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Biology of Reproduction 59, 53-61 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Oocyte Sensitivity to Serotonergic Regulation during the Follicular Cycle of the Teleost Fundulus heteroclitus1

Joan Cerdà2,a, Nishikant Subhedar3,a, Gerhard Reicha, Robin A. Wallacea,b, , and Kelly Selmana,b

a The Whitney Laboratory, University of Florida, St. Augustine, Florida 32086 b Department Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the teleost Fundulus heteroclitus, serotonin (5-HT) reversibly inhibits oocyte maturation induced in vitro by the maturation-inducing steroid (MIS) 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP). The 5-HT inhibition of 17,20ßP-induced meiotic maturation was examined in ovarian follicles at different developmental stages or isolated at different times during the follicular cycle. Steroid treatment of late vitellogenic and early maturing follicles (1.2- to 1.7-mm diameter) promoted oocyte maturation in a size-dependent manner, and this maturation was inhibited by 5-HT in follicles of < 1.6- to 1.7-mm diameter. Thus, the 5-HT inhibition progressively decreased as follicles developed the ability to mature in the absence of 17,20ßP. The effectiveness of 5-HT to increase follicular cAMP remained similar within the same developmental stages, indicating that the reduction of 5-HT inhibitory action was not related to the competence of 5-HT to activate inhibitory signals in the oocyte. During the follicular cycle, fully grown follicles (1.3- to 1.4-mm diameter) showed a decreased maturational competence in response to gonadotropin or MIS stimulation after the follicular recruitment into maturation and spawning occurred, which coincided with an increase of the effectiveness of 5-HT at inhibiting 17,20ßP-induced maturation. In further experiments, preincubation of follicles with hCG was found to reduce 5-HT inhibitory action, but when follicles were incubated with either hCG in the presence of a steroidogenesis inhibitor or estradiol-17ß (E2), the 5-HT inhibition was unaffected. These findings suggest that 5-HT inhibition of the MIS-induced meiotic maturation is not under direct gonadotropin or E2 regulation but that it might be regulated in vivo by changes in the competence of the oocytes to undergo oocyte maturation after MIS stimulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The involvement of neurotransmitters in the control of reproduction in vertebrates, through the regulation of the release of pituitary gonadotropins, is well known. However, direct and indirect evidence has been accumulated during the last decade suggesting that biogenic amines, and particularly serotonin (5-hydroxytryptamine, 5-HT), also influence gonadal tissues directly, being effective regulators of oocyte maturation either by promoting the ability of follicle cells associated with the oocyte to secrete maturation-inducing steroids (MIS) in response to a gonadotropin surge, or by affecting the oocyte maturation responsiveness to MIS [1, 2].

In the mammalian ovary, the extensive innervation by both sympathetic and sensory fibers, as well as the presence of 5-HT and its receptors, has been well documented [1, 3, 4]. High levels of 5-HT were found in bovine corpora lutea [5], and in the rat reproductive tract [6, 7] and human follicular fluid [8], in which the 5-HT concentrations changed in association with the ovulatory cycle. Ovarian 5-HT does not seem to be produced by follicular cells [7] but is thought to be locally released by mast cells within the ovary, oviduct, and uterus [7, 9, 10], or to be transported to reproductive organs through nerve terminals [3, 7, 11–13] and blood leukocytes (e.g., [14]). The direct effect of 5-HT on reproductive organs has been shown by experiments in vitro, in which this monoamine was found to stimulate progesterone and estradiol-17ß (E2) secretion in rat and hamster preovulatory follicles [15, 16], dissociated bovine luteal cells [5, 17], and porcine and human granulosa cells [18, 19]; 5-HT receptors located on the granulosa or lutein cells, related to the 5-HT1 and 5-HT2 receptor subtypes, seem to be involved in these actions [15, 16, 20]. Furthermore, a serotonergic regulation of oxytocin and vasopressin secretion by cultured human and porcine granulosa cells [1, 18], as well as a direct inhibition of oocyte maturation by 5-HT in bovine oocytes [1], have been observed.

Although in lower vertebrates, such as fish and amphibians, there are similarities to mammalian species regarding the effects of 5-HT on ovarian functions, the specific mechanisms leading to oocyte maturation that are affected by 5-HT seem to be different among the species studied to date. For example, in medaka (Oryzias latipes), 5-HT stimulates the synthesis of E2 and the MIS 17{alpha},20ß-dihydroxy-4-pregnen-3-one (17,20ßP) by the granulosa cells [21, 22], thus promoting oocyte maturation, whereas in another teleost, Fundulus heteroclitus, 5-HT reversibly inhibits both gonadotropin- and MIS-induced meiosis reinitiation without affecting the steroidogenesis pathways in the granulosa cells [23]. In F. heteroclitus, the inhibitory effect by 5-HT on oocyte maturation appears to be mediated by oocyte-associated 5-HT receptors with a unique pharmacological profile (as defined by germinal vesicle breakdown (GVBD) assays [24]), whereby a cAMP-protein kinase A transductional pathway is activated [25]. In the amphibians Xenopus laevis and Bufo viridis, a similar inhibitory effect on progesterone-induced meiotic maturation by 5-HT has been observed, which is believed to take place through the activation of multiple 5-HT receptor subtypes (as yet unidentified) located on the granulosa cells, on the oocyte, and in the ooplasm, coupled to transductional pathways yet unknown [2, 26–28].

The teleost F. heteroclitus presents a group-synchronous ovary with a semilunar spawning activity, in which follicle recruitment, maturation, and ovulation are overlapped in a tight sequence [29, 30]. Under laboratory conditions, the follicular cycle of F. heteroclitus can be consistently reproduced with a periodicity of approximately 2 wk all year around, and this can be monitored by daily egg collection and frequent ovarian sampling [30, 31]. Thus, this species offers an excellent nonmammalian model of cyclic reproductive activity, recently proposed as "the Fundulus model" [30], for the study of cellular and hormonal mechanisms controlling follicular development and recruitment in lower vertebrates. In the present work, we have used this model and combined observations in vivo with observations in vitro to investigate oocyte sensitivity to 5-HT both during late oocyte growth and throughout the follicular cycle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fish and Chemicals

Killifish, F. heteroclitus, were collected from salt marshes close to the Whitney Laboratory (St. Augustine, east coast of Florida). The fish were immediately transported to the laboratory, treated with 300 ppm Paracide-F (Argent Chemical Laboratories, Inc., Redmond, WA) for 1 h, and placed in three different spawning tanks with approximately 200 fish in each (female:male ratio approximately 2:1). The spawning tanks contained a plastic horizontal screen adapted to a tray (54 x 28 x 6 cm) kept at the bottom of the tank, since the fish tend to spawn against the screen [30, 31]. Thus, the spawning cycles in captivity were monitored by daily egg collection from these tanks (see below). Other groups of fish were located in similar tanks, but spawning was not monitored. All groups of fish were maintained under controlled temperature (25–28°C) and photoperiod (14L:10D) [31] for approximately 4 mo.

Reagents, culture medium, and hormones were purchased from Sigma (St. Louis, MO), unless indicated otherwise.

Solutions

The culture medium for incubation of ovarian follicles in vitro was 75% Leibovitz L-15 medium with L-glutamine, containing 100 µg gentamicin/ml and adjusted to pH 7.5 with HCl [32]. Fundulus pituitary extract (FPE), as a homologous source of gonadotropin, was prepared at a concentration of 10 pituitary equivalents (PEq)/ml in 75% L-15 culture medium [33], and hCG (10 000 IU) was dissolved in 3.3 ml of culture medium. Both solutions were kept at -20°C in 100-µl aliquots that were thawed only once before each experiment. The solutions of E2 and 17,20ßP were prepared in 95% ethanol at a concentration of 4 µg/µl and 0.1 µg/5 µl, respectively. The stock solution of DL-aminoglutethimide (AGI) was dissolved in dimethyl sulfoxide (DMSO) at 10 mg/ml and kept at -20°C. A stock solution of 5-HT (5-HT hydrochloride) was prepared as 1.4 mM in distilled water adjusted to pH 3 with acetic acid and was stored in aliquots at -20°C. Before each experiment, serial dilutions of the 5-HT stock were prepared with distilled water, pH 3; these were added to the culture medium in a volume of 5-10 µl [23]. The same stock solution of each hormone or drug was used for all the experiments, and the final concentrations of their vehicles, i.e., ethanol, DMSO, or acid water, in the culture medium did not exceed 1%; this concentration had no effect on oocyte maturation or on the inhibition of steroid-induced GVBD by 5-HT (data not shown).

Collection of Ovarian Follicles throughout the Spawning Cycle

In order to define the spawning cycle in each tank, the eggs produced were collected daily from each tray and counted. Although the eggs spawned against the screen represent a fraction of the total number spawned, they can be reliably used to define the periodicity of the spawning cycle [29–31]. After the first or second well-defined spawning peak was observed by daily egg collection, which occurred shortly after fish were moved into the laboratory or after 30–40 days in captivity (data not shown), 8–15 females from each of the three groups were sampled at days randomly selected throughout the next spawning cycles in order to perform follicle size-frequency profiles and to collect the ovarian follicles for the experiments in vitro. The sampling days selected in each group (6–8 different days for each tank) were assumed to approximately represent different periods throughout the follicular cycle based on the egg collection data, but they were precisely defined by employing statistical analysis when all the spawning cycles were recorded at the end of the experiment (see Statistics).

On each sampling day, the females were randomly collected from each tank between 1000 and 1200 h and killed, and the ovaries were dissected and transferred to a 35 x 10-mm Petri dish (Falcon 3001; Becton, Dickinson and Co., Oxnard, CA) containing 3 ml of 75% L-15. Within a few minutes, a piece of the ovary was separated for the assessment of oocyte size-frequency distribution of ovarian follicles, by randomly measuring approximately 100 follicles >= 0.45-mm diameter at a magnification of x25 to the nearest 0.01 mm, using an Olympus stereomicroscope (Olympus America Inc., Melville, NY) [31]. Follicles remaining in the ovary that were not used for the oocyte size-frequency profiles were used for incubations in vitro. For most of the sampling days, however, follicles were collected for the experiments in vitro, and oocyte size-frequency profiles were not carried out.

Incubations of Ovarian Follicles and Induction of Final Oocyte Maturation In Vitro

Follicle-enclosed oocytes from 1.1 to 1.7 mm in diameter, with visible germinal vesicle, were manually isolated from the rest of the ovary using watchmaker forceps. These follicles represent mid and late stages of follicular development in F. heteroclitus [32]: 1.1- to 1.2-mm diameter, late vitellogenic stages; 1.3- to 1.4-mm diameter, fully grown follicles; and 1.5- to 1.7-mm diameter, early maturational stages. Follicles from different females in one tank sampled during one day were pooled together according to their size. In experiments in which the specific day of the spawning cycle was not considered, follicles of the same size were isolated from females in different tanks during the same day and were pooled together. In both types of experiments, oocytes were washed several times in fresh culture medium, equilibrated for 1 h at room temperature (22–25°C), and placed in groups of 15–20 healthy follicles that were transferred to 24-well tissue culture trays (Corning No. 25820; Becton, Dickinson) containing 1 ml of medium. Follicles were then treated with different hormones (see below) and incubated at 23–25°C in a humidified, temperature-controlled incubator.

The competence of follicle-enclosed oocytes to undergo final maturation in vitro was determined by following previous protocols (e.g., [33, 34]). The effect of gonadotropin on oocyte maturation was tested by using FPE at 0.5 PEq/ml or hCG at 50 IU/ml; hCG was selected for some experiments since these preparations are broadly active on F. heteroclitus [35] and are free of 5-HT or other putative inhibitors that may be present in pituitary extracts. Final oocyte maturation was also induced by exposure of follicles to the MIS 17,20ßP at 1 or 100 ng/ml. Since previous studies have shown that 5-HT does not affect follicular steroidogenesis in F. heteroclitus [23], only the effect of 5-HT on 17,20ßP-induced GVBD was tested. In these cases, 5-HT was added to the culture medium 5-10 min before the addition of the steroid, at a concentration of 0.05 µM or 0.2 µM, the latter being the IC50 dose for 5-HT (the 5-HT concentration that reduces by 50% the oocyte maturation induced by 100 ng/ml 17,20ßP [23]). The control follicles were treated only with steroid and 5-HT vehicles, 95% ethanol (0.1%) and distilled water pH 3 (1%), respectively.

The incidence of resumption of meiosis (oocyte maturation) was measured as the percentage of GVBD [36]. These measurements were performed at approximately 12-h intervals up to 54–72 h of culture, enough time for reaching maximum GVBD in the follicles exposed to 17,20ßP alone. In some experiments, the percentage of oocyte maturation induced by exogenously added 17,20ßP was calculated after subtraction of the incidence of GVBD observed in the control follicles not exposed to hormonal treatment, i.e, spontaneous maturation. The percentage of inhibition of oocyte maturation by 5-HT was calculated when the incidence of maturation in follicles treated with 17,20ßP in the presence of 5-HT was significantly lower than that in follicles treated with 17,20ßP alone. Both calculations were performed by considering the incidence of GVBD in the groups treated with 17,20ßP alone to be 100%.

Cyclic AMP Assays

To measure the 5-HT-induced production of cAMP by ovarian follicles of various-sizes, groups of approximately 20 individual follicles were incubated in 1 ml 75% L-15 containing 0.1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) for 1–2 h at 23–25°C. After this period, follicles were treated with 100 µM 5-HT or 5-HT vehicle (Control) and incubated for 1 h at 23–25°C; within this period of time and dose, 5-HT has been found to induce maximum increase of the follicular cAMP levels [25]. Both the control and 5-HT-treated follicles were then transferred to a tube containing 500 µl of cold 95% ethanol, containing 10 mM theophylline and 2.5 mM IBMX, and were frozen in liquid nitrogen. The tubes were kept at -20°C until the RIA was carried out according to a protocol already described [25]. The levels of cAMP in the control follicles were expressed as fmol/100 µg protein, and the levels of cAMP in the 5-HT-treated follicles were expressed as relative to the control values in order to normalize the data [25].

Statistics

The data are presented as the mean ± SEM. A statistical analysis of the spawning cycles from the three tanks, which provided a basis to define different days during the ovarian follicular cycle in F. heteroclitus, was performed by using statistical programs (SAS Institute, Cary, NC) described elsewhere [29–31]. Briefly, after 4–6 consecutive spawning episodes occurred in each group, the data from daily egg collections were examined with Spectral analyses to search for spawning cycles defined by amplitude, phase, and period. These parameters were introduced into a nonlinear regression to search for a sine curve significantly (p < 0.01) matching the data and thus dividing it into individual cycles, which provided the chronological basis for pooling data from separate cycles (and from separate tanks) [30, 31]. The nonlinear regression of the daily egg collection data indicated periods of 10.8 ± 0.2 days, 13.7 ± 0.8 days, and 15.3 ± 0.4 days for the three groups, and since the mean was approximately 13 days, each cycle was dated with a sequence of -6 to 6 Days, with the cycle peak in egg production as Day 0. Accordingly, the sampling days on which the ovarian follicles were collected were chronologically defined by their temporal relation to Day 0, and the data were pooled into a composite spawning cycle.

The frequency of follicles within the ovary was observed through ovarian sampling done on Days -4, -3, -2, -1, 0, 1, 3, 4, and 5 of the composite spawning cycle, and their variations were analyzed by the Kruskal-Wallis nonparametric, ranked test (significance at p < 0.05). In the rest of experiments, one- or two-way ANOVA was used to detect the significance of the variation in a set of data collected across the cycle or throughout follicle development, and Tukey's test was further applied to detect differences among the means (significance at p < 0.05). In order to generate data from the three tanks for the ANOVA, the data on the follicle responsiveness to the hormones were pooled on Day -3 (including Days -4, -3, and -2), Day 0 (including Days -1, 0, and 1), or Day 4 (including Days 3, 4, and 5), corresponding to ovarian recrudescence, spawning, and postspawning periods, respectively, for each follicle-size group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of 5-HT on MIS-Stimulated Oocyte Maturation and Follicular cAMP Levels during Late Oocyte Growth

In order to study the effectiveness of 5-HT at inhibiting MIS-induced resumption of meiosis during follicle development, ovarian follicles of various-sizes (from 1.1- to 1.7-mm diameter) were isolated from the ovaries and exposed to 17,20ßP (1 ng/ml), in the presence or absence of 5-HT (0.05 µM). The results depicted in Figure 1 show that follicles >= 1.2-mm diameter progressively matured in response to 17,20ßP in a size-dependent manner, with the maximum incidence of oocyte maturation observed in 1.6- and 1.7-mm follicles. The presence of 5-HT significantly (p < 0.05) inhibited 17,20ßP-triggered oocyte maturation in 1.2- to 1.5-mm follicles, although this monoamine was ineffective at inhibiting steroid-induced oocyte maturation in 1.6- and 1.7-mm follicles. Control follicles >= 1.4-mm diameter displayed an increased ability to mature spontaneously in the absence of steroid as they increased in size, indicating that a number of these follicles were already at very early stages of oocyte maturation when they were isolated; interestingly, this maturation was not inhibited by 5-HT.



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  		FIG. 1. Effect of 5-HT (0.05 µM) on 17,20ßP (1 ng/ml)-induced oocyte maturation in F. heteroclitus follicle-enclosed oocytes at different stages of development, corresponding to late vitellogenesis, fully grown, or early maturation stages. Values are the incidence of GVBD (mean ± SEM) from 7–9 different experiments using different batches of follicles that were performed on different days; each experiment consisted of 2 replicates per treatment (n = 15–20 follicles per replicate). For a given follicle-size group, values with different superscripts are significantly different (ANOVA, p < 0.05).

To detect possible differences in 5-HT inhibitory action during late oogenesis, the percentage of maturation induced by exogenously added steroid and the percentage of GVBD inhibition by 5-HT were further calculated from the previous data (Fig. 2). The results indicated that both oocyte maturation promoted by exogenously added 17,20ßP and 5-HT inhibition were progressively and dramatically reduced as follicles increased in size.



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  		FIG. 2. Percentage of oocyte maturation in vitro induced by exogenously added 17,20ßP, and percentage of steroid-induced GVBD inhibition by 5-HT, in follicle-enclosed oocytes of increasing size. The values (mean ± SEM) were calculated from data shown in Figure 1.

Another set of experiments was carried out to evaluate the ability of 5-HT to stimulate the production of follicular cAMP in follicles of increasing size. Figure 3 shows the levels of cAMP in control 1.1- to 1.7-mm follicles not exposed to 5-HT (inset), as well as the relative increase in cAMP production by these follicles when they were treated with 5-HT (100 µM). In control follicles, the levels of cAMP were found to decrease significantly (p < 0.05) in 1.1- to 1.5-mm follicles, but they increased thereafter in 1.6- and 1.7-mm follicles to reach levels similar to those found in 1.1-mm follicles. In contrast, the 5-HT-induced increase in cAMP remained unchanged in 1.1- to 1.6-mm follicles (2.5- to 3.5-fold increase with respect to the values in the controls), although the 1.7-mm follicles showed a slightly lower elevation of the cAMP levels after 5-HT treatment (1.4-fold increase) when compared with smaller oocytes.



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  		FIG. 3. Levels of cAMP in control follicles of various sizes (inset), and effect of 5-HT (100 µM) on cAMP levels in the same range of follicle sizes. The data on cAMP levels in control follicles are the mean ± SEM of 3 different experiments using different batches of follicles isolated from 10-15 females, and they were performed on different days. The content of cAMP after 5-HT treatment was normalized with respect to the control values for each follicle-size group. Different letters indicate statistically significant differences (ANOVA, p < 0.05).

Inhibition of MIS-Induced Oocyte Maturation by 5-HT during the Follicular Cycle

To define the follicular cycle, spawning episodes from fish maintained in three different tanks were monitored by daily egg collection and ovarian sampling on selected days (see Materials and Methods). Based on the previous experiments that indicated significant changes in the percentage of GVBD inhibition by 5-HT in follicles of 1.3-, 1.4-, and 1.5-mm diameters, the distribution of these follicle populations across the composite spawning cycle was independently analyzed (Fig. 4). Fully grown follicles, 1.3 to 1.4 mm in diameter, were found to be present throughout the cycle, although their numbers were significantly (p < 0.05) higher on Day -4 and Day 5 relative to Days -3 and -2 close to the spawning peak (Fig. 4, a and b). The decrease in fully grown follicles in the early part of the cycle was followed by an increase (p < 0.05) in the population of 1.5-mm follicles (early-maturing follicles) by Days -3 and -2, but they drastically declined (p < 0.05) thereafter from Day -1 to Day 5 (Fig. 4c). Thus, the increase in 1.5-mm follicles in the ovary by Days -3 and -2, and their decrease around Day 0, indicated that these follicles were quickly recruited into maturation and ovulation, since this observation coincided with the increase in the number of eggs around Day 0 (Fig. 4d).



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  		FIG. 4. Distribution of follicles with 1.3- (a), 1.4- (b), and 1.5-mm (c) diameters within the ovary, and numbers of eggs collected from the tanks (d) across the composite spawning cycle of F. heteroclitus in captivity. Values in a, b, and c are the mean ± SEM (n = 2–3 different tanks; 10–15 females sampled in each tank); values in d are the numbers of eggs per tank throughout 4–6 spawning cycles. Means with different superscripts are significantly different (Kruskal-Wallis, p < 0.05). Note below d the three general chronological periods of the follicular cycle. Note in c (arrow) the recruitment of fully grown follicles into maturation that occurred on Days -3 and -2.

The responsiveness of 1.3-, 1.4-, and 1.5-mm follicles to FPE (0.5 PEq/ml) and 17,20ßP (1 ng/ml) in the presence or absence of 5-HT (0.05 µM) by undergoing oocyte maturation was then examined across the cycle. The results from these experiments (Fig. 5) indicated that 1.3- and 1.4-mm control follicles displayed low levels of spontaneous maturation on Day -3, which appeared to decrease (p < 0.05) as Days 0 and 4 approached (Fig. 5, a and b), whereas in 1.5-mm follicles the spontaneous maturation remained high on Days -3 and 0, and decreased (p < 0.05) thereafter by Day 4 (Fig. 5c). As seen in previous experiments, treatment with 5-HT had no effect on the incidence of spontaneous maturation in any of the follicle-size groups. Treatment of follicles with FPE induced oocyte maturation in all groups, although a more potent maturation response was observed in 1.4- and 1.5-mm follicles when compared with that in 1.3-mm follicles (Fig. 5, d–f). In both 1.3- and 1.4-mm follicles, the FPE-induced GVBD was higher (p < 0.05) on Day -3 with respect to that on Days 0 and 4 (Fig. 5, d and e), whereas the 1.5-mm follicles showed no differences across the cycle in their responsiveness to FPE (Fig. 5f). All follicle-size groups also underwent GVBD after 17,20ßP treatment, and this response was stronger according to the size of the follicles (Fig. 5, g–i). Similar to the results observed for FPE, the 1.3- and 1.4-mm follicles significantly (p < 0.05) decreased their response to 17,20ßP by undergoing GVBD on Days 0 and 4 (Fig. 5, g and h), whereas the 1.5-mm follicles did not show significant differences in the 17,20ßP-induced GVBD across the cycle (Fig. 5i).



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  		FIG. 5. Responsiveness of 1.3-, 1.4-, and 1.5-mm follicles to FPE (0.5 PEq/ml) or 17,20ßP (1 ng/ml), and effect of 5-HT (0.05 µM), shown by the incidence of GVBD in vitro during ovarian recrudescence (Day -3), spawning (Day 0) or postspawning (Day 4) periods (a–i). Each value is the mean ± SEM of the incidence of GVBD (n = 3 different tanks; 3–4 different experiments, consisting of 2–3 replicates per treatment with 15–20 follicles per replicate, were carried out in each tank). Values with different superscripts are significantly different (ANOVA, p < 0.05).

The presence of 5-HT was found to significantly (p < 0.05) inhibit 17,20ßP-induced GVBD in all follicle-size groups at any day of the follicular cycle (Fig. 5, g–i). In order to detect differences in the effectiveness of 5-HT at inhibiting steroid-induced GVBD throughout the cycle, the percentage of inhibition of 17,20ßP-induced oocyte maturation by 5-HT in each follicle-size group during different days was calculated and analyzed (Fig. 6). This analysis indicated that the 5-HT action was more potent on Day 4 when compared with Days -3 and 0 in both 1.3- and 1.4-mm follicles, coinciding with their lower responsiveness to FPE and 17,20ßP stimulation by these Days. In contrast, 5-HT displayed lower (p < 0.05) effectiveness at inhibiting 17,20ßP-initiated GVBD in 1.5-mm follicles than in 1.3- and 1.4-mm follicles on Day -3 and Day 4, and this inhibition remained relatively constant throughout the cycle.



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  		FIG. 6. Effectiveness of 5-HT at inhibiting 17,20ßP-induced oocyte maturation during the follicular cycle in each follicle-size group. The percentage of oocyte maturation inhibited by 5-HT was calculated from the data shown in Figure 5. Values with different superscripts are significantly different (ANOVA, p < 0.05).

Effect of Gonadotropin on 5-HT Inhibitory Action

To test the possible regulation of 5-HT action by gonadotropin during the follicular cycle, another set of experiments was designed to determine the effects of hCG on the inhibition of 17,20ßP-induced oocyte maturation by 5-HT in follicles of 1.3- and 1.4-mm diameter. To ascertain whether the gonadotropic regulation of 5-HT activity required steroid production, we simultaneously used AGI, an inhibitor of cholesterol side-chain cleavage [37] known to abolish gonadotropin-induced accumulation of steroids in F. heteroclitus [38, 39]. Accordingly, groups of follicles were preincubated in culture media in the presence or absence of AGI (50 µg/ml) for 1 h, and subsequently treated or not with hCG (50 IU/ml) for up to 6–10 (Fig. 7a) or 17-22 (Fig. 7b) h. After these periods of time, the follicles from each group were washed three times in fresh culture medium and treated with steroid and 5-HT vehicles, 5-HT (0.2 µM), 17,20ßP (100 ng/ml), or 5-HT plus 17,20ßP for an additional 54 h. These further treatments were applied in hCG-free culture medium, and AGI was added again in those groups that were initially preincubated in the presence of this inhibitor.



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  		FIG. 7. Effect of preincubation of follicles with hCG (50 IU/ml), in the presence or absence of the steroidogenesis inhibitor AGI (50 µg/ml), on the serotonergic (0.2 µM 5-HT) inhibition of 17,20ßP (100 ng/ml)-induced GVBD in vitro. Follicles were preincubated with hCG, in the presence or absence of AGI, for 6–10 (a) or 17–22 (b) h. The data are the levels of GVBD (mean ± SEM) from 4 separate experiments performed on different batches of follicles and different days; each experiment consisted of 2–3 replicates per treatment (n = 15–20 follicles per replicate). Values with different superscripts for a given preincubation group indicate statistical differences (ANOVA, p < 0.05).

The results of these experiments shown in Figure 7 indicated that the steroid-induced GVBD in follicles initially preincubated in hCG-free culture medium or in medium containing only AGI for 6–10 or 17–22 h was effectively inhibited by approximately 50% by the IC50 concentration of 5-HT. Similar inhibition of steroid-induced maturation occurred in groups incubated continuously in medium containing only AGI. In both hCG-free groups incubated without or with AGI, the spontaneous maturation was not affected by 5-HT.

Initial preincubation of follicles with hCG for 6–10 h in the absence of AGI was sufficient to initiate a potent maturational response in follicles not subsequently exposed to 17,20ßP, and this response appeared to be inhibited by approximately 50% by 5-HT (Fig. 7a). However, 5-HT did not inhibit GVBD in follicles from the same group that were subsequently treated with 17,20ßP. In contrast, when follicles were preincubated with hCG for 17–22 h, inhibition of hCG-induced GVBD by 5-HT did not occur (Fig. 7b). Finally, hCG-preincubated follicles in the presence of AGI did not show a markedly increased ability to mature without steroid treatment (indicating that AGI effectively inhibited hCG-induced synthesis of MIS), and under these conditions 5-HT appeared to inhibit the 17,20ßP-initiated GVBD to an extent similar to that found in follicles not exposed to hCG or in follicles exposed only to AGI (Fig. 7, a and b).

The previous experiments indicated that the preventive effect by hCG on 5-HT inhibition of 17,20ßP-induced GVBD required further follicular steroid production. To determine whether this effect could be mediated by E2 (the major steroid also produced by the follicle cells in response to gonadotropin but lacking maturation-inducing activity [32, 33, 38]), we tested the ability of E2 to reduce the 5-HT effect on 17,20ßP-promoted GVBD. Table 1 shows the comparison of 5-HT inhibition in follicles previously preincubated in the presence or absence of E2 for 9–10 h and subsequently treated as described in the previous experiments. The results indicated that 5-HT was equally effective at inhibiting 17,20ßP-initiated GVBD in follicles preincubated in E2-free media and in those preincubated in media containing 2–20 µg/ml E2: GVBD inhibition by 5-HT was approximately 50% in both groups of follicles. The same results were obtained with identical experiments in which the follicles were preincubated with the same doses of E2 for approximately 20 h (data not shown).


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  		TABLE 1. Effect of follicle preincubation with E2 (2–20 µg/ml) for up to 9–10 h on the serotonergic inhibition of the 17,20ßP–induced GVBD in vitro.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Follicle-enclosed and defolliculated oocytes > 1.0-mm diameter isolated from the F. heteroclitus ovary and arranged according to size have been found to be increasingly competent to respond to 17,20ßP by undergoing oocyte maturation in a size-dependent manner ([32, 40, 41], present work). They thus appear to be similar to oocytes from X. laevis, which are self-differentiating cells and do not require an external signal to acquire competence to respond to MIS [42, 43]. As a corollary, F. heteroclitus oocytes also apparently do not require a previous treatment with gonadotropin to acquire maturational competence, as has been suggested for other teleosts [44–47]. In this work with F. heteroclitus, we initially used a low dose of 17,20ßP closer to the physiological levels (1 ng/ml, instead of 100 ng/ml used in previous studies [32]), and we have confirmed competence to MIS as oocytes underwent maturation during late vitellogenesis (Fig. 1). We also found that the effectiveness of 5-HT at inhibiting 17,20ßP-induced oocyte maturation progressively decreased as follicles enlarged from late vitellogenic into early maturational stages (Figs. 1 and 2). This decrease in the effectiveness of 5-HT occurred concomitantly with increased "spontaneous" maturation that follicles underwent as they advanced into the final maturational stages, characterized by a progressive increase in oocyte diameter due to oocyte hydration (Figs. 1 and 2).

In previous studies, it has been reported that 5-HT inhibition of MIS-induced meiosis reinitiation in F. heteroclitus may be mediated by an increase in the levels of cAMP in the oocyte, which serves to counteract the MIS-induced small and transient reduction in the levels of cAMP and subsequent induction of oocyte GVBD [25]. Thus, the cAMP-mediated inhibitory effect by 5-HT would be dependent on the extent to which oocytes have been exposed to MIS: 5-HT-activated high levels of intracellular cAMP are able to inhibit oocyte GVBD within 6–8 h of 17,20ßP exposure, but they are ineffective after longer periods of treatment with 17,20ßP [23]. In the present study, the 5-HT-induced increase in follicular cAMP levels in follicles developing from vitellogenesis into early maturation was found to be similar (Fig. 3), suggesting that the number or affinity of oocyte-associated 5-HT receptors was likely to be unchanged despite the progressively reduced effectiveness of 5-HT inhibitory action that was observed throughout these developmental stages. On the basis of these observations, it seems likely that spontaneously maturing and early maturing follicles (>= 1.6-mm diameter) had already reached a point downstream from the transient decrease in cAMP during the steroid-transduction pathway when they were isolated from the ovary, which would be consistent with the absence of 5-HT inhibition observed for these follicles (Fig. 1). It thus appears that the inhibitory action by 5-HT on oocyte meiosis may be mainly dependent through late oocyte growth on the extent to which oocytes have advanced along the MIS-activated maturational pathways, rather than on changes in the number or activity of 5-HT receptors.

The regularity of cyclic spawning episodes in F. heteroclitus provides a chronological basis for pooling data from different cycles occurring in different groups of fish. Thus, ovarian sampling from monitored populations provides sufficient size-frequency data to construct a dynamic profile of follicle development occurring within the ovary throughout the cycle [30, 31]. Accordingly, data were pooled from three populations in order to analyze both the frequency of 1.3-, 1.4-, and 1.5-mm follicles during the ovarian cycle and the responsiveness of these follicle populations to FPE and 17,20ßP by undergoing oocyte maturation. Similar to the case in previously reported studies [30], late vitellogenic follicles (1.3- and 1.4-mm diameter) were observed to be recruited into maturation (by the formation of 1.5-mm follicles) early in the cycle, around Days -3 and -2 (Fig. 4c), and this 1.5-mm follicle population was quickly depleted from the ovaries, giving rise to ovulated eggs and a heavy spawning at midcycle (Fig. 4d). Interestingly, in these females the competence of 1.5-mm follicle to respond to either FPE or 17,20ßP by undergoing oocyte maturation was unchanged across the cycle (Fig. 5, f and i), whereas the 1.3- and 1.4-mm follicles isolated at spawning and postspawning (Day 0 and Day 4, respectively) showed lower levels of GVBD after FPE or 17,20ßP treatment than those collected during ovarian recrudescence (Day -3) (Fig. 5, d, e, g, and h). These changes in follicle competence to hormonal stimulation, present in 1.3- and 1.4-mm follicles but absent in 1.5-mm follicles, suggest that once follicles are recruited into maturation they are irreversibly committed to subsequently ovulate. In contrast, oocyte sensitivity to gonadotropin and MIS in 1.3- and 1.4-mm follicles appears to be differentially regulated during the follicular cycle by mechanisms that have yet to be defined but which serve to control follicular recruitment into maturation and ovulation with a semilunar periodicity characteristic of F. heteroclitus. Comparable findings have been recently reported for the amphibian B. viridis, in which the oocyte sensitivity to 5-HT inhibition of progesterone-induced GVBD exhibits seasonal variations related to annual reproductive activity [28].

When the effectiveness of 5-HT inhibition of 17,20ßP-induced oocyte maturation was analyzed across the follicular cycle, it was found that the percentage of GVBD inhibition by 5-HT was most pronounced in 1.3- and 1.4-mm follicles during postspawning, which coincided with the low responsiveness of these follicles to FPE and 17,20ßP (Fig. 5, g and h). For 1.5-mm follicles, however, the percentage of GVBD inhibition by 5-HT was lower than in 1.3- and 1.4-mm follicles and remained similar throughout the follicular cycle. In teleosts as in mammals, the switch in follicle physiology from a prematurational stage (leading to the acquisition of maturational competence) to maturation has led to the suggestion that the pituitary releases an FSH-like gonadotropin first and an LH-like gonadotropin second (e.g., [48]). Therefore, a gonadotropic regulation of 5-HT action during the follicular cycle may be possible. We tested in vitro to see if such regulation of 5-HT inhibitory action existed, either directly by gonadotropin itself, or indirectly through the production of non-maturation-inducing steroids such as E2, but we found that hCG or E2 preparations (even at higher levels than those produced by gonadotropin-stimulated follicles) had no effect on the inhibition of steroid-induced GVBD by 5-HT (Fig. 7 and Table 1). Further, prolonged but not brief treatment by hCG was required to counteract subsequent 5-HT inhibition (Fig. 7), suggesting that only the appearance of 17,20ßP, and possibly other maturation-inducing steroids synthesized by the follicles after hCG stimulation [49], were able to down-regulate the 5-HT inhibition.

Taken together, these findings suggest that the function of oocyte-associated 5-HT receptors in F. heteroclitus are not likely to be under direct gonadotropic regulation in vivo. Rather, inhibitory activity by 5-HT may be related to the self-generated acquisition of competence by the oocyte to undergo GVBD as a response to MIS stimulation, and once the response is generated beyond a cAMP-dependent step, 5-HT can no longer inhibit the process. Although an increase in the population or affinity of 5-HT receptors in vitellogenic follicles during postspawning cannot be ruled out at this point, the observation that the effectiveness of 5-HT at inhibiting oocyte maturation increased in 1.3- and 1.4-mm follicles at postspawning, coincident with their lower responsiveness to MIS stimulation by undergoing oocyte maturation, is consistent with our hypotheses. However, the sensitivity of follicle-enclosed oocytes to gonadotropin also appeared to be differentially regulated throughout the follicular cycle (Fig. 5, d and e). These latter changes may involve fluctuations in the competence of granulosa cells to release steroids in response to gonadotropic stimulation in addition to variations in the oocyte maturational competence to respond to MIS. Therefore, the intrafollicular concentration of 17,20ßP and other maturation-inducing steroids released by the granulosa cells during the gonadotropin-triggered recruitment of follicles into maturation may be another mechanism that regulates the 5-HT inhibitory action in vivo during the follicular cycle.

Serotonin has been found in rat and human follicular fluid, and its concentration seems to change during the ovulatory cycle [6–8]. In F. heteroclitus, however, preliminary efforts to identify 5-HT in ovarian preparations have been unsuccessful thus far (data not shown), although a dramatic depletion of pituitary 5-HT during the spawning and postspawning periods has been observed in reproductively cycling females [50]. Therefore, even though this study shows differences in the oocyte sensitivity to 5-HT (which do not necessarily involve fluctuations in the levels of 5-HT within the ovary), it is clear that the demonstration of ovarian 5-HT in F. heteroclitus will be essential to support the role of this monoamine in the cyclic spawning scenario of this species.

In conclusion, the present study indicates that the 5-HT inhibition of MIS-induced oocyte maturation in the teleost F. heteroclitus is differentially regulated throughout the follicular cycle. Inhibitory action by 5-HT was not regulated by gonadotropin directly or through the gonadotropin-stimulated release by the granulosa cells of non-maturation-inducing steroids, such as E2. Rather, it appeared to be related to cyclic changes in the competence of oocytes to respond to MIS. Further studies will be required to understand the cellular and hormonal mechanisms involved in the regulation of follicle responsiveness to gonadotropin, MIS, and 5-HT, and thus to elucidate the specific role(s) that 5-HT may play during follicular recruitment into maturation and ovulation in F heteroclitus as in other lower vertebrates.


   FOOTNOTES
 
1 Financial support was provided by a grant from ORTGE at UF awarded to K.S. and by an NSF grant IBN-93036123 awarded to R.A.W. Participation of J.C. was financed by a postdoctoral fellowship from the Ministry of Education and Science (Spain). Back

2 Correspondence and current address: Division for Cell Biology (0110), German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. FAX: 49-6221-423404;j.cerda{at}dkfz-heidelberg.de Back

3 Permanent address: Department of Pharmaceutical Sciences, Nagpur University Campus, Nagpur 440010, India. Back

Accepted: December 19, 1997.

Received: February 20, 1998.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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