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Biology of Reproduction 61, 1608-1615 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Ontogeny of Follicle-Stimulating Hormone and Luteinizing Hormone Gene Expression During Pubertal Development in the Female Striped Bass, Morone saxatilis (Teleostei)1

Shimon Hassin3,a, M. Clairea, H. Hollanda, and Yonathan Zohar2,a

a Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pubertal development in teleost fish is characterized by gonadal growth that is directly stimulated by the pituitary gonadotropins, FSH and LH. We used a quantitative ribonuclease protection assay to provide, for the first time, the developmental profiles of the {alpha}-, ßFSH-, and ßLH-subunit gene expression in a seasonal breeding fish, the female striped bass (3-yr study, n = 207). Two-year-old females were sexually immature, although a transient rise in all gonadotropin subunit mRNAs was measured in the pituitary. Pubertal ovarian development occurred in 65% of 3-yr-old females, characterized by the appearance of lipid droplets within the oocytes. This reproductive phase, termed pubertal development, was associated with a 34-fold increase in the mRNA levels of ßFSH and a rise in the pituitary concentration of LH. The first sexual maturation took place in 4-yr-old females and coincided with a 218-fold increase in the mRNA levels of ßFSH. During this time period, the mRNA levels of the {alpha} and ßLH subunits increased by 11- and 8-fold, respectively. At the final stages of vitellogenic growth, mRNA levels of ßFSH declined to basal levels, whereas the mRNA levels of the {alpha} and ßLH subunits remained elevated. Throughout the study, pituitary LH concentration was positively correlated to the mRNA levels of ßLH, but plasma levels of LH remained low and unchanged (0.4–0.8 ng/ml) despite increasing levels of pituitary LH concentration, suggesting a regulated secretion pathway. Taken together, the data show that the profiles of ßFSH and ßLH mRNAs appear to follow an annual rhythm that is associated with developmental events in the growing oocytes. In particular, increasing levels of ßFSH mRNA appear to underlie the first sexual maturity in the female striped bass.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sexual maturity in female teleosts is characterized by an increase in oocyte diameter and ovarian size, resulting from the uptake of lipid and protein yolk into the growing oocytes. Similar to the situation in other vertebrate classes, the growth and development of the ovary are controlled by the brain via the hypothalamic-pituitary-gonadal axis [1]. Multiple forms of GnRH are present in fish brains, but the hypothalamic variant is probably the endogenous releasing hormone [2, 3]. The pituitary gland synthesizes the gonadotropic hormones, which are heterodimers containing noncovalently linked {alpha} and ß subunits. Within a species, the {alpha} subunits are identical, while the ß subunits differ and confer the physiological specificity of the hormone [4]. Two distinct gonadotropins, termed gonadotropin-I (GtH-I) and GtH-II, were initially isolated from chum salmon pituitaries [5]. The duality of gonadotropic hormones in teleost fish has now been confirmed in a variety of species, including the striped bass [6]. On the basis of structural [7, 8] and functional (see below) studies, teleost GtH-II is believed to be homologous to tetrapod LH, but the homology of GtH-I and FSH is still debated and awaits the characterization of GtH-I from more fish species. Nevertheless, in this paper, GtH-I and GtH-II are referred to as FSH and LH.

Most actions of fish gonadotropins are mediated through steroids produced by the follicle cells of the oocytes. One of these is estradiol-17ß (E2), which promotes oocyte growth and the production of vitellogenin, the precursor to yolk proteins. Vitellogenins are phospholipoproteins that are produced in the liver, transported via the bloodstream to the ovary, and sequestered by a receptor-mediated mechanism into the growing oocytes. Tyler et al. [9] have demonstrated that salmon FSH is about 100 times more potent than LH in stimulating uptake of vitellogenin into rainbow trout ovarian follicles in vitro. This ability to stimulate vitellogenin uptake is consistent with the fact that FSH levels are elevated during vitellogenesis in salmonids, whereas LH levels are very low or undetectable [10–12]. FSH is also expressed first in ontogeny, whereas LH appears in the later stages of the reproductive cycle [13, 14]. Just before final oocyte maturation and ovulation, FSH levels decline and LH levels rise. LH stimulates the meiotic maturation of oocytes through the enhanced synthesis by the ovarian follicles of hydroxylated progestins called maturation-inducing steroids [15]. LH is also essential for the acquisition of maturational competence of oocytes [16].

Only fragmentary information is available regarding the ontogeny of ßFSH and ßLH mRNA levels during pubertal growth in fish. In the rainbow trout, ßFSH mRNA predominated in the pituitary of immature fish, with ßLH mRNA being only weakly expressed [13, 17, 18]. During the advanced stages of vitellogenic growth, the levels of ßLH mRNA were more abundant than those of ßFSH. In contrast, both ßFSH and ßLH mRNAs were weakly expressed in immature goldfish, and both became strongly expressed with the progression of ovarian maturity [19]. These studies indicate that the ontogeny and regulation of the gonadotropin genes probably differ among teleost species.

Striped bass are seasonal breeders, spawning once a year during the spring [20]. Age at maturity is variable, ranging from 4 to 7 yr, depending on the environmental conditions. The recruitment of previtellogenic follicles into vitellogenic growth and the associated increase in oocyte size begins in the fall and continues throughout the winter. Plasma levels of LH are low or undetectable in immature and vitellogenic females, whereas a surge in plasma concentration of LH occurs in periovulatory females [21]. Although the circulating levels of FSH in the striped bass during the reproductive cycle are still unknown (because of the lack of specific antisera), it is presumed that FSH is involved in the early stages of the reproductive cycle, similar to the situation in salmonid species. We hypothesized that the ability of the pituitary gonadotrophs to secrete increasing amounts of gonadotropins during the reproductive cycle results from transcriptional and/or translational control of their genes. Also, in most mammals, the amount of mRNA for ßLH subunit and the secretion of LH are tightly coupled [22], but this relationship has not yet been evaluated in fish.

The goals of the present study were 1) to describe the developmental profiles of {alpha}-, ßFSH-, and ßLH-subunit mRNAs in the pituitary of striped bass females until sexual maturation and 2) to investigate the correlation between the steady state levels of ßLH mRNA, pituitary content of LH, and the plasma levels of this hormone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Sampling Protocol

Two stocks of domesticated striped bass (Morone saxatilis, Moronidae, Teleostei), produced in 1991 and 1993 from a captive brood stock of Chesapeake Bay origin, were raised at the Aquaculture Research Center of the Center of Marine Biotechnology. Fish were used according to a protocol approved by the Institutional Animal Care and Use Committee. The animals were maintained in 2.5- or 11.8-m3 fiberglass tanks (depending on animal size) recirculated with 10 ± 1 parts per thousand custom-made salt water. The tanks were kept at a thermo- and photo-period 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% body weight per day, depending on age and size. A routine husbandry regime maintained fish in a healthy state throughout the year.

Monthly sampling of both populations started in February 1994 and continued until April 1995 for the 1991 year class and until January 1996 for the 1993 year class (n = 207). Approximately 10–15 females were killed at 1- to 2-mo intervals. At sampling time, fish were anesthetized in a solution of 0.25 ml/L 2-phenoxyethanol (JT Baker, Phillipsburg, NJ), weighed, bled, and killed by decapitation. The pituitaries were quickly extracted and frozen in liquid nitrogen. The ovaries were separated and weighed for the calculation of the gonadosomatic index (GSI = gonad weight/total BW x 100), and small pieces of the ovary were removed for histological preparation. Blood samples were collected using heparinized syringes, and the plasma was separated by centrifugation at 2500 x g for 15 min at 4°C. The pituitaries and plasma were kept frozen at -80°C and later assayed for LH concentration. The abundance of gonadotropin subunit mRNAs in the pituitaries was measured by a ribonuclease protection assay (RPA).

Determination of Ovarian Developmental Stage

Ovarian samples were placed in 4% formaldehyde/1% glutaraldehyde fixative, dehydrated through a 75–90% ethanol series, and embedded in JB-4 Plus (Polysciences, Warrington, PA). Sections of 2–3 µm were cut and stained with Polychrome I and II [23]. Oocyte morphology was examined under a light microscope, and oocytes were classified as oogonia, primary growth (PG), secondary growth (SG)-I, SG-II, and SG-III oocytes, as described in detail by Holland et al. [24]. Briefly, oogonia and PG oocytes (under 150 µm in diameter) contained little ooplasm and no cellular inclusions; SG-I oocytes (150–250 µm) contained a few small ooplasmic inclusions; SG-II oocytes (260–700 µm) contained large amounts of lipid droplets; and SG-III oocytes (400–800 µm) contained yolk granules.

RNA Extraction and Quantitative RPA

Gene expression was measured by total RNA extraction followed by a quantitative RPA, in which the mRNA levels of the {alpha}, ßFSH, and ßLH subunits are measured simultaneously in the same pituitary gland as described in Hassin et al. [25]. To compensate for tissue size and procedural errors, the data were normalized to ß-actin mRNA levels.

Immunoassays

Pituitary content of LH was measured by a homologous striped bass ELISA [26] in an aliquot removed during the RNA isolation protocol [25]. The intra- and interassay coefficients of variation for this assay are 7.7% and 8.7%, respectively. Plasma levels of LH were measured in duplicate by a homologous striped bass RIA [27], and all plasma samples were run in a single RIA to eliminate interassay variation. The intraassay coefficient of variation for the LH RIA is 4.6%. Total protein content in the pituitary was measured by the Micro BCA kit (Pierce, Rockford, IL).

Statistical Analyses

All data are untransformed and are presented as mean ± SEM. Annual peaks in means of GSI, gonadotropin subunit mRNA, and LH concentration in the pituitary were examined by ANOVA followed by Duncan's New Multiple Range test. Differences in these parameters between pubertal and juvenile females were analyzed by two-way ANOVA. The analyses were performed using the SuperANOVA statistical software (Abacus Concepts, Berkeley, CA). Significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ovarian Development

A detailed account of the reproductive cycle in this stock of domesticated striped bass is given in Holland et al. [24]. Briefly, oocytes in 2-yr-old fish were at the PG stage, and all females were classified as juveniles. A significant increase in GSI was recorded in January (Fig. 1) but was not associated with any morphological changes in the oocytes. In the third year, SG-I oocytes were present in all females during the summer and fall seasons. In December, the oocytes in 50% of the females proceeded to SG-II, mirrored by the dichotomy in GSI values (see Fig. 3E). During February and April, the ovaries of these females grew in size and the oocytes became filled with lipid droplets, but yolk globules were only rarely observed. On average, 65% of the 3-yr-old females exhibited this type of partial ovarian development, termed pubertal. In the fourth year, yolk globules appeared at the beginning of September in a large number of oocytes (SG-III stage), and 100% of the females became sexually mature for the first time. Although sexually mature, these fish were termed adolescent because the GSI and yolk accumulation in their oocytes were lower than those observed in adult (i.e., older) females.



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  		FIG. 1. Changes in the values of the GSI (mean ± SEM) in female striped bass throughout the study period. Consecutive reproductive cycles are indicated at the bottom, and asterisks designate peak values for each cycle. The period of pubertal growth in 3-yr-old fish is highlighted by the hatched area



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  		FIG. 3. mRNA levels of pituitary gonadotropin subunits (mean ± SEM) during pubertal development in 3-yr-old females. The asterisks represent significant differences between pubertal and juvenile females (P < 0.05)

Ontogeny of Gonadotropin Subunit Gene Expression

Total RNA extracted from the pituitaries at the beginning of the study (March; 2 yr old) was 2.7 ± 0.2 µg, and increased gradually to 39.4 ± 3.3 µg at the last sampling time (April; 4 yr old). The average amount of total RNA extracted from the pituitary throughout the study period was 1 ± 0.1 µg/mg tissue. The levels of ß-actin mRNA (per microgram total RNA extracted from the pituitary) did not change significantly during the study period; thus they were suitable for internal normalization of gonadotropin subunit mRNA levels (data not shown).

Figure 2, A–C, illustrates the changes in the expression of gonadotropin subunits (normalized to ß-actin) during the 3-yr study period. In the second year, the abundance of all gonadotropin subunit mRNAs was relatively low; these levels were thus termed basal levels. During September and November, the period corresponding to vitellogenic growth in adult fish, there was a significant rise in the gene expression levels of the {alpha}, ßFSH, and ßLH subunits—in particular, an 18-fold increase (over basal levels) in the expression of ßFSH (see inset in Fig. 2B). After November, the mRNA levels of ßFSH declined, but the levels of the {alpha} and ßLH subunits remained moderately elevated.



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  		FIG. 2. Developmental profiles of pituitary gonadotropin subunit mRNA levels (mean ± SEM) in female striped bass sampled for 3 yr (n = 207). A) {alpha}-Subunit mRNA; B) ßFSH mRNA (inset shows the first 2 yr); C) ßLH mRNA; D) LH concentration. In the boxes beneath the curve (A,B,C), letters with a common underline indicate means that are not significantly different (P > 0.05). Consecutive reproductive cycles are indicated at the bottom, and asterisks designate peak values for each cycle. The period of pubertal growth in 3-yr-old fish is highlighted by the hatched area

In the third year, pubertal development (as determined by histological examination of the ovary [24]) took place in 65% of the females, whereas the rest of the fish remained at the juvenile phase (Fig. 3E). The period of pubertal ovarian development is highlighted by the hatched area in Figure 2 and shown in detail in Figure 3, A–E. During the winter, there was a significant rise in the gene expression levels of the {alpha}, ßFSH, and ßLH subunits, in particular a 34-fold increase in ßFSH mRNA levels (inset in Fig. 2B). In December and January, the levels of ßFSH in pubertal females were twice as high as those measured in the juvenile females (Fig. 3B). In contrast, there were no significant differences in ßLH mRNA levels between juvenile and pubertal females.

The first sexual maturation occurred in 4-yr-old females and was associated with a dramatic increase in the gene expression of all gonadotropin subunits (Fig. 2). Between July and September, the mRNA levels of the {alpha}, ßFSH, and ßLH subunits increased 11-, 218-, and 8-fold, respectively. Since the basal levels of the ßFSH-subunit gene expression were about 50-fold lower than that for the {alpha} and ßLH subunits, the net result was that the mRNA levels of all gonadotropin subunits were in the same order of magnitude. The mRNA levels of the {alpha} subunit peaked in October (16-fold over basal levels) and remained elevated throughout April. ßFSH mRNA levels reached maximal values in October (370-fold increase) and returned to basal levels in April. Peak levels of ßLH mRNA were measured in February (18-fold increase), 4 mo after the peak in ßFSH levels.

Figure 4A shows that there was a positive relationship between the {alpha}- and ßFSH-subunit mRNA levels (r = 0.92, P < 0.001). The correlation between the {alpha}- and ßLH-subunit mRNA levels was even higher (Fig. 4B; r = 0.97, P < 0.001).



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  		FIG. 4. Correlation between gonadotropin {alpha}- and ß-subunit mRNA levels in the pituitaries of female striped bass (n = 207). A) ßFSH; B) ßLH. A linear function describes this relationship

Pituitary LH Content and Its Relationship to ßLH mRNA Levels

The changes in pituitary LH concentration are shown in Figure 2D. The profile appears to follow an annual rhythm, characterized by an increasing amplitude of the annual peak. In pubertal (3 yr old) females, pituitary LH concentration was about 2-fold higher than in their juvenile cohorts (Fig. 3D). Maximal concentrations of pituitary LH were measured in 4-yr-old females in December during vitellogenesis (Fig. 2D).

The correlation between ßLH mRNA abundance and pituitary LH content is shown in Figure 5. The relationship was calculated separately for the following oocyte developmental stages: A) PG and SG-I, B) SG-II (lipid droplet stage), C) SG-III (vitellogenic growth). There was a positive correlation at all stages (P < 0.01), but oocyte growth was associated with an increase in the correlation coefficient (r), from r = 0.33 in PG and SG-I oocytes to r = 0.79 in SG-III oocytes.



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  		FIG. 5. Correlation between ßLH mRNA levels and pituitary content of LH in female striped bass during oocyte development. A) PG and SG-I stages (n = 127); B) SG-II stage (n = 26); C) SG-III stage (n = 54). The relationships are described by linear functions

Plasma Levels of LH

Throughout the study period, the circulating plasma levels of LH remained low and unchanged (0.4–0.8 ng/ml; data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The hormonal events underlying the transitional period between the juvenile and adult reproductive phases have been studied in a number of model fish species [14, 28, 29]. Unfortunately, the terms puberty, adolescence, sexual maturation, and adulthood are often used interchangeably, complicating the interpretation of data for comparative studies. The term "puberty" is used here to describe partial ovarian development that does not lead to sexual maturity, whereas "adolescence" is reserved for the first sexual maturity. Although capable of emitting mature germ cells, adolescent striped bass and other fish species are compromised in their reproductive competence since they exhibit lower egg and sperm quality and produce smaller eggs and larvae that are less likely to survive [30–32]. Thus during their life cycle, striped bass females proceed through the following reproductive phases: juvenile, pubertal, adolescent, and adult.

At the beginning of the study, basal levels of all gonadotropin subunit genes were expressed in the pituitary of juvenile females. We found that the basal mRNA levels of the {alpha} and ßLH subunits were about 50-fold higher than for ßFSH, but the difference between the two decreased at puberty. The earliest ovarian activity was recorded in the second year of life, 2 yr prior to sexual maturation. This activity occurred in the winter, which is the period of vitellogenic growth in adults, and was marked by a slight increase in ovarian size. However, we did not observe any changes in the oocytes by histological examination [24]. During this time period, we found a transient increase in the gene expression of all gonadotropin subunits, in particular that of ßFSH. There was also an increase in the pituitary concentration of LH, but circulating levels of LH remained low and unchanged. At present, we do not have an explanation for the increased synthesis of LH during this early developmental phase; but it is possible that in addition to its recognized role during final oocyte maturation, LH may have other endocrine functions in juveniles. This hypothesis is supported by two lines of evidence. First, the pituitary gland of other juvenile fish species contains the ßLH mRNA [13, 17], and LH is present in the pituitaries [10, 13, 29] and plasma [12] of immature fish. Second, the type-I gonadotropin receptors present in the ovaries of juvenile salmon [33] show affinity for both FSH and LH, with only a slightly higher affinity for FSH. The precise role of LH during the juvenile reproductive phase, if any, remains to be determined.

An incomplete ovarian cycle was observed in 65% of 3-yr-old females. In these fish, a small number of oocytes grew in size (reflected by the increase in GSI) and became filled with lipid droplets (SG-II stage). However, yolk globules were only rarely observed, probably due to the low levels of plasma vitellogenin [24]. This reproductive phase was classified as pubertal and was easily distinguishable from the adolescent phase using the following criteria: 1) ooplasmic inclusions in pubertal fish appeared in December, whereas in adolescent and adult fish they appeared 3 mo earlier, in September; and 2) the ovaries of pubertal fish contained primarily SG-II and only a marginal number of SG-III oocytes, whereas adolescent females contained a leading clutch of SG-III oocytes. We have previously described pubertal (i.e., incomplete) ovarian development in domesticated populations of the European sea bass [34] and the white grouper [35]. However, it appears that incomplete ovarian development is not related to domestication, since partial ovarian growth also occurs in wild stocks of striped bass [36]. Pubertal ovarian growth, also known as a "dummy run" in the fishery literature, is probably common to many fish species; but the physiological and endocrine mechanisms underlying this developmental phase are still poorly understood.

Pubertal development in the striped bass coincided with increased mRNA levels of ßFSH, although the absolute amounts of ßFSH mRNA were low compared to the tremendous increase in ßFSH mRNA concentration that would take place a year later during the first sexual maturation. Unfortunately, it could not be determined whether the pubertal rise in ßFSH mRNA levels was associated with an increase in the pituitary or plasma levels of FSH, since an immunoassay for striped bass FSH has not been established yet. It is presumed that the low mRNA levels of ßFSH in the pituitaries of pubertal striped bass may be indicative of low plasma levels of this hormone. The low levels of E2 and vitellogenin in the plasma of pubertal females [24] suggest that this is indeed the case. Interestingly, in the masu salmon, a pubertal rise in pituitary FSH content did not coincide with any histological changes in the ovary [29], suggesting that the hormone was not released into the bloodstream, or that the ovary was insensitive to FSH stimulation.

Sexual maturation in adolescent females was characterized by the appearance of a leading clutch of SG-III (vitellogenic) oocytes in September and a concomitant rise in GSI values. The cytological changes coincided with an increase in the mRNA level of both ßFSH and ßLH, but several differences were noted between them. First, between July and September, steady state levels of ßFSH increased 218-fold, compared to a mere 6-fold rise in ßLH mRNA levels. Second, the levels of ßFSH mRNA peaked in October, whereas maximum levels of ßLH mRNA were detected only 4 mo later, in February. And third, during the final stages of ovarian development (April), the levels of ßFSH gene expression declined to basal levels, while mRNA levels of ßLH remained elevated. The pituitary content of LH mirrored the rise in the levels of ßLH mRNA, but plasma LH concentrations remained low and unchanged throughout the reproductive cycle. It has already been reported that in the striped bass, plasma levels of LH are low during vitellogenic growth and that a plasma LH surge occurs only during final oocyte maturation and ovulation [21]. It will be interesting to find out whether the preovulatory surge in plasma LH results from a further increase in ßLH mRNA abundance or from increased translational activity of existing mRNA.

The developmental profiles of gonadotropin gene expression in the striped bass showed that during the juvenile phase, ßLH mRNA levels were about one order of magnitude higher than those of ßFSH. This is unlike the situation in the immature rainbow trout, where pituitary ßFSH predominated over ßLH mRNA concentration [13, 17, 18]. Nevertheless, the data in the striped bass are consistent with a study in the goldfish, where both ßFSH and ßLH were weakly expressed in immature fish and became strongly expressed with the progression of ovarian maturity [19]. Although there is still a paucity of data regarding gonadotropin gene expression in fish, these studies indicate that the ontogeny and regulation of the gonadotropin genes may differ among teleost species.

The regulation of LH synthesis and release by GnRH and steroids has been the subject of intense research, but it is still unclear whether these factors also regulate FSH. For example, in contrast to its effect on ßLH, E2 had no effect on ßFSH mRNA levels, either in vitro [37] or in vivo [38]. The recent findings that GnRH and E2 were ineffective in stimulating FSH secretion at any reproductive stage in the rainbow trout [12, 39] indicate that the regulation of FSH release is probably different from that of LH. The mechanisms underlying sexual maturation in striped bass females and the associated rise in ßFSH gene expression are an active area of research in our laboratory. The involvement of E2 and testosterone (T) seems unlikely because the onset of sexual maturation was not associated with changes in the plasma levels of these steroids [24]. Moreover, the chronic administration of T and/or GnRH agonist did not affect ßFSH mRNA levels, although it did stimulate a moderate rise in the mRNA levels of ßLH (unpublished results). The involvement of ovarian peptides such as activin/inhibin in the regulation of FSH synthesis and release in fish remains to be determined [40]. Whatever the regulators of ßFSH mRNA may be, it appears that they act in a circannual rhythm. These factors are active at least 2 yr prior to sexual maturation, manifested by the annual peaks in ßFSH mRNA levels in juvenile and pubertal striped bass. A similar phenomenon was described in the female masu salmon, where seasonal peaks in the pituitary content of FSH occur during the juvenile reproductive phase [29].

The only data available for comparison with mammals comes from studies on the rat and sheep estrous cycles. In the rat, preovulatory surges of pituitary gonadotropins take place in the evening of proestrus. A rise in ßLH and ßFSH mRNA levels occurs on proestrus, presumably in response to increased GnRH release [41, 42]. On metestrus, there is a selective increase in ßFSH mRNA, probably reflecting input from ovarian peptides. In the sheep, estrus (i.e., female receptivity) is followed by preovulatory surges in LH and FSH. The levels of ßLH mRNA tend to rise in parallel with the gonadotropin surge, whereas ßFSH mRNA concentrations decline during the preovulatory surge, reaching nadir values when serum FSH levels are maximal [43]. ßFSH mRNA levels then reach maximal levels 24 h after the onset of estrus. The level of complexity of the hypothalamus-pituitary-gonadal axis in mammals is obviously greater than in teleost species; nonetheless, it appears that the major regulators of the gonadotropin genes—GnRH, sex steroids, and perhaps activin/inhibin—were already functional 800 million years ago in teleost fish.

It has been suggested that expression of the {alpha} subunit is the limiting factor in the synthesis of FSH in rainbow trout females [13]. In contrast, our data show that in the striped bass, the levels of the {alpha} subunit were highly correlated to the levels of ßFSH mRNA (r = 0.920) and almost parallel to ßLH mRNA (r = 0.973). Such parallelism may indicate that these genes share similar regulatory elements, which is not surprising given the common ancestral origin of the {alpha}, ßFSH, and ßLH subunits [7].

Once translated and assembled, there are two pathways for gonadotropin secretion from the pituitary: regulated and constitutive [44]. In the regulated pathway, the hormone is stored in globules and released in pulses in response to secretagogues. In contrast, secretion via the constitutive pathway is continuous and temporally coupled to hormone biosynthesis, and consequently the level of mRNA is a major determinant of the mass of hormone released. In rats, there is evidence that FSH secretion occurs via both the regulated and constitutive pathways, whereas LH secretion is regulated primarily by GnRH [45]. In the present study, pituitary content of LH was correlated to the mRNA levels of ßLH throughout the progression of ovarian maturity. The correlation coefficient increased gradually during oocyte development, perhaps due to increased coupling of the transcription/translation mechanisms. However, plasma levels of LH remained unchanged despite increasing levels of pituitary LH concentration, suggesting that similarly to the release of LH in mammals, LH is released through the regulated pathway, probably in response to an endogenous rise in GnRH input to the pituitary.

In summary, the correlation between ßFSH and ßLH mRNA levels and the reproductive phases of the female striped bass suggests that they play a major role in the acquisition of sexual maturity. In particular, the dramatic changes in ßFSH-subunit gene expression during the first sexual maturation suggest that a rise in plasma FSH underlies the onset of sexual maturation, although a role for LH cannot be excluded. It is reasonable to assume that the manipulation of ßFSH mRNA levels in juvenile fish will accelerate sexual maturation, but the endogenous regulators of ßFSH transcription in fish are still poorly understood. Perhaps study of the ßFSH promoter region will provide the necessary insight to achieve that goal.


   ACKNOWLEDGMENTS
 
We are grateful to Steve Rogers and Tina D'Alesio at the Aquaculture Research Center, University of Maryland Biotechnology Institute, for taking care of the experimental fish. Thanks are also extended to Kathy Kight and to Drs. Alok Deoraj, Yoav Gothilf, and John Trant for their comments on earlier versions of the manuscript.


   FOOTNOTES
 
1 This research was supported in part by grant #IS-2634-95-C from the US-Israel Binational Agricultural Research and Development Fund (BARD) and by grant #NA46RG0091 (Amnd. 6) from Maryland Sea Grant College Program (to Y.Z.). This publication is contribution no. 328 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute. Back

2 Correspondence: Yonathan Zohar, 701 E. Pratt St., C.O.M.B., Baltimore, MD 21202. FAX: 410 234 8896; zohar{at}umbi.umd.edu Back

3 Current address: SARS International Center for Molecular Marine Biology, University of Bergen, Thormøhlensgt 55, 5008 Bergen, Norway. Back

Accepted: July 22, 1999.

Received: May 6, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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