisol levels that were induced by the temperature stress (see Results). On the last day of the treatment, blood samples were taken for cortisol measurement by radioimmunoassay (RIA).
Experiment 2: Long-Term Steroid Treatment
Two hundred animals, 63 dph, were divided over two groups. Group 1 served as controls and was fed with control food, while group 2 received four times daily the cortisol-containing food.
At the onset of the experiment, 63 dph, a start control group was sampled. At 90, 95, 101, and 106 dph, covering the pubertal development of this strain of common carp, 20 fish per group were sampled. The fish were caught and anesthetized within 1 min in tricaine methane sulfonate (Crescent Research Chemicals, Phoenix, AZ). As shown by Weyts et al. [15], a cortisol stress response due to handling is avoided in this procedure.
Body weight was determined and blood was collected by puncturing the caudal vasculature, using 1-ml syringes (needle: 26-gauge x 1/2'') rinsed with a solution of 7% sodium EDTA (pH 7.2). Plasma samples were stored at -20°C until use. After blood sampling, fish were immediately decapitated. Brains and pituitaries were collected, snap frozen in liquid nitrogen, and stored at -80°C until use for hormone measurements, by means of RIA, or for mRNA quantification by RNase protection analysis (RPA). Testes were taken, weighed for determining the gonadosomato index (GSI = testes weight x 100/[bodyweight - testis weight]) and fixed for histological determination of the testicular development.
Testicular Histology
For determination of the spermatogenetic stages, testis tissue of 10 fish per control and cortisol-treated group, respectively, was processed for histology. Spermatogenesis was subdivided into four stages according to Cavaco et al. [16]. In short these were stage I, spermatogonia only; stage II, spermatogonia and spermatocytes; stage III, spermatogonia, spermatocytes, and spermatids; and finally stage IV, all germ cells including spermatozoa. The numbers of animals per group with the same stage of testicular development are counted and expressed as a percentage of the total group.
Steroid RIAs
Plasma levels of cortisol were determined by an RIA according to de Man et al. [17] and van Dijk et al. [18]. The plasma levels of the steroids 11KT, 11-ketoandrostenedione (OA), and T were measured in an RIA as described by Schulz [19]. In most male teleosts, 11KT is considered to be the most dominant androgen in the plasma [20]. Also in the male common carp 11KT has been found to be the major androgen produced by the testes [21, 22]. However, in immature common carp OA is the main androgen (Komen, personal communication). Testosterone was included because Cavaco et al. [23] showed that this androgen is essential for gonadotroph development during puberty.
Plasma and Pituitary LH
Luteinizing hormone was quantified in the plasma and the pituitaries using an homologous RIA (slightly modified from Goos et al. [24]). Purified carp LHß subunit (a gift from Dr. E. Burzawa-Gerard) was used for the preparation of standards and for 125I-labeling. Anti-LHß (6.3) was used as a first antibody.
Ten pituitaries per treatment group were individually homogenized and assayed. Plasma LH levels were measured in all animals. In common carp, as in many species, the presence of an FSH has also been demonstrated [25]. However, an FSH-specific assay is not available.
Salmon GnRH Content in the Brain
Salmon GnRH, which is the native hypothalamic form for carp, was measured by RIA using an sGnRH-specific antibody and iodinated sGnRH [26, 27]. Ten brains per treatment group were individually homogenized in 2 N acetic acid, heated at 90°C for 10 min, snap frozen, and sonicated. The homogenates were centrifuged 3500 x g at 4°C for 30 min. The supernatants were collected. The pellets were resuspended in 2 N acetic and centrifuged. The second supernatants were added to the previous ones and stored at -70°C. Before assaying, the samples were lyophilized. The residues were reconstituted to a smaller volume with 2 N acetic acid, sonicated, and centrifuged at 3500 x g and 4°C for 30 min. The supernatants were neutralized with 5 N NaOH, centrifuged at 3500 x g and 4°C for 5 min and further diluted with the assay buffer.
Ribonuclease Protection Analysis
To quantify the steady-state mRNA levels for glycoprotein hormone 
subunit (GP), LHß, and FSHß subunits, 10 pituitaries per treatment group were used for RPA, based on the method described by Rebers et al. [28]. However, the homogenization was performed in 50 µl lysis buffer to account for the low amounts of mRNA present in the pituitaries of immature common carp. For the quantification of the GP, LHß, and FSHß subunit mRNA levels, 45 µl of this homogenate was used. For the quantification of the 28S rRNA (internal standard) levels, 42.5 µl lysis buffer was added to 2.5 µl of the remaining homogenate.
The following oligodeoxynucleotide primers were used (Life Technologies, Breda, The Netherlands): GP Fw, 5'-GAGGTCCAAGAAAACCATGCT-3'; GP Rv, 5'-TTTAACTGTAATACGACTCACTATA
AACACAAGCAAATCTTGAATGTC-3' (based on Huang et al. [29]); LHß Fw, 5'-TCCGACTGTACGATTGAAAGCC-3'; LHß Rv, 5'-TTTAACTGTAATACGACTCACTATA
TCTTCAGCTCAATATCCACGCC-3' (based on Chang et al. [30]); FSHß Fw, 5'-
GCTCACCAATATCTCCATTACCG-3'; FSHß Rv, 5'-TTTACCTGTAATACGACTCACTATA
GCATGTTATATTTATTGATGCTTGCA-3' (based on Kobayashi, unpublished, DDBJ accession no. AB003583); 28S rRNA Fw, 5'-
GTGAAAGCGGGGCCTCACGATCCT-3'; 28S rRNA Rv, 5'-GGTACCTGTAATACGACTCACTATA
CCAGCTCACGTTCCCTATTAGTGGGT-3' (based on conserved sequences found in other 28S rRNA sequences). In all primers, the sequences in italics represent the T7 RNA polymerase promoter sequence used for cRNA probe synthesis (Rebers et al. [28]). The underlined sequences are unable to hybridize to the mRNA to be detected and yield the difference in length between the cRNA probe and the protected fragment in the assay.
Statistics
All results are expressed as mean ± SEM. Plasma levels of the different steroids are given as ng per ml plasma. The LH levels are given as ng per ml plasma or as ng per pituitary. Salmon GnRH content is expressed in pg/brain. Messenger RNA levels for the GP, LHß, and FSHß subunits are corrected for 28S rRNA levels and expressed as percentage of the control. All results on the treatment effect of cortisol were processed for statistical analysis by Student t-test (P < 0.05). Differences between time intervals were processed by one-way ANOVA, followed by Fisher's least significant difference test (P < 0.05).
RESULTS
Plasma Cortisol
Plasma cortisol levels during and after a single or a four times daily cortisol food application are depicted in Figure 1, A and B, respectively. At the onset of the single treatment, plasma cortisol levels of the control group are elevated but decrease to basal within 1 h. This profile reflects the normal stress reaction to the expectation of food. A single feeding with cortisol-containing pellets caused a significant increase in plasma cortisol level that reached a peak value of 140 ng/ml plasma after 1 h and was returned to basal level after 3 h (Fig. 1A). Repeated application of the cortisol-treated food, four times daily with intervals of 1.5 h, induced an elevated cortisol profile over 7 h. Cortisol peak values up to 150 ng/ml plasma were observed after each meal. In the control group, cortisol plasma values were all below 50 ng/ml plasma, except the first time point (Fig. 1B).
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Growth, GSI, and Testicular Histology
The growth curve (Fig. 2A) for the control group and cortisol-treated group demonstrates that cortisol causes a slight retardation in growth that becomes significant from 101 dph onward. The increase in GSI, observed in the control animals reflects the normal testicular development during puberty (Fig. 2B). In contrast, the cortisol-treated animals show an impaired testicular development as follows from the significantly lower GSI at 90 and 106 dph. This reflects the retardation in spermatogenesis as observed after histological analysis of the testis. Due to the somewhat higher variation in the control group, this difference is not statistically significant at 95 and 101 dph (Fig. 2C). At 90 dph most of the control fish are in stage II, whereas the cortisol-treated fish remain in the first stage of spermatogenesis. When at 95 dph the control group is already in stage III–IV, all cortisol-treated fish are still in stage II–III.
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Plasma Levels of Sex Steroids
Prolonged feeding with cortisol prevented the significant increase of 11KT plasma levels as observed in the control animals during the course of the experiment (Fig. 3A). The T levels were significantly lower at 106 dph only (Fig. 3C). Plasma OA levels are lower in cortisol treated animals. Statistical significance could not be calculated at 95 and 101 dph because most levels in cortisol-treated animals were below the detection limit of the assay (8 out of 10 at 95 dph and 6 out 9 at 101 dph, respectively) (Fig. 3B).
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Plasma and Pituitary LH
Plasma LH levels in both the control and cortisol-treated fish gradually decreased during the experiment (Fig. 3D). However, the LH plasma levels for the treated groups are significantly lower than the levels measured in the control group, except at day 90 dph, due to the larger variation in the control group.
The pituitary LH content shows a steady increase in the control group that reflects the normal elevation of LH content during the pubertal development (Fig. 4A). This rise in LH content can also be observed in the cortisol-treated group. However, cortisol treatment resulted in a slightly retarded elevation that caused a significant difference at 101 dph.
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Salmon GnRH Content in the Brain
Salmon GnRH content of the brain gradually decreased in the control animals from 90 to 101 dph but was strongly increased at 106 dph. Prolonged feeding with cortisol resulted in lower brain contents of sGnRH (Fig. 4B) at 90, 95, 101, and 106 dph. Due to the somewhat larger variation in the cortisol group at 101 dph, there was no statistical difference between groups.
Steady-State mRNA Levels for GP, LHß, and FSHß Subunits
Ribonuclease protection analysis showed that cortisol treatment had different effects on the mRNA levels for GP, LHß, and FSHß depending on the age of the animals at time of the sampling (Fig. 5). At 90 dph, FSHß subunit mRNA levels are significantly lower compared to control values. Messenger RNA levels for GP and LHß tend to be lower in the cortisol-treated group, but the differences are not significant. At 95 dph and 101 dph no differences are observed between the control and treated group. However at 106 dph GP, FSHß, and LHß subunit mRNA levels are significantly lower in the cortisol-treated group.
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DISCUSSION
In the present study we showed that long-term cortisol treatment caused an inhibition of pubertal development, affecting directly or indirectly all components of the BPG axis. To our knowledge this is the first time that negative effects of cortisol on spermatogenesis during pubertal development have been described.
In this study we defined puberty as the period that spans the onset of spermatogonial multiplication until the appearance of the first flagellated spermatozoa. In the isogenic common carp strain that was used, puberty occurs between 90 and 110 dph. Feeding with cortisol-containing food pellets from 63 dph onward caused retardation in weight gain between 90–100 dph. Similar results have been observed before for several species (reviewed in Van Weerd and Komen [31]). Stress has been shown to have adverse effects on growth. In Atlantic salmon parr repeated stress once or twice daily over a period of 30 days reduced the growth rate significantly [32], and chronic crowding for a 9-mo period in rainbow trout reduced the bodyweight as well [33].
Growth of the testis was affected by the cortisol treatment, reflected by lower GSIs at all sampling points. Due to the somewhat larger variation at 90 and 95 dph the difference was not statistically significant at these time points. Identical results have been obtained in other fish species. Hydrocortisone acetate treatment of male Labeo gonius caused a reduction in volume and length of the testes and in GSI [34]. Cortisol-implanted maturing male brown trout had smaller gonads [9]. Female tilapia treated with cortisol showed reductions in GSI and oocyte size [35]. In mammals, reduction of testicular development was shown in stressed Siberian dwarf hamsters [36]. A causal relationship with declined T and estradiol (E2) levels has been suggested.
Reduction of plasma sex steroid levels due to stress or cortisol treatment has been reported for a variety of vertebrate species (mammals [7, 37]; reptiles [38, 39]; amphibians [40], and fish [8, 9, 41]).
In African catfish, it has been shown by Cavaco et al. [42] that an important function of 11KT during sexual maturation is the stimulation of spermatogenesis. In Japanese eel, Miura et al. [43] demonstrated that in vitro complete spermatogenesis could be induced by the application of 11KT to the culture medium. In the present study we show that cortisol caused a decline of 11KT levels that is accompanied by an inhibition of the first wave of spermatogenesis, suggesting a causal relationship between the retardation of spermatogenesis and the decrease in 11KT secretion. Whether this is a direct effect of cortisol on the testicular androgen production or an indirect action via the hypothalamic-pituitary gonadotropic system cannot be deduced from the present results. In mammals, T is secreted by the Leydig cells under LH stimulation, and T may be considered to be the functional homologue of the fish androgen 11KT for promoting spermatogenesis (significance of T for mammalian spermatogenesis reviewed by McLachlan et al. [44] and Griswold [45]). Mammalian Leydig cells are known to express glucocorticoid receptors (GRs) [46], and in vitro experiments suggest that stress or corticosteroids decrease the Leydig cell sensitivity to gonadotropins [7, 47] either by reducing the LH receptor content [48] or by inhibiting the 17-hydroxylase and/or C17,20-lyase activity [49]. In fish, the data on the direct effect of cortisol on steroidogenesis are less consistent compared to mammals. Carragher and Sumpter [50] and Pankhurst et al. [51] found a reduction of E2 and T secretion by cultured ovarian follicles. In other species (goldfish, common carp, and the sparid Pagrus auratus), however, Pankhurst et al. [52] found no evidence that the inhibitory effects of stress on reproduction are mediated by the action of cortisol on ovarian steroidogenesis directly.
From the present study, we do not have evidence for direct effects on the secretion of 11KT. However, in an earlier study [53] we have shown that testes of cortisol-treated common carp have a decreased OA and 11KT basal and LH-induced secretory capacity in vitro, indicating that a direct effect of cortisol on the Leydig cells may occur. Furthermore, in the same study we have shown that the addition of the nonmetabolizable cortisol agonist, dexamethasone, to the incubation medium has similar effects.
In a successive study, whether cortisol competitively inhibits the conversion of 11ß-hydroxyandrostenedione (OHA) into OA or has an effect on testicular steroid-synthesizing capacity will be investigated.
Cortisol may also have its effect on spermatogenesis via an action on Sertoli cells. In fish as in mammals, one of the functions of Sertoli cells is to mediate the action of androgens on spermatogenesis [54]. Because GRs have been demonstrated in Sertoli cells in mammalian testes [55] and these cells respond to glucocorticoids [56, 57], an effect of cortisol on spermatogenesis via Sertoli cells cannot be excluded. The presence of GR in the testis of fish has been confirmed by reverse transcription-polymerase chain reaction [58], but the exact localization is unknown yet.
Like in the African catfish [59], we observed an activation of the gonadotrophs in the pituitary during pubertal development, reflected by the increasing LH content. Schulz et al. [59] suggested that a signal of testicular origin was responsible for the activation of the LH gene transcription and translation and LH storage. Indeed, several studies have shown that T stimulates the maturation of gonadotrophs and the expression and storage of LH in various teleost species [23, 60–63]. This is supported by studies of Cavaco et al. [16] showing that T is produced by the testis before sexual maturation. Moreover, castration slowed down gonadotroph maturation, a process that could be restored by T replacement [64].
There is no evidence yet whether gonadotropin gene expression, storage, and release are directly influenced by cortisol. The observed effect may be indirect via a reduced secretion of T [64]. Pituitary LH content was suppressed in the cortisol-treated group only at 101 dph. There seems to be no relation with any of the other parameters that limits the relevance of this observation. The GP, FSHß, and LHß mRNA steady-state levels, however, were significantly decreased at 106 dph, which corresponds to the reduced T plasma content.
In the present experiments we found reduced plasma LH levels. Although a suppression of gonadotropin levels in fish by cortisol has been observed earlier [9, 65], the data are not always consistent. Some studies showed no effect [66], others even an increase [8].
The reduced expression and release of gonadotropins after cortisol treatment may be related to the impaired testicular androgen secretion, but again, a direct effect of cortisol on the pituitary or via the hypothalamus cannot be excluded. In mammals, it has been demonstrated that corticosteroids inhibit the GnRH-induced LH release by inhibiting the responsiveness to GnRH [67, 68]. The inhibitory effects on LH release may, however, also be caused by a suppression of hypothalamic GnRH release [69]. Both these pathways suppose the presence of GRs on GnRH neurons (or on neural elements controlling the GnRH neurons) or the gonadotrophs. Indeed, in fish GRs have been found in the hypothalamic GnRH neurons and in pituitary gonadotrophs of the rainbow trout [70]. Studies on the GnRH gene of several teleost species have shown that the GnRH promoter contains putative glucocorticoid responsive elements (sGnRH: Klungland et al. [71], Higa et al. [72] and seabream GnRH and chicken GnRH-II: Chow et al. [73]). In vitro experiments with immortalized GnRH-secreting cell lines, expressing a functional GR, showed that dexamethasone repressed both the endogenous mouse GnRH gene by decreasing steady-state levels of GnRH mRNA and the transcriptional activity of transfected rat GnRH promoter-reporter gene constructs [74]. The same author identified negative regulatory elements in the mouse GnRH encoding gene that bind heteromeric complexes containing GR and mediate the repressive action of glucocorticoids [75]. In addition, Attardi et al. [76] showed that dexamethasone affected GnRH secretion from GT1-7 cells as well.
In the present study, sGnRH content of the brain of control animals shows a gradual decrease from 90 to 101 dph and a sudden increase on 106 dph. We have no indication yet whether this profile reflects changes in synthesis, storage, or release or a combination of these processes. However, our experiments demonstrate that prolonged cortisol treatment resulted in lower sGnRH levels in the brain, suggesting that the observed effects on the gonadotrophs may indeed be caused by a reduction of sGnRH secretion.
In conclusion, we show that cortisol inhibits pubertal development in common carp and that this inhibition is present at all levels of the BPG axis. The present results are not sufficient to elucidate whether cortisol acts directly or indirectly in the different parts of the BPG axis. Current investigations are designed to answer this question.
FOOTNOTES
1 This project was supported by grant 805-33.103P from the Netherlands Organization for Scientific Research (NWO). 
2 Correspondence: D. Consten, Graduate School for Developmental Biology, Research Group for Comparative Endocrinology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 31 30 253 2837; d.consten{at}bio.uu.nl
Accepted: November 9, 2000.
Received: June 2, 2000.
REFERENCES
and gonadotropin IIß subunit mRNA levels in the pituitary gland of mature male African catfish, Clarias gariepinus. Fish Physiol Biochem 1997; 17:99–108.[CrossRef] subunit genes. Biochim Biophys Acta 1992; 1129:239–242.[Medline]This article has been cited by other articles:
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  T. Kitahashi, S. Ogawa, T. Soga, Y. Sakuma, and I. Parhar Sexual Maturation Modulates Expression of Nuclear Receptor Types in Laser-Captured Single Cells of the Cichlid (Oreochromis niloticus) Pituitary Endocrinology, December 1, 2007; 148(12): 5822 - 5830. [Abstract] [Full Text] [PDF]
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