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Ovary |
Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activin, follicle, follistatin, oocyte development, ovary, puberty, zebrafish
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the ovary of mammals and fish, activin ßA and ßB have been localized in the follicle cells, particularly the granulosa cells, by in situ hybridization, immunohistochemistry, or reverse-transcription polymerase chain reaction (RT-PCR) [9–16]. In mammals, treatment with activin in vitro not only stimulates follicular growth [17] and granulosa cell proliferation [18], but also increases the production of FSH receptor and FSH-induced luteinizing hormone (LH) receptor in cultured granulosa cells [19–21]. Moreover, activin also regulates ovarian steroidogenesis including stimulation of estradiol production via enhancing aromatase activities in the granulosa cells [22–24] and modulation of androgen production in the theca cells [25]. In addition, activin has also been demonstrated to stimulate oocyte maturation in several species, including zebrafish [26–32]. Interestingly, follistatin is also expressed predominantly in the granulosa cells of mammalian ovarian follicles [11, 33, 34]. As a binding protein of activin, follistatin antagonizes most of the actions of activin in the ovary [2].
Although activin subunits and follistatin are coexpressed in the ovarian granulosa cells, they seem to have specific temporal expression profiles during ovarian follicle development in mammals. Activin ßB appears to be expressed abundantly in the granulosa cells of small antral follicles, and the expression diminishes in later large dominant follicles, whereas the expression of activin ßA is abundant in the dominant follicles [9, 11]. Follistatin mRNA has been demonstrated in the granulosa cells of the secondary and antral follicles, but its level decreases in the preovulatory follicles [34–36]. In the chicken ovary, the temporal expression patterns of activin and follistatin have also been characterized during follicle development. Activin ßB is predominantly expressed in the follicles of early stages, whereas activin ßA expression is abundant at the late stage before ovulation [37–40].
We have previously demonstrated that both activin subunits (ßA and ßB) and follistatin are expressed in the zebrafish ovary, and that the ovarian activin-follistatin system may play an important role in mediating the effects of gonadotropin(s) on zebrafish oocyte maturation and maturational competence [30, 32, 41, 42]. This is supported by the evidence that the expression of activin and follistatin is regulated by gonadotropin(s) in cultured zebrafish follicle cells. Gonadotropin(s) stimulates activin ßA and follistatin expression but suppresses ßB expression through different signal transduction pathways. The stimulation of activin ßA and follistatin by human chorionic gonadotropin (hCG) is mediated by the cAMP-PKA pathway in cultured zebrafish follicle cells, whereas hCG inhibition of activin ßB expression is signaled through a cAMP-dependent but PKA-independent pathway [41, 43, 44]. The differential expression of activin ßA and ßB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during the development of the ovary and ovarian follicles. Studies on this issue will not only offer support for the differential regulation of activin subunits by gonadotropin(s), but also promise to provide clues to the physiological roles played by different forms of activin as well as follistatin in the ovary.
Three experiments were conducted in the present study. Using sexually immature zebrafish, we first examined the temporal patterns of activin (ßA and ßB) and follistatin expression at the ovary level during sexual maturation, which was characterized by the recruitment and vitellogenic growth of the first cohort of developing follicles. Using sexually mature gravid zebrafish, we further investigated the stage-dependent expression of activin ßA, ßB, and follistatin at the follicle level. Since the zebrafish is a daily spawner, the third experiment was performed to investigate the expression profiles of these proteins during the daily ovulatory cycle, with particular emphasis on the pre- and postovulatory stages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexually mature or immature zebrafish, Danio rerio, were purchased from local pet stores and maintained in flow-through aquaria (36 L) at 25°C on a 14L:10D photoperiod with the light on at 0800 h and off at 2200 h. The fish were fed twice per day with the commercial tropical fish food with a supplement of live brine shrimp once or twice per week. All experiments were performed under license from the government of the Hong Kong SAR and endorsed by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong.
Sampling of Ovaries or Follicles and RNA Isolation
To examine activin and follistatin expression at the ovary level during sexual maturation, we performed two independent experiments using about 200 sexually immature zebrafish in each experiment. Since we were not sure about the exact age of fish after purchasing them from the local suppliers, we first examined the stage of ovarian development by histological observation to ensure no signs of vitellogenesis in the ovary. The day of the first sampling was designated Day 0, and afterward the sampling was performed on Days 3, 6, and 10. At each time point, six female zebrafish were sampled and both ovaries of each fish quickly removed. One of the ovaries was fixed with Bouin solution for histological sectioning and microscopic observation of follicle development, and the other one was subjected to total RNA extraction with Tri-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol.
In the second experiment studying the expression profiles of activin and follistatin at the follicle level, the ovaries were removed from 20 sexually mature gravid zebrafish after decapitation and placed in a 90-mm culture dish containing 60% medium Leibovitz L-15 (GIBCO Invitrogen, Carlsbad, CA). The follicles of different stages were manually separated and divided into five groups based on their size and vitellogenic state [45]: primary growth stage (PG, 
0.15 mm); previtellogenic or cortical alveolus stage (PV, 0.25 mm); early vitellogenic stage (EV, 0.35 mm); midvitellogenic stage (MV, 0.45 mm); and full-grown immature stage (FG, 0.65 mm). The isolated healthy follicles were homogenized in Tri-Reagent to extract total RNA.
To investigate the temporal expression of activin and follistatin during the daily ovulatory cycle, 60 sexually mature female zebrafish and 60 males of similar body size were selected and randomly divided into six groups for sampling. Under our aquarium conditions, the mature gravid fish normally spawn within 1 h after light on; therefore, the fish were sampled at different times (1800, 2200, 0100, 0400, 0700, and 1200 h) to cover pre- and postovulatory periods. Six to 10 females were sampled at each time point. Similarly, one of the ovaries from each fish was fixed for histological observation and the other one was used for RNA extraction.
Follicle Incubation
The isolation and incubation of full-grown follicles were carried out according to our previous report [30]. Briefly, the ovaries were removed from 15 to 20 female zebrafish and placed in a dish containing 60% medium Leibovitz L-15. The full-grown immature follicles (0.65 mm) were isolated and incubated in 24-well plates (30–40 follicles per well in 1 ml medium). After 6 or 16 h incubation at 28°C, the follicles were examined microscopically for germinal vesicle breakdown (GVBD), a visible morphological marker for oocyte maturation.
Validation of Semiquantitative RT-PCR Assays for Activin ßA, ßB, Follistatin, and ß-Actin
Reverse transcription (RT) was performed at 42°C for 2 h in a total volume of 10 µl consisting of 3 µg total RNA, 1x Single Strand Buffer (GIBCO Invitrogen), 10 mM dithiothreitol, 0.5 mM each dNTP, 0.5 µg oligo-dT, and 100 U SuperScript II (GIBCO Invitrogen). The semiquantitative PCR assays were optimized according to our previous reports except that the total RNA used for optimization was isolated from the whole ovary instead of the cultured follicle cells [41, 44]. The cycle numbers optimized were 33 cycles for activin ßA, ßB, and follistatin (Fig. 1), and 21 cycles for ß-actin (data not shown). The primers used for PCR amplification are listed in Table 1.
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Data Analysis
The mRNA level of each gene in each sample was first calculated as the ratio to that of ß-actin, which was amplified as the internal control and then expressed as the percentage of the control group. The data were analyzed by Student t-test or one-way ANOVA followed by Dunnett test using GraphPad Prism 4.0 for Macintosh OS X (GraphPad Software, San Diego, CA). We performed each experiment at least twice using different batches of animals to confirm the results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the expression patterns of activin ßA, ßB, and follistatin in vivo, we first examined their temporal expression profiles at the ovary level during sexual maturation from prepubertal to postpubertal stage. Histological examination showed that the ovarian development was highly synchronous among the fish used. On Day 0 of sampling, all ovaries collected contained oocytes at PG stage with no signs of vitellogenic activity, a clear indication of sexual immaturity. On Day 3, the first cohort of follicles had grown significantly and accumulated a substantial amount of cortical vesicles (cortical alveoli) in the oocytes, marking the start of vitellogenesis; however, no yolk granules were observed in any oocytes at this stage (PV stage). A substantial amount of yolk granules appeared around the germinal vesicle (GV) in the first cohort of vitellogenic oocytes on Day 6 (MV stage). On Day 10, the follicles had grown to submaximal size or late vitellogenic (LV) stage with yolk granules occupying most of the space and GV located in the middle of the oocytes (Fig. 2).
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During this 10-day period of ovarian development that covers the onset of puberty and much of vitellogenesis of the first cohort of developing follicles, we observed phenomenal changes in activin and follistatin expression, particularly activin ßA (Fig. 2). The expression of activin ßA was extremely low on Day 0 before puberty when all of the follicles in the ovary were at the PG stage. A significant increase in activin ßA expression was noticed on Day 3 when the first cohort of follicles had been recruited and started to produce and accumulate cortical vesicles, representing the beginning of vitellogenesis. The expression of activin ßA continued to increase with vitellogenesis and reached the maximal level on Day 6, which was characterized by the accumulation of yolk granules. The high expression level was maintained through Day 10 or late vitellogenic stage. In comparison, the expression of follistatin was easy to detect at all stages of ovarian development, and its level also steadily went up during the development, although the increase was not as dramatic as that of activin ßA. In contrast to activin ßA, the expression of activin ßB could be easily detected at all stages, but it showed little variation during the development from the PG to the LV stage.
Stage-dependent Expression of Activin ßA, ßB, and Follistatin in the Ovarian Follicles of Mature Gravid Zebrafish
The experiment described above strongly implicated activin ßA and follistatin, but not activin ßB, in the initiation of follicle recruitment at the onset of puberty and subsequent vitellogenic growth. However, since the expression of these proteins was analyzed at the organ level, it remained unknown whether the increased activin ßA and follistatin expression was associated with the development of the first cohort of recruited follicles. To address this issue, we further examined the stage-dependent expression of activin ßA, ßB, and follistatin at the follicle level in sexually mature zebrafish.
The expression of activin ßA was very low in the follicles of the PG stage (0.15 mm), in contrast to that of activin ßB, which could be easily detected at the same stage. However, during follicle growth or vitellogenesis, the expression of activin ßA increased dramatically, and it reached the maximal level in the midvitellogenic follicles (0.45 mm) followed by a slight but significant drop in the full-grown follicles (0.65 mm; Fig. 3).
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In comparison with activin ßA, the mRNA of activin ßB could be easily detected at all stages examined, and its levels remained more or less constant during vitellogenesis. When normalized to ß-actin, the expression of activin ßB showed a slight but significant increase in the late stages compared with the PG stage. It should be noted that this increase is largely due to the relatively higher expression level of ß-actin at the PG stage. However, we did notice that activin ßB expression in the full-grown follicles tended to increase in vitro during the procedure of follicle isolation, especially when the procedure lasted for a long time (data not shown).
Similar to activin ßA, the expression of follistatin also increased significantly from the PG to the MV stage. However, its expression showed a sharp decline at the FG stage (Fig. 3).
Expression of Activin ßA, ßB, and Follistatin During Daily Ovulatory Cycle
To investigate if activin subunits and follistatin in the ovary are also differentially expressed in short-term processes, we performed the present experiment to study the temporal expression profiles of these molecules during the daily ovulatory cycle, with emphasis on the periods before and after natural oocyte maturation and ovulation.
Histological observation showed that at 0100 h, the GV in most full-grown follicles had started to migrate toward the periphery of the oocytes. At 0400 h, the migration had finished and the GV was located at the periphery of oocytes in most full-grown maturing follicles. GVBD was observed histologically in most fish sampled at 0700 h, and this event could also be easily seen while dissecting the fish for the ovary as the mature follicles turned translucent. The ovarian samples collected at 1200 h were at postovulatory stage containing empty follicles with follicle layers only (Fig. 4).
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Analysis of mRNA expression again revealed differential expression of activin ßA and ßB during this period of time. Similar to the previous observations, the expression of activin ßA and follistatin steadily increased during GV migration from 1800 h onward and peaked around 0400 h when the GV had reached the periphery of the oocytes. Their expression slightly declined from 0400 through 1200 h. Meanwhile, the expression of activin ßB exhibited a trend of decline from 1800 through 0400 h, albeit statistically insignificant. In sharp contrast to activin ßA and follistatin, activin ßB expression showed a phenomenal surge at 0700 h, the time when GVBD and possibly ovulation happened (Fig. 4). These results were highly reproducible in two independent experiments.
Further Evidence for Activin ßB Involvement in the Final Stage of Oocyte Maturation
The dramatic increase in activin ßB expression at 0700 h strongly implicated activin ßB but not ßA in GVBD, the late stage of oocyte maturation, and/or ovulation. At 0700 h, we sampled ovaries from 15 zebrafish in two separate experiments. Four of the 15 fishes sampled did not show any signs of GVBD or ovulation in their ovaries, whereas 11 fishes had mature or ovulated oocytes. These were confirmed by histological examination of the ovaries from all sampled individuals. Interestingly, in the ovaries of the four fishes without GVBD, the GV in all of the full-grown follicles had migrated to the periphery of the oocytes, suggesting that they had been undergoing maturation. To ascertain that the surge of activin ßB expression at 0700 h is related to GVBD, we reanalyzed the data from the fishes with or without GVBD separately. As shown in Figure 5, there was no difference in activin ßA and follistatin expression between the two groups of individuals. In contrast, the expression levels of activin ßB in the fishes with GVBD were significantly higher than those without GVBD (Fig. 5).
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To substantiate the correlation between increased activin ßB expression and GVBD, we performed another experiment. Full-grown follicles were isolated from sexually mature spawning zebrafish and incubated for 6 h without any treatment. The follicles were scored for GVBD at the end of incubation, and total RNA was extracted from the immature follicles with no signs of GVBD and those that had undergone spontaneous maturation and completed GVBD. Analysis of mRNA expression showed that both activin ßA and follistatin declined their expression when the follicles underwent spontaneous maturation characterized by GVBD; in sharp contrast, the expression of activin ßB was significantly higher in the mature follicles (Fig. 6), consistent with our in vivo finding at 0700 h of the ovulatory cycle at the organ level (Figs. 4 and 5).
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Blockade of Spontaneous Oocyte Maturation In Vitro by Follistatin
Our previous study demonstrated that activin promoted zebrafish oocyte maturation in vitro [30]. The significant increase in activin ßB expression and the concurrent decrease of follistatin during spontaneous oocyte maturation in vitro (Fig. 6) strongly implicated activin in the event. To test this idea, we examined the effect of recombinant human follistatin on the rate of spontaneous oocyte maturation in vitro. Administration of follistatin significantly suppressed the level of spontaneous maturation in a clear dose-dependent manner after a 16-h incubation (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the human and primate ovaries, activin ßB expression is predominant in the granulosa cells of small antral follicles and decreases to a nondetectable level in the dominant follicles, whereas the expression of activin ßA reaches the highest level in the dominant and preovulatory follicles [9, 11]. Similar patterns of activin ßA and ßB expression have also been reported in the chicken ovary. Activin ßB is expressed predominantly in the nonhierarchy small yellow follicles, but the expression decreases to nondetectable levels in the granulose cells of the large preovulatory follicles of higher order (F1–F4). In contrast, activin ßA is abundantly expressed in the granulosa cells of preovulatory follicles [37–40, 46]. As for follistatin expression, its expression has been reported to be high in the granulosa cells of the growing secondary and tertiary follicles followed by a decrease in the mature preovulatory follicles in mammals [34, 35]. Similarly, follistatin in chickens is highly expressed in the small yellow and large white follicles, but the expression dramatically decreases in the granulosa cells of the hierarchy preovulatory follicles [39]. These findings together with our observations in the zebrafish suggest a conserved scheme of activin-follistatin expression and regulation, and possibly function as well, in vertebrate ovaries, with variation among different organisms with different modes of reproduction.
The significant increase of activin ßA but not ßB during the period of zebrafish ovarian growth strongly correlates activin ßA with the process of follicle growth or vitellogenesis, and its activity is likely modulated by follistatin, whose expression showed a concurrent increase. This is further supported by the expression levels of these molecules at the follicle level in adult gravid zebrafish. Activin ßA expression dramatically increased in the vitellogenic follicles as compared with that at the PG stage; once again, its increase was not accompanied by activin ßB, which maintained relatively constant expression levels in the follicles from the PG to the FG stage. Although the regulatory mechanisms for the increased expression of activin ßA and follistatin are not known, pituitary gonadotropin(s) is likely one of the factors involved because the expression of both proteins is stimulated by hCG and goldfish pituitary extract in vitro through a cAMP-PKA pathway [41, 44]. What is interesting to address in the future is the physiological relevance of the increased activin ßA expression during zebrafish follicle growth. In mammals, activin has been demonstrated to promote the formation of follicle-like structures in the presence of FSH and the growth of early follicles [17, 18, 47], and it stimulates FSH receptor biosynthesis and aromatase activities [19, 22–24].
To provide in vivo evidence for the involvement of activin ßA and ßB in short-term events such as oocyte maturation and ovulation, we carried out an experiment to examine the expression of activin ßA, ßB, and follistatin in the ovary during the ovulatory cycle with emphasis on the periovulatory period involving oocyte maturation and ovulation. It is generally believed that oocyte maturation in many fishes is marked by the onset of GV migration from its central or eccentric location toward the periphery (animal pole) of the oocyte, which is followed by GVBD and ovulation [48]. In the present study, we found that with the migration of GV initiated, the expression of activin ßA and follistatin increased steadily and their expression levels peaked at the time when the GV reached the periphery. Meanwhile, the expression of activin ßB exhibited a trend of decline, although it was not statistically significant. Considering that gonadotropin(s) is the key factor that initiates oocyte maturation and ovulation in vertebrates including fish, and it stimulates activin ßA and follistatin but suppresses activin ßB expression in cultured zebrafish ovarian follicle cells, we hypothesize that the preovulatory gonadotropin surge could be responsible for the differential patterns of activin ßA and ßB as well as follistatin expression during the periovulatory period, although we do not know the exact time of the surge secretion in the zebrafish. In the goldfish maintained on a 16L:8D photoperiod with light off from 2000 to 400 h, the plasma gonadotropin concentration reaches the maximal level at 2000 h and maintains a high level through 0400 h when ovulation happens [49].
Since gonadotropin(s) promotes oocyte maturation and enhances oocyte maturational competence (the responsiveness to maturation-inducing hormone 17
, 20ß-dihydroxy-4-pregnen-3-one) in the zebrafish and these effects can be blocked by follistatin [32], we have hypothesized that gonadotropin(s) probably acts in the ovary by activating the ovarian activin-follistatin system. Evidence from the present study implies that activin ßA but not ßB is likely one of the ovarian factors activated by gonadotropin(s) to promote oocyte maturational competence and final maturation. This is supported by the in vitro findings that hCG and goldfish pituitary extract stimulate zebrafish activin ßA but inhibit ßB expression in the follicle cells [41] and an injection of hCG into the adult zebrafish increases the production of activin A protein [31]. In the rat, a similar bell-shaped expression profile has also been reported for activin ßA during the gonadotropin surge [50, 51]. As for activin ßB, it does not seem to participate directly in the early stage of maturation involving acquisition of oocyte maturational competence and GV migration as suggested by its constant or even lower expression level during this period. However, the expression of activin ßB surged dramatically at the time when the GVBD and possibly ovulation happened. When its expression levels in the fishes that showed obvious GVBD or ovulation at 0700 h were compared with those in the fishes that failed to show GVBD at the time, the correlation between the two events became even clearer. The expression levels of activin ßB in the ovulating fishes were significantly higher than those of nonovulating ones, which showed no difference from those of preovulatory stages. Interestingly, when the ovaries from the nonovulating fishes collected at 0700 h were examined by histology, the full-grown follicles in all individuals had finished GV migration with the nuclei remaining intact at the periphery of the oocytes. This suggests that the full-grown follicles in these nonovulating fishes had embarked on the course of oocyte maturation and undergone the early stage of the process, but failed to finish the final event of GVBD and ovulation at the time of sampling, and this failure was strongly correlated with the lack of activin ßB surge.
The phenomenal surge of activin ßB expression at the time of GVBD or ovulation in vivo immediately prompted us to think of such a correlation in vitro when the isolated full-grown follicles are going through spontaneous maturation. We therefore did an experiment in which we incubated the full-grown zebrafish follicles without any treatment for 6 h and then analyzed the expression of activin ßA, ßB, and follistatin in the follicles with or without GVBD. Consistent with the in vivo data, activin ßB expression increased significantly in the follicles showing GVBD; however, the expression of activin ßA in these follicles was significantly lower than that in the immature ones. Interestingly, there was a significant drop in follistatin expression in the mature follicles, which obviously would enhance the activin activity in these follicles. The idea that there is an increased activin activity in the maturing follicles, probably due to the increased activin ßB expression and decreased follistatin expression, is substantiated by the evidence that recombinant human follistatin dose-dependently suppressed the rate of spontaneous maturation of zebrafish oocytes in vitro. As demonstrated both in vivo and in vitro, the correlation between the final stage of oocyte maturation (i.e., GVBD and ovulation) and a surged activin ßB expression is strong; however, the cause-effect relationship between the two events remains to be further elucidated. This finding has brought up a few interesting questions to address in the future. What causes the increase in activin ßB expression at the final stage of oocyte maturation, the endocrine hormones such as gonadotropins or local factors? What exactly does activin ßB do at this stage? In what form does activin ßB function, as activin B (ßBßB), activin AB (ßAßB), or even the putative inhibin B (ßB)? Since the full-grown follicles undergo spontaneous maturation after being isolated from the ovary, one possibility for the increased activin ßB expression would be the removal of gonadotropin inhibition on the subunit, which we have demonstrated in vitro [41]. This could also be the cause for the decreased activin ßA and follistatin expression in the mature follicles because the expression of both proteins has been demonstrated to be stimulated by hCG and goldfish pituitary extract in vitro [41, 44]. Is this also the cause for increased activin ßB expression in vivo at the time of GVBD/ovulation? We do not have any clues for this question yet, and the answer would have to rely on the availability of information on gonadotropin profiles in vivo.
In summary, we have characterized the temporal expression patterns of activin ßA, ßB, and follistatin in the zebrafish ovary. The expression of activin ßA increased dramatically with the vitellogenic growth of oocytes at both the ovary and follicle levels. Follistatin also increased its expression together with activin ßA. In comparison, the expression of activin ßB seemed to be more or less constant during vitellogenesis. During the periovulatory period in the adult gravid zebrafish, activin ßA and follistatin both went up during the early phase of oocyte maturation marked by GV migration, whereas the expression of activin ßB remained unchanged or even lower in the same period. However, a surge increase in activin ßB expression was observed at the final stage of oocyte maturation marked by GVBD and ovulation, and this was not accompanied by activin ßA. The implication of activin ßB in the final stage of oocyte maturation was further confirmed by the results from an in vitro experiment using full-grown follicles that underwent spontaneous maturation. The present study not only provides evidence for differential expression of activin subunits and follistatin in the ovary, but also offers important clues to the functions of these proteins during the ovary and follicle development. However, since the present study only examined the expression profiles of these molecules at the mRNA level, more studies at the protein level will be essential for better understanding this important family of growth factors in the ovary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Wei Ge, Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. FAX: 852 2603 5646; weige{at}cuhk.edu.hk
Received: 29 May 2004.
First decision: 16 June 2004.
Accepted: 3 August 2004.
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  E. S. Clelland, Q. Tan, A. Balofsky, R. Lacivita, and C. Peng Inhibition of Premature Oocyte Maturation: A Role for Bone Morphogenetic Protein 15 in Zebrafish Ovarian Follicles Endocrinology, November 1, 2007; 148(11): 5451 - 5458. [Abstract] [Full Text] [PDF]
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Y. Wang, J. Li, C. Y. Wang, A. H. Y. Kwok, and F. C. Leung Identification of the Endogenous Ligands for Chicken Growth Hormone-Releasing Hormone (GHRH) Receptor: Evidence for a Separate Gene Encoding GHRH in Submammalian Vertebrates Endocrinology, May 1, 2007; 148(5): 2405 - 2416. [Abstract] [Full Text] [PDF]
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 A. H. Y. Kwok, Y. Wang, C. Y. Wang, and F. C. Leung Cloning of Chicken Glucocorticoid Receptor (GR) and Characterization of its Expression in Pituitary and Extrapituitary Tissues Poult. Sci., February 1, 2007; 86(2): 423 - 430. [Abstract] [Full Text] [PDF]
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 W.-K. So, H.-F. Kwok, and W. Ge Zebrafish Gonadotropins and Their Receptors: II. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Subunits--Their Spatial-Temporal Expression Patterns and Receptor Specificity Biol Reprod, June 1, 2005; 72(6): 1382 - 1396. [Abstract] [Full Text] [PDF]
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H.-F. Kwok, W.-K. So, Y. Wang, and W. Ge Zebrafish Gonadotropins and Their Receptors: I. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Receptors-- Evidence for Their Distinct Functions in Follicle Development Biol Reprod, June 1, 2005; 72(6): 1370 - 1381. [Abstract] [Full Text] [PDF]
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