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Laboratory of Fish Reproductive Physiology, Faculty of Agriculture,3 Ehime University, Ehime 790-8566, Japan
Aomori Prefecture Fisheries Research Center, Inland Water Fisheries Institute,4 Aomori 034-0041, Japan
ABSTRACT
Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.
17
, 20ß-dihydroxy-4-pregnen-3-one, estradiol, estradiol-17ß, first meiotic division, gametogenesis, in vitro culture, meiosis, ovary, progesterone, Spo11
During early oogenesis, oogonia proliferate by mitosis and subsequently develop into primary oocytes that have initiated meiosis. In general, during oogenesis, primary oocytes arrest division at the diplotene stage in the prophase of the first meiotic division and accumulate yolk during meiotic arrest. Thereafter, oocytes resume the first meiotic division and differentiate into mature eggs through final maturation. However, it is very difficult to analyze the endocrine control of early oogenesis, since in most organisms oocytes attain the prophase of the first meiotic division at a very early stage of embryogenesis. For a detailed analysis of early oogenesis, it is therefore important to select an animal model in which oogenesis is initiated at a comparatively late developmental stage. Japanese huchen (Hucho perryi) belongs to the family Salmonidae. Although this species used to occur in the northern part of Honshu, Japan's main island, it currently inhabits only the northern island Hokkaido. Since females take 6–8 yr to mature and the 1-yr-old juvenile females contain many oogonia, this species is considered to be a suitable experimental model for studying early oogenesis. There is little information on early oogenesis in Japanese huchen, it is not known when meiosis is initiated, and females are not easy to obtain. On the other hand, common carp (Cyprinus carpio) females are very common, the primordial germ cells began to proliferate at 7–9 wk postfertilization, and male and female gonads can be clearly distinguished histologically from 11 wk onwards. Oogenesis, including meiosis, starts at 16 wk, a few weeks before the onset of spermatogenesis at 20 wk postfertilization [1]. Using gonads with progressed oocytes, we were able to develop a new culture method for oogonia. Using these experimental models, we investigated whether sex steroid hormones are involved in early oogenesis.
In fish, differentiation of primary oocytes into maturing oocytes that resume meiosis requires several steroid hormones. As in other vertebrates, oogenesis is primarily regulated by pituitary gonadotropins (GTHs) and ovarian endocrine factors, including estrogens and progestins. The roles of estrogens and progestins have been elucidated largely through correlating seasonal changes in circulating hormone levels with the different stages of the annual ovarian cycle in a variety of fish species [2–5]. However, it is not clear how the progression of germ cells through different stages of oogenesis is regulated.
Estradiol-17ß (E2), which is a major estrogen in female vertebrates, including fish, stimulates the liver of fish to synthesize vitellogenin, a yolk protein precursor [6–9]. This steroid also induces the proliferation of isolated rat fetal gonocytes [10], primary spermatogonia in the frog Rana esculenta [11, 12], spermatogonial stem cell renewal in Japanese eel [13, 14] and DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation, especially during the mitotic phase, in male Japanese huchen [15]. The compound 17,20ß-dihydroxy-4-pregnen-3-one (DHP) is the meiotic maturation-inducing steroid of salmonids and a number of other teleosts [16], and its secretion by the gonads is regulated by GTHs in both sexes [17–19]. DHP also plays an important role in the acquisition of sperm motility in several teleost fishes [20–22]. Recently, we have shown that DHP is an essential factor for the initiation of meiosis in spermatogenetic cells of Japanese eel [23]. In the present in vitro study, we tested the hypothesis that the control mechanisms of early oogenesis are similar to those that regulate the early stages of spermatogenesis, and involve E2 and DHP.
Immature female huchen (April to May, 2002; 11–12 mo posthatch) with total body lengths that ranged from 10 to 15 cm were used in the in vitro experiments, and immature female huchen (11–13 mo and 24 mo posthatch) were used in the in vivo experiments. The fish were obtained from the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University. Juvenile common carp (1-yr-old) with total body lengths that ranged from 10 to 15 cm were obtained from a commercial supplier (Masai Yorijyo, Awaji City). The fish were kept in a circulating fresh water tank at 25°C for 1 day before culture. The experiments were conducted in accordance with the institutional animal ethics guidelines of Ehime University.
Light Microscopic Observations
Ovaries of huchen were excised from 1-yr-old fish (in April to June, 2002) and 2-yr-old fish (in April to May), and fixed in Bouin solution. The ovaries were dehydrated with ethanol and acetone, and embedded in Historesin Plus (Leica, Nussloch, Germany). Carp ovaries were fixed in Bouin solution and embedded in paraffin wax. Sections were cut at 5-µm thickness and stained with Delafield hematoxylin and eosin.
Assays of Serum Steroid Hormones
The serum levels of DHP and E2 in 1-yr-old (in April, May, and June) and 2-yr-old fish were measured by time-resolved fluoroimmunoassays (TR-FIA) according to the methods of Yamada et al. [24].
Ovarian Organ and Epithelium Culturing
The organ culture technique employed was the floating method described by Miura et al. [25, 26], with some modifications. Ovaries of immature female huchen were excised and cut into 1 x 1 x 0.5-mm fragments. For each treatment, two ovarian fragments from each fish were placed on 2% agarose gel (cylinder of diameter 1 cm) covered with a nitrocellulose membrane (5 x 5 x 5 mm) in 24-well plastic culture dishes. Each culture well contained 1 ml of culture medium.
The ovarian epithelium culture technique was modified from a pellet culture technique developed to study spermatogenesis [27]. To remove previtellogenic and vitellogenic oocytes, ovarian fragments of carp were excised and treated with 0.2% collagenase (236 U/mg; WAKO, Osaka, Japan) and 0.6 U dispase (Boehringer Mannheim, Mannheim, Germany). After shaking for 2 h, the fragments were placed on a stainless steel mesh (pore size of 75 µm), and the pieces were rubbed through the mesh with flat forceps. The residual ovarian fragments retained by the mesh were precultured for 1 mo on 2% agarose gel covered with a nitrocellulose membrane in 6-well plastic culture dishes. For both culture methods, the basal culture medium consisted of Leiboviz-15 (L-15) medium that was supplemented with 0.1 mM glutamic acid, 1.7 mM L-proline, 0.5% (w/v) BSA fraction V (Sigma Chemical Co., St. Louis, MO), 1 mg/L bovine insulin (Sigma), and 10 mM Hepes, adjusted to pH 7.4 with 1 M NaOH. Ovarian explants were cultured in media with or without 0.01, 0.1, or 1 ng/ml E2, 0.1, 1, or 10 ng/ml DHP, 100 µg/ml fadrozole with or without 0.1 ng/ml E2, and 1 ng/ml of one of following progestins: 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S), 17-hydroxyprogesterone (17-OHP4), progesterone (P4), 17-hydroxypregnenolon (17-OHP5), and pregnenolone (P5) for 15 days in humidified air at 10°C for huchen and 25°C for carp. The media were changed weekly.
5-Bromo-2-deoxyuridine Labeling
5-Bromo-2-deoxyuridine (BrdU) is incorporated into replicating DNA, making detection of proliferating cells possible. Labeling with BrdU was carried out according to the manufacturer's instructions (Amersham Biosciences, England), with a minor modification. Ovarian fragments were incubated with BrdU (1 µl/well) for the last 12 h of culture. After culturing, the ovarian fragments were fixed in Bouin solution, and 5-µm-thick paraffin sections were cut and immunohistochemically stained with an anti-BrdU monoclonal antibody (1:1000 dilution). Sections were then counterstained with hematoxylin. Five random sections from the fixed fragments of each treatment for each animal were examined under a light microscope. The number of immunolabeled germ cells was counted and expressed as a percentage of the total number of germ cells (BrdU index; immunolabeled germ cells/total germ cells x 100). Histological observations showed that there were no differences in the ratios of germ cells at different stages of development.
Electron Microscopic Observations
For electron microscopy, cultured ovarian fragments were fixed overnight in 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h, and embedded in Epok 812 (Oken, Japan) using standard procedures. Ultrathin sections were stained with uranyl acetate and lead citrate. Five random sections from the fixed fragments of each treatment for each animal were examined under an electron microscope. The number of oocytes with synaptonemal complexes (SCs) was counted and expressed as a percentage of approximately 50 germ cells, excluding peri-nucleolus stage oocytes (percentage of oocyte with SC; oocytes with SC/total germ cells counted x 100).
Imunohistochemistry Using Anti-Eel Spo11 Antibody
Cultured carp fragments were fixed in Bouin solution, embedded in paraffin wax, and cut into 5-µm-thick serial sections. Immunohistochemical analysis of sections using an anti-eel Spo11 antibody (1:1000 in PBS), which also specifically detects carp Spo11, was performed using the Histofine SAB-AP kit (Nichirei, Tokyo). As a negative control, immunohistochemical analysis was performed using a 1:1000 dilution of normal rabbit serum in PBS.
The results were expressed as mean ± SEM. Differences between means were analyzed by one-way ANOVA, and if a significant difference was found (P < 0.05), the Bonferroni multiple-comparison test was performed. Significance was set at P < 0.05.
Histological Changes in Ovary and Serum DHP and E2 Levels at Several Stages of Ovarian Development in Japanese Huchen
Histological observations were made of the ovaries of 1-yr-old (both April and June) and 2-yr-old huchen (Fig. 1), along with measurements of the serum DHP and E2 levels at each stage (Fig. 2). Many oogonia were observed in the ovaries of 1-yr-old fish in April (Fig. 1A) and the serum DHP levels were low (Fig. 2A). By June, many chromatin-nucleolus stage oocytes were observed in the ovaries of 1-yr-old fish (Fig. 1B) and the serum DHP levels were significantly higher than those of the 1-yr-old fish in April and 2-yr-old fish (Fig. 2A). In the 2-yr-old fish, almost all the germ cells had peri-nucleolus stage oocytes (Fig. 1C). The serum E2 levels ranged from 0.38 to 2.0 ng/ml and did not change significantly during the experimental period (Fig. 2B).
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BrdU Incorporation into Ovarian Germ Cells of Japanese Huchen
The effects of E2 and DHP on the ovarian germ cells of Japanese huchen were assayed by the immunohistochemical detection of BrdU in ovarian organ cultures (Fig. 3, A–C). Numerous germ cells were labeled with BrdU after 15 days of culture of ovarian sections with E2 or DHP, compared with the controls, indicating that DNA was synthesized in the germ cells.
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For DHP, the highest BrdU indices were found in the 1 ng/ml DHP-treated fragments. The BrdU index of the ovarian fragments incubated with 0.1–1.0 ng/ml DHP was significantly higher than those of the initial, control, and 10 ng/ml DHP-treated fragments (Fig. 4). The BrdU index for the 0.1 ng/ml E2 treatment was significantly higher than those for the initial controls, controls, and other concentrations of E2. Fadrozole, which is an aromatase inhibitor, also inhibited DNA synthesis in ovarian germ cells, although this could be recovered by treatment with E2 (Fig. 5).
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Comparison of Percentages of Oocytes with SC Between E2- and DHP-Treated Ovarian Fragments of Japanese Huchen
Ultrastructural analysis of the E2- and DHP-treated fragments of huchen revealed that some germ cell nuclei contained SC characteristic of cells in meiosis (Fig. 3D). For the 1 ng/ml DHP-treated fragments, the percentage of cells that displayed SC was significantly higher than those for the initial controls, controls, and 0.1 ng/ml E2-treated fragments (Fig. 6).
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BrdU Incorporation into Ovarian Germ Cells of Common Carp Using the Ovarian Epithelium Culture Technique
Histological observations were made of the ovaries of 1-yr-old carp. In these ovaries, many peri-nucleolus stage oocytes and a few oogonia were observed (Fig. 7A). After removal of peri-nucleolus stage oocytes and culturing of the carp ovarian epithelium for 1 mo in basal medium, numerous oogonia were observed (Fig. 7B). After preculture, these fragments were treated for 15 days with E2 or DHP. BrdU was incorporated into numerous ovarian germ cells of E2- and DHP-treated ovarian fragments compared with the controls, indicating that DNA was synthesized in the germ cells. The BrdU indices for the 0.1 ng/ml E2 and 1 ng/ml DHP treatments were significantly higher than those for the initial control and control treatment (Fig. 8).
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Effects of Various Progestins on DNA Synthesis in Germ Cells
To investigate the effects of other progestins on early oogenesis, ovarian epithelium fragments of common carp were cultured with different progestins (20ß-S, 17-OHP4, P4, 17-OHP5, and P5) for 6 days, and BrdU incorporation into the germ cells was analyzed (Fig. 9). A significant increase in DNA replication occurred only when ovaries were cultured with DHP.
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Effects of DHP on Common Carp Ovarian Fragment Cultures
The effects of DHP on the ovarian germ cells of the common carp were evaluated in vitro using ovarian epithelium cultures. Many chromatin-nucleolus stage oocytes, which have larger nuclear size than control fragments, were observed in the DHP-treated fragments (Fig. 10, A and B). At the ultrastructural level, DHP-treated fragments displayed some germ cell nuclei that contained SC, which is characteristic of cells in meiosis (Fig. 10C). Furthermore, stronger signals were detected in the DHP-treated carp ovarian fragments that in the control or E2-treated fragments using the anti-eel Spo11 antibody (Fig. 11). The use of normal rabbit serum did not result in positive staining (data not shown).
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The control of early oogenesis, from the proliferation of oogonia to the initiation of meiosis, involves many factors [28]. Using hypophysectomy [29, 30], unilateral ovariectomy [31], and ovarian tissue culture, gonadotropins, especially FSH, have been implicated in the regulation of oogonia [32–35]. With regard to sex steroid hormones, E2, testosterone, and progesterone have been reported to be involved in the regulation of mitosis or meiosis [36, 37]. Furthermore, numerous growth factors, such as activin A, SCF, IGF, and retinoids, may also be involved in this process [38–40]. Thus, the control of early oogenesis involves many factors and its mechanisms seem rather complex. In the present study, using a newly developed, simple experimental system, we analyzed aspects of the endocrine control of early oogenesis, focusing on steroid hormones progestin and E2.
Histological observations of the early stages of oogenesis suggest that DHP is involved in the initiation of the first meiotic division of Japanese huchen oogonia. In 1-yr-old fish in April, the DHP levels were relatively low. At this time, oogonia appeared to proliferate. By June, the DHP levels had increased two-fold, coincident with the initiation of the first meiotic division, as indicated by the many chromatin-nucleolus stage oocytes found in the ovary. In 2-yr-old fish, it appeared that most of the germ cells had entered meiosis, since numerous peri-nucleolus stage oocytes were present. Moreover, the DHP levels were significantly lower in the 2-yr-old fish than in the 1-yr-old fish in June. In sockeye salmon (Oncorhynchus nerka), the serum levels of DHP have been shown to increase from about 1 ng/ml to 2 ng/ml [41] in immature females before the spawning season.
To understand the role of steroids in early oogenesis in two teleost fish species, huchen and carp, we determined whether E2 and DHP were able to induce oogonial proliferation or initiation of meiosis in vitro using an organ culture system. We found that 0.1 ng/ml E2 induced mitotic activity in the oogonia of both species. In huchen, treatment with fadrozole, which is an inhibitor of aromatase activity, inhibited DNA synthesis in ovarian germ cells. This result suggests that E2 is produced in the ovary during the early stage of oogenesis. Furthermore, the inhibitory effect of fadrozole was overcome by adding E2, which suggests that not only exogenous, but also endogenously synthesized E2 has direct effects on early oogenesis. Recently, 0.01 ng/ml of E2 has been shown to be sufficient to induce spermatogonial stem cell proliferation in Japanese eel testes maintained in vitro [13]. Similarly, in male Japanese huchen testes, 0.1–1 ng/ml E2 induced spermatogonial mitotic activity in vitro [15]. We did not find significant changes in the serum E2 levels during the experimental period. While the relationship between serum E2 levels and oogonial proliferation remains unclear, 0.38 ± 0.08 ng/ml of E2 in April may be enough to allow oogonial mitosis, which is ongoing in April. During sexual maturation, the plasma levels of E2 are approximately 20 ng/ml in female Atlantic salmon (Salmo salar) in the context of vitellogenesis [42], while in coho salmon (Oncorhynchus kisutch) changes occur at a much lower level of E2 during an early stage of oogenesis, when the serum levels of E2 increase from about 0.05 ng/ml to 0.2 ng/ml [43]; a similar trend was observed for female huchen in the present study. Thus, a low level of E2 may play an important role in gonial proliferation.
In the present study, DHP also induced DNA synthesis in ovarian germ cells. Using in vitro cultures, we found that a low concentration (1 ng/ml) of DHP was sufficient to promote DNA synthesis in ovarian germ cells. Since five other progestins were ineffective or had minimal effects, DHP appears to be the most effective progestin for inducing DNA synthesis in ovarian germ cells. However, as mentioned above, E2 also induced DNA synthesis in ovarian germ cells. What is the difference between DNA synthesis induced by DHP and E2? We have shown that DHP is an important steroid in the initiation of meiosis in spermatogenetic cells of the Japanese eel [23]. Although this steroid induces meiotic maturation of oocytes of salmonid fish and some other species [16], there is no information regarding the role of DHP at earlier stages of oogenesis. During oogonial proliferation and the initiation of meiosis, germ cells undergo DNA synthesis [44]. BrdU incorporation can be used to detect DNA synthesis stage but other techniques are needed to determine whether meiosis has been initiated. Therefore, we focused on two markers of meiosis, the occurrence of SC and the expression of Spo11. The SC is one of the most useful markers for the initiation of the first meiotic division. The SC is a feature of homologous chromosomal synapsis [45–47], and this type of ultrastructure is observed only at the zygotene and pachytene stages of meiosis. In the present study, the percentage of oocytes displaying SC was higher in the DHP-treated fragments than in the control or E2-treated fragments in huchen. Furthermore, Spo11, which is involved in meiotic recombination in several species [48–52], is also a good marker of meiosis. Recently, we isolated a cDNA that encodes Spo11 from the Japanese eel, raised a specific antibody, and detected Spo11 in early meiotic germ cells of the Japanese eel testes [53]. In the present study, the meiosis-specific marker Spo11 was expressed in germ cells in the ovarian fragments of DHP-treated carp. Collectively, these data suggest that the main action of DHP is to induce oogonia to enter the meiotic prophase. Therefore, DHP is an important factor for the initiation of meiosis in both spermatogenesis [23] and oogenesis. In other words, DHP may be the initiator of the first meiotic division in both males and females, while E2 induces oogonial proliferation.
ACKNOWLEDGMENTS
We thank Dr. G. Young (University of Washington) and Dr. R.W. Schulz (Utrecht University) for their valuable comments on the manuscript. We also thank Dr. Kohei Yamauch and Dr. Akihiko Hara (Hokkaido University) for their suggestions and assistance.
FOOTNOTES
1Supported by grants-in-aid from the Ministry of Agriculture, Forestry and Fisheries of Japan, the Global COE program of Japan's Society for the Promotion of Science (JSPS), and the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. 
Correspondence: 2Takeshi Miura, Laboratory of Fish Reproductive Physiology, Faculty of Agriculture, Ehime University, 3-5-6 Tarumi, Matsuyama, Ehime 790-8566, Japan. FAX: 81 89 946 9818; e-mail: miutake{at}agr.ehime-u.ac.jp
Received: 14 March 2007.
First decision: 11 April 2007.
Accepted: 6 August 2007.
REFERENCES
, 20ß-dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorhynchus rhodurus): correlation with plasma gonadotropin and in vitro production by ovarian follicles Gen Comp Endocinol 1983 5196–105[CrossRef][Medline]
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