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Regular Article |
a Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
ABSTRACT
Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60–64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary ßFSH and ßLH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of ßFSH were elevated during early spermatogenesis, whereas ßLH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in ßFSH mRNA and a rise in ßLH mRNA abundance. Immature males had lower levels of ßLH mRNA than maturing males, but there were no differences in ßFSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in ßFSH and ßLH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on ßFSH and ßLH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.
anterior pituitary, FSH, gene regulation, hormone action, LH, puberty, seasonal reproduction
INTRODUCTION
Testicular development in teleost fish is controlled by the gonadotrophic hormones, GtH-I and GtH-II [1]. Due to growing evidence of structural and functional homology of GtH-I to tetrapod FSH and GtH-II to LH [2, 3], this nomenclature will be used in this report. Based on their plasma levels and biological activity, it is widely accepted that in salmonid fish species, FSH is involved in the initiation of meiosis and early spermatogenesis, whereas LH controls the final maturation of spermatozoa and spermiation [4–6].
Only recently have we begun to understand the molecular basis underlying FSH and LH synthesis in teleost males [6, 7]. In salmonids, the mRNA levels of ßFSH increased gradually during spermatogenesis, leading to a peak at spermiation. Low levels of ßLH mRNA were expressed during spermatogenesis, followed by a dramatic increase at spermiation. Only fragmented data are available regarding seasonal changes in ßFSH and ßLH gene expression in males of nonsalmonid species [8].
Studies carried out in teleost males have established that GnRH and steroids stimulate ßLH gene expression. For example, GnRH was effective in increasing ßLH gene expression in goldfish [9] and sockeye salmon [10]. Evidence for an in vivo stimulatory effect of testosterone (T) on ßLH mRNA levels in males was also demonstrated in the goldfish [11]. A similar effect was shown to occur in vitro, indicating that androgens exert their effect through direct action at the level of the pituitary. Data on the effects of GnRH and steroids of ßFSH mRNA levels in male teleost are still scarce. The available data suggest that, at least during specific testicular developmental stages, these hormones stimulated ßFSH gene expression, both in vivo [12] and in vitro [13].
In some teleosts, dopamine (DA) is known for its powerful inhibition of LH release. This neurotransmitter is released from synapses directly innervating the pituitary, where it activates DA2 receptors on the gonadotrope [14]. Dopaminergic inhibition of LH release has been shown to be minimal in sexually mature perciform fish, including the striped bass [15], but some authors have suggested that it may be involved in pubertal development [16, 17]. In the present study, the possible involvement of DA in the onset of sexual maturity was tested using pimozide (P), a potent DA inhibitor.
Striped bass are seasonal breeders, reproducing in the spring in response to increased day length and elevated water temperatures [18]. In captivity, 60–64% of the males attain sexual maturity as 1 or 2 yr olds, although their body weight (BW) is not different from their immature cohorts [19, 20]. All males attain sexual maturity in their third year of life. Consequently, fish that mature as 1 or 2 yr olds are considered as early maturing males. A similar plasticity in the age of first maturation was described in other captive populations of striped bass [18].
The age of first maturation is influenced by genetic [21, 22] and environmental [23] factors. Nevertheless, it is well established that hormonal intervention can induce spermatogenesis, overriding the genetic basis of puberty [24–27]. The mechanism involved in the differentiation of 2-yr-old striped bass into maturing males was investigated in two experiments. In a preliminary study, we examined the effects of chronic administration of GnRH analogue (GnRHa), T, and their combination on the incidence of sexual maturity [28]. Although none of the treatments increased the incidence of sexual maturity, the combined administration of GnRHa and T resulted in a moderate stimulation of gonadotropin synthesis and testicular growth in some immature fish. In the present (follow-up) study, we tested the hypothesis that a higher dose of GnRHa, or perhaps the removal of a dopaminergic inhibition, may be required to stimulate further the brain-pituitary-gonadal axis of immature fish. The effects of these treatments on spermatogenesis are presented elsewhere [29]. This report describes changes in gonadotropin subunit gene expression, including 1) the profiles of the 
-, ßFSH-, and ßLH-subunit mRNAs during testicular development in a population of 2-yr-old striped bass males, 2) the effects of chronic administration of T and GnRHa (with and without P) on gonadotropin gene expression in this species, and 3) the differences between immature and maturing males in terms of gonadotropin gene expression.
MATERIALS AND METHODS
Animals
A stock of 2-yr-old striped bass (Morone saxatilis, Moronidae, Teleostei), produced in the spring of 1993 from captive broodstock of Chesapeake Bay origin, were raised at the Aquaculture Research Center of the Center of Marine Biotechnology, University of Maryland. The animals were maintained in 4000-L fiberglass tanks recirculated with 10 ± 1 ppt of custom-made salt water. The tanks were kept at a thermo- and photoperiod regime simulating the environmental changes in their natural habitat: a gradual change from 13 ± 1°C and 8L:16D in the winter to 23 ± 1°C and 16L:8D in the summer. The fish were fed twice daily with Trout Growers pellets containing 38% crude protein (Zeigler, Gardners, PA), at a ratio of 1–3% BW per day, depending on age and size. A routine husbandry regime maintained fish in a healthy state throughout the year. Fish were used according to a protocol approved by the Institutional Animal Care and Use Committee of the Center of Marine Biotechnology, University of Maryland.
Annual Profile of Gonadotropin Subunit Gene Expression
Fish were sampled from September 1994 until October 1995, at a 1- to 2-mo interval. At each sampling time, 7–14 fish were anesthetized in a solution of 0.25 ml/L 2-phenoxyethanol (J.T. Baker, Phillipsburg, NJ), weighed, and killed by decapitation. The pituitaries were quickly extracted, frozen in liquid nitrogen, and later stored at -80°C for analysis of ßFSH and ßLH mRNA abundance by a ribonuclease protection assay (RPA). The testes were separated and weighed for the calculation of the gonadosomatic index (GSI = gonad weight/total BW x 100), and small pieces of the testicular tissue were removed for histological preparation.
Regulation of Gonadotropin Subunit Gene Expression by GnRHa, T, and P
Long-term release of hormones into the bloodstream was achieved via i.m. injection of sustained-release microspheres. The GnRHa-containing microspheres were shown to release GnRHa into the bloodstream for about 60 days [30]. The GnRHa used was (D-Ala6,Pro9)-LHRH, because this analogue is effective in releasing LH into the bloodstream in spermatogenic striped bass [12]. The T-containing microspheres were shown to maintain physiological levels of circulating T (1–3 ng/ml) for about 60 days [31]. The dopaminergic antagonist P was suspended in a vehicle of 0.1% sodium metabisulfite and injected at a dose of 10 mg/kg BW.
In January 1997, 144 striped bass of mixed sex were selected for experimentation. The fish were 21 mo old, with an average BW of 387 ± 4 g. They were randomly divided into four groups of 36 fish each. The initial ratios of male to female and immature to maturing males in each group were unknown. Each group received one of the following treatments: T + GnRHa (T + G), T + G + P, T + a high dose of GnRHa (T + high G), and a control group (C) that received microspheres devoid of any hormones. Testosterone and G were administered at the beginning of the experiment and after 6 wk at a dose of 4 mg T/kg BW and 300 µg GnRHa/kg BW, respectively. The high G treatment was achieved by three injections of GnRHa at 3-wk intervals. Pimozide injections were administered five times at 2-wk intervals. Immediately after receiving the initial treatment, the fish were transferred to the experimental tanks supplied with 16 ± 1°C water at 10 ± 1 ppt salinity. Fish from all experimental groups were sacrificed after approximately 11 wk (75 days) and their pituitaries and gonads collected as described above.
Determination of Testicular Developmental Stage
The testicular samples were embedded in JB4Plus, and sections of 2–3 µm were cut and stained with Polychrome I and II. The histological sections were examined under a light microscope, and cells were identified as spermatogonia, spermatocytes, spermatids, or spermatozoa according to Holland et al. [19]. A testicular developmental stage was assigned to each sample according to the dominant type of germ cells. Testes consisting of at least 50% spermatocytes, spermatids, or spermatozoa were classified as maturing. Fish were categorized as spermiating when sperm could be expelled by gentle abdominal pressure. A detailed description of the annual spermatogenic cycle in this population of striped bass is given elsewhere [20].
Gonadotropin Subunit Gene Expression
Gonadotropin gene expression was measured by an optimized RPA, in which the mRNA levels of the -, ßFSH, and ßLH subunits are measured simultaneously in the same pituitary gland [12]. The assay is highly reproducible, with a sensitivity of 0.3 fmol for the -subunit, ßLH and ß-actin, and 0.03 fmol for the ßFSH subunit. Because the mRNA levels of ß-actin fluctuated throughout the reproductive cycle and in response to hormonal stimulation, gonadotropin gene expression levels were normalized to total RNA content. Total RNA levels were not affected by the hormonal treatments (data not shown).
Plasma Levels of LH
Circulating LH levels were measured in 100 µl plasma (in duplicate) using a specific striped bass LH RIA [32]. All plasma samples were run in a single assay to eliminate interassay variation. The intra-assay coefficient of variation for the LH RIA is 4.6%, whereas the detection limit is 0.4 ng/ml.
Statistical Analysis
All data are untransformed and presented as means ± 1 SEM. The statistical analyses were performed using the SuperANOVA statistical software (Abacus Concepts, Berkeley, CA). The significance level was set at P < 0.05.
Differences in means of gene expression were analyzed by one-way ANOVA followed by Duncan's new multiple range posthoc test. Differences between immature and mature fish were determined by a two-way ANOVA followed by the method of least squares.
RESULTS
Testicular Development and Plasma Levels of LH
A detailed description of spermatogenesis in this population of striped bass males is given in Holland et al. [20]. Briefly, maturing males became distinguishable from their immature cohorts by histological examination in December 1994, when the testes of maturing males became filled with spermatocytes. In contrast, the testes of immature fish were filled with spermatogonia, although scattered nests of spermatocytes were observed in about 50% of the males. Spermatids and spermatozoa appeared in January, and spermiation (release of sperm into the sperm duct) was observed between February and July. The incidence of mature 2-yr-old males was 64%. The changes in GSI values during the annual reproductive cycle and the period of spermiation are shown in Figure 1D. The plasma levels of LH were under the detection limit throughout the reproductive cycle.
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Annual Profile of Gonadotropin Subunit Gene Expression
RNA extraction Total RNA extracted from single pituitaries increased gradually from 4.7 ± 0.3 µg in September 1994 to 19.8 ± 2.1 µg in October 1995 (data not shown). There were no differences in total RNA content between immature and maturing/mature males.
-Subunit (Fig. 1A)
In maturing males, the levels of -subunit mRNA increased gradually and plateaued during the spermiation period. In contrast, the mRNA levels in immature fish declined during the same period and were 1.5- to 2-fold lower than those measured in maturing males. The appearance of spermatozoa in the testis was associated with a 1.7-fold increase in the mRNA levels of the -subunit (Fig. 2A).
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ßFSH subunit (Fig. 1B)
In general, throughout the study period the mRNA levels of ßFSH subunit were about one order of magnitude lower than the - and ßLH-subunit mRNAs. In maturing males, ßFSH-subunit mRNA levels increased during the winter (November to January), followed by a gradual decline to basal levels during the spermiation period. There were no differences in ßFSH-subunit levels between immature and maturing fish throughout the study period. The appearance of spermatocytes in the testis was associated with a 2.6-fold increase in ßFSH mRNA, whereas the appearance of spermatids coincided with a 4.2-fold increase in ßFSH mRNA (Fig. 2B). Spermatozoa were associated with low levels of ßFSH-subunit mRNA.
ßLH subunit (Fig. 1C) In maturing fish, ßLH-subunit mRNA levels rose gradually and reached maximum levels at the beginning of the spermiation period. Thereafter, the mRNA levels gradually declined to basal levels. The profiles of ßLH gene expression were similar in immature fish, although the mRNA levels were 1.5- to 3-fold lower compared to their mature counterparts. The appearance of spermatozoa was associated with a 3.3-fold increase in pituitary ßLH-subunit mRNA (Fig. 2C).
Regulation of Gonadotropin Gene Expression by GnRHa, T, and P
The average GSI for immature and mature males at the end of the experiment was 0.21 ± 0.1% and 4.9 ± 0.4%, respectively. None of the treatments affected the incidence of maturing males.
Immature males The effects of the hormonal treatments on gonadotropin gene expression levels in immature males are shown in Figure 3, A–C. The T + G treatment was effective in stimulating a three- to fivefold increase in the mRNA levels of all gonadotropin subunits. The administration of a higher GnRHa dose did not further stimulate gonadotropin gene expression, whereas the coadministration of P suppressed the stimulatory effect of T + G.
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Mature males The effects of the hormonal treatments on gonadotropin gene expression levels in mature males are shown in Figure 4, A–C. None of the treatments had a significant (P < 0.05) effect on gonadotropin subunit gene expression.
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In summary, the data show seasonal, reproductive stage-related changes in the expression of the gonadotropin genes. In addition, we found two major differences between immature and early maturing striped bass males: 1) Immature fish had lower levels of ßLH mRNA than mature fish but had comparable levels of ßFSH mRNA; and 2) ßFSH and ßLH mRNA levels were increased by the combined administration of GnRHa and T in immature fish but not in maturing fish.
DISCUSSION
Early Sexual Maturity
During their second year of life, 64% of striped bass males raised in our facility reached sexual maturity. Because all males are sexually mature in the third year of life [20], maturation in the second year is considered early maturity. Early testicular maturity is quite common in fish and has been extensively studied in the Atlantic salmon [33, 34]. In this species, precociously mature males (known as parr) mature in their natal rivers, skipping the anadromous spawning migration of their cohorts. Salmon parr lack secondary sex characteristics, and their behavior is highly adapted to their alternative reproductive strategy. In contrast to the partial spermatogenesis described in striped bass, precocious maturation in the salmon parr is an all-or-nothing event, meaning that an individual either matures fully or not at all [33]. In many species, early testicular maturation can also be the result of the natural plasticity of age-at-maturity [35–37], a phenomenon that does not involve an alternative reproductive strategy. Early maturing juveniles of these fish species exhibit abortive or partial spermatogenesis, similar to our observations in the striped bass [19, 20].
Annual Profile of Gonadotropin Gene Expression
In maturing males, the mRNA levels of both ßFSH and ßLH increased gradually during the reproductive season. Maximal levels of ßFSH mRNA were measured during early spermatogenesis, followed by a peak in ßLH mRNA levels during spermiation. Gene expression of both ßFSH and ßLH declined to basal levels at the end of the reproductive season. A similar pattern of ßLH mRNA levels was reported in the rainbow trout [6]. In contrast, trout ßFSH mRNA levels increased gradually during gametogenesis, reaching peak levels during spermiation. Perhaps this discrepancy reflects fundamental differences in gonadotropic function between salmonid and perciform fish species.
In immature striped bass males, the gene expression patterns of ßFSH and ßLH mRNAs were similar to those exhibited by maturing males—a rise during the reproductive season and a decline to basal levels thereafter. However, although the pattern was similar, lower amounts of ßLH-subunit mRNA were synthesized in the pituitaries of immature fish from February to June (the spermiation period in mature fish). This may be related to the low plasma levels of steroid hormones in juvenile male striped bass [20]. It thus appears that a seasonal cycle of LH synthesis is imprinted in immature males, and that threshold levels of LH synthesis may be necessary for maturity to occur. In the third year this pattern is amplified, leading to maturity. Similar findings were described in pubertal female striped bass [20, 38].
In the present study, the plasma levels of LH remained low and unchanged during spermatogenesis. This is in contrast to a dramatic rise in circulating LH that was associated with spermiation in salmonid species [39, 40]. Nevertheless, our data are in agreement with an earlier study in the domesticated striped bass, in which low circulating levels of LH were measured in spermiating males [41]. It thus appears that even low circulating levels of LH are sufficient to maintain spermatogenesis in captive striped bass.
In contrast to ßLH levels, ßFSH mRNA levels did not differ between immature and mature males throughout the reproductive cycle. It has already been reported in salmonids that plasma FSH levels of immature and maturing males were comparable [40, 42]. Unfortunately, we could not determine whether such differences occur in the striped bass, because an immunoassay for striped bass FSH has not yet been developed. In this context, it is worth mentioning that spermatogenesis in mammals may be completely independent of FSH [43, 44].
It is also possible that the mechanism responsible for the differentiation of juveniles into maturing males lies downstream of gonadotropin synthesis and/or secretion, for example, in the capability of the testes to respond to gonadotropin stimulation. A similar hypothesis was put forward by Miura et al. [45] to explain partial spermatogenesis in hCG-treated juvenile eels. These authors assumed that the production of a threshold amount of a yet unknown testicular factor might be required to achieve complete spermatogenesis.
The present study also describes strong correlations between gonadotropin gene expression and distinct developmental stages of the germ cells. An increase in the abundance of pituitary ßFSH-subunit mRNA was associated with the transition from mitotic to meiotic divisions (appearance of spermatocytes), suggesting a role for FSH in this transformation. An increase in ßLH-subunit mRNA levels was correlated to the appearance of spermatozoa, supporting the importance of LH for sexual maturation [5, 46].
Regulation of Gonadotropin Gene Expression by GnRHa, T, and P
The stimulatory actions of GnRHa and T on ßLH and ßFSH gene expression in immature males are consistent with other reports [13, 47]. In contrast to their stimulatory effect in immature males, the chronic administration of GnRHa and T had no effect on gonadotropin gene expression in maturing fish. It thus appears that sexual maturation in the striped bass is associated with reduced pituitary sensitivity to stimulation by GnRH and T. We have previously reported that the acute injection of GnRHa to maturing (i.e., early spermatogenesis) striped bass males stimulated ßLH and ßFSH gene expression [12]. The discrepancy with the present study is unclear; however, we hypothesize that it is related to the different mode of GnRHa administration used in these studies (acute vs. chronic administration).
The coadministration of P (a DA antagonist) had no effect on gonadotropin gene expression in mature striped bass, similar to results obtained in the tilapia [47]. Interestingly, in immature males, P suppressed the stimulatory effect of GnRHa and T on gonadotropin mRNA levels. This suggests that DA may have a novel function in regulating gonadotropin gene expression, in addition to its well-described role as an inhibitor of LH release.
ACKNOWLEDGMENTS
We are grateful to Steve Rodgers and Tina Dalesio at the Aquaculture Research Center, University of Maryland Biotechnology Institute, for taking care of the experimental fish. Thanks are also extended to John Stubblefield for comments on earlier versions of the manuscript.
FOOTNOTES
1 This research was supported in part by grants (to Y.Z.) IS-2634-95-C from the U.S.-Israel Binational Agricultural Research and Development Fund (BARD), NA46RG0091 (Amnd. 6) from Maryland Sea Grant College Program, and 58-6420-7-049 from the United States Department of Agriculture. This publication is contribution no. 522 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute. 
2 Correspondence: Yonathan Zohar, 701 E. Pratt St., Baltimore, MD 21202. FAX: 410 234 8896; zohar{at}umbi.umd.edu
3 Current address: SARS International Center for Molecular Marine Biology, University of Bergen, Thormøhlensgt. 55, 5008 Bergen, Norway.
Accepted: July 25, 2000.
Received: May 2, 2000.
REFERENCES
-methyltestosterone on spermatogenesis in immature milkfish, Chanos chanos. J Fish Biol 1986; 29:567–572.[CrossRef]This article has been cited by other articles:
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  B. Vidal, C. Pasqualini, N. Le Belle, M. C. H. Holland, M. Sbaihi, P. Vernier, Y. Zohar, and S. Dufour Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty Biol Reprod, November 1, 2004; 71(5): 1491 - 1500. [Abstract] [Full Text] [PDF]
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H.F. Vischer, A.C.C. Teves, J.C.M. Ackermans, W. van Dijk, R.W. Schulz, and J. Bogerd Cloning and Spatiotemporal Expression of the Follicle-Stimulating Hormone {beta} Subunit Complementary DNA in the African Catfish (Clarias gariepinus) Biol Reprod, April 1, 2003; 68(4): 1324 - 1332. [Abstract] [Full Text] [PDF]
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