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Effects of non-steroidal aromatase inhibitor letrozole on sex inversion and spermatogenesis in yellow catfish Pelteobagrus fulvidraco.

Introduction

It has been demonstrated in a variety of fish species that phenotypic sex differentiation can be influenced by administration of exogenous estrogens or androgens during or before sex differentiation, revealing the involvement of sex steroids in sex differentiation (Pandian and Sheela. 1995: Nakamura et al., 1998; Devlin and Nagahama, 2002; Strussmann and Nakamura, 2002). To induce sex inversion effectively, an exogenous sex steroid treatment must be administered during the critical period (i.e., labile period), which includes both anatomical and cytological phases of sex differentiation.

The natural endogenous inducers of sex differentiation in fish are considered to be the sex steroids. Estrogens acting as female inducers and androgens functioning as male inducers were initially postulated by Yamamoto (1969) from the results of his pioneering research on medaka (Oryzias latipes). Thereafter, increasing evidence has accumulated on the key function of sex steroid hormones in fish sex differentiation (Hunter and Donaldson, 1983; Pandian and Sheela. 1995; Nakamura et al., 1998; Baroiller et al., 1999; Guiguen et al., 1999). Growing evidence demonstrates that aromatase (eypl9a 1 a) mRNA is expressed before sex differentiation both in fish and reptiles (Ramsey et al., 2007; Fernandino et al., 2008; Cao et al., 2012), indicating that sex steroids may play a role in sex differentiation from a very early stage. Additionally, Nakamura (2010) hypothesized that sex steroids, rather than sex-determining genes, may be key factors for sex determination in some species of vertebrates. The hormonal balance between estrogens and androgens, which relies on the availability and activity of the steroid-synthesizing enzymes and, in particular, on the cytochrome P450 aromatase complex, appears to be critical in the process of sex differentiation. The importance of P450arom in sex differentiation in fish is manifested by the fact that inhibition of the aromatase activity during the labile period caused sex inversion of genetically female fish to phenotypic males (Piferrer et al., 1993; Kitano et al., 2000; Kroon and Liley, 2000; Kwon et al., 2002; Komatsu et al., 2006).

Aromatase is responsible for the final step in estrogen biosynthesis, catalyzing the aromatization of androstenedione and testosterone into estrone and estradiol, respectively (Haynes et al., 2003), and its activity determines the androgen-to-estrogen ratio in developing gonads (Navarro-Martin et al., 2009). Guiguen et al. (2010) highlight the pivotal role of estrogens and cyp 19a 1 a in both gonochoristic and hermaphrodite fish species, and suggest their involvement not only in ovarian but in testicular differentiation as well. Moreover, cyp 19a 1 a downregulation is the only necessary step for inducing testicular differentiation. Consequently, aromatase inhibitor (AI)-induced masculinization has been recognized as one of the most effective means for verifying the involvement of endogenous estrogen in ovarian differentiation and sex inversion of genotypic females. Letrozole (LZ; CGS 20267), a non-steroidal inhibitor with high potential and selectivity for aromatase, has been shown to inhibit aromatase activity and concomitantly suppress estrogen synthesis effectively. It has been reported that LZ is more potent than fadrozole in maculinizing gonads in turtles (Dorizzi et al., 1994). For clinical application, LZ is able to inhibit 98%-99% of aromatase activity and reduce serum concentration of estrone and estradiol (E2) to below the detection limit in humans (Smith, 1999).

The yellow catfish (Pelteobagrus fulvidraco [Richardon, 1846)], which is gonochoristic, is a commercially important edible fish (Wang et al., 2006). This species exhibits sexual dimorphism in growth, with males growing faster and reaching a larger ultimate size (three times) than females (Park et al., 2004; Liu et al., 2007, 2013). In an attempt to elucidate the involvement of aromatase activity in gonadal differentiation, we examined the effects of LZ on gonadal differentiation in yellow catfish.

Materials and Methods

Fry production

Eight female and two male yellow catfish broodstock were selected from a fishing ground at the National Research Centre for Freshwater Fisheries Engineering (Wuhan, PR China) and artificially induced to spawn after one week of temporary rearing. Newly hatched fry were gradually weaned from a rotifer diet to a micro-bound diet exclusively for juvenile fish (purchased from Shengsuo Fishery Feed Research Centre of Shandong Province, China; main nutrient components: raw protein 50%, raw fat 8%, raw fiber [less than or equal to]3%, raw ash[less than or equal to] 16.5%, moisture [less than or equal to]12%) from 4 days post-hatching (DPH) until AI treatment. LZ (>99.0%) was purchased from Wuhan Yuancheng Technology Development Co., Ltd, China.

Rearing, Al administration, and sampling

Five doses of LZ (0, 20, 50, 150, and 300 mg [kg.sup.-1] micro-bound diet; referred to as control, L20, L50, L150, and L300 in the following sections) were prepared following the methods of Gao et al. (2010). One hundred twenty of 10-DPH fish (13.4 mm mean total length and 0.03 g mean body weight) were randomly assigned to one of fifteen 120-1 experiment tanks with flow-through water (10 1 [h.sup.-1]). The control plus four treatment groups constituted a total of five groups, each having three replicates. The larvae received their dosages of LZ feed three times daily from 10 to 23 DPH, and twice daily from 24 to 59 DPH. Residual feeds were removed half an hour after the end of the feeding. Mortality was monitored at the end of AI treatments in all groups. No dead fish were found after 50 DPH. From the termination of Al administration until 105 DPH, all the fish were fed twice daily with a normal commercial expanded diet for yellow catfish. Twenty fish were sampled randomly from each tank, fixed in Bouin's solution for 48-96 h, and moved to 70% ethanol for histological examination after body weight had been measured (precision 0.01 g) at 60, 75, and 105 DPH, respectively. The period of LZ treatments, sampling timetable, and approximate labile period of gonad in yellow catfish (Park et al., 2004) are presented in Figure 1.

Histological examination

The gonad, connected with the kidney, was separated from each fish and processed for histological sectioning (6-8 [micro]m for each section) by routine dehydration and paraffin embedding procedures. For each fish, successive cross-sections covering the whole gonad tissue were cut using a Leica microtome. At least 10 sections from each fish were stained with hematoxylin and eosin. The slides were examined carefully and photographed with a Nikon 80i microscope to determine phenotypic sex and gonad structure. Phenotypic sex was classified as three types: female, male, and intersex (ovotestis).

Data analysis

All data are presented as means [+ or -] SD. Data of survival rates and growth performances were analyzed with ANOVA followed by Duncan's post hoc multiple comparisons, and sex proportions were determined with chi-square tests. Normality was ensured after logarithmic, sinusoidal, or arcsine transformation of data when required. Differences are considered significant at the level of P < 0.05.

Results

Survival and growth performance

Survival and growth performance are presented in Figure 2. The survival was about 80% in all groups, and no difference was found between the groups. At 60 DPH, body weight in the L20 treatment was significantly higher than that of the control and other LZ treatments. The increased growth in the L20 treatment lasted to 75 DPH, 15 days after the end of LZ treatment, then disappeared later at 105 DPH.

Sex ratios

Sex proportions (male, female, and intersex) in the control and LZ groups at the end of the treatments (60 DPH) are shown in Figure 3. The efficiency of masculinization by LZ was dose-dependent: LZ treatments produced 68.3%, 80.00%, 88.3%, and 96.7% males, respectively, values that were significantly higher than that of the control (38.3% [+ or -] 2.9%). Intersex gonads were observed in all experimental groups except the control. In the L300 treatment group, no female gonads were observed in any of the three replicates, and one out of three replicates produced 100% phenotypic males.

Sex proportions at 75 DPH and 105 DPH were also investigated. No change was observed in the control group from 60 DPH to 105 DPH, with 38.3% [+ or -] 2.9%, 39.9% [+ or -] 1.9%, and 39.4% [+ or -] 3.3% of male proportions being produced respectively. The male proportion at 105 DPH decreased significantly compared with the male proportion at 60 DPH, while intersex increased significantly in the L20 group (Fig. 4). However, the only significant increase in intersex was found in the L50 group from 75 to 105 DPH, and no significant change was found in the two higher doses of LZ (Fig. 4). These results indicate that masculinization is reversibly induced by a low dose of LZ, while it is persistent with a high-dose treatment.

Gonad histology

The testicular development of male yellow catfish exposed to LZ from 10 to 59 DPH was dramatically advanced, since testes from fish treated with LZ displayed abundant spermatozoa in lobule lumens and had enlarged lumens as well (Fig. 5a). In contrast, testes were mainly composed of spermatocytes and spermatogonia in the control group (Fig. 5b). Intersex gonads (Fig. Sc). consisting of female and male germ cells simultaneously, were observed in all LZ treatments. Testicular development in intersex gonads was similar to that of the control group, which lagged developmentally with respect to testis of the LZ-treated fish. A proportion of intersex gonads were composed of testicular tissue as well as oocytes and vacuolate tissues simultaneously (Fig. 5c). Furthermore, degenerating oocytes were found in some of the intersex gonads (image not shown). It is interesting that oocytes and ovarian structures were indistinguishable between the control (Fig. 5d) and normal females in the LZ treatment groups (image not shown).

Figure 6 shows the histological examination of gonads at 105 DPH, 45 days after the termination of LZ treatments. Increased density of spermatozoa as well as enlarged lumens were observed from normal males in LZ treatments (Fig. 6A) when compared with the control (Fig. 6B) and intersex gonads (Fig. 6C). The developmental stage in intersex testes lagged behind the normal males in LZ-treated fish; however, it was similar to that of the control. No visible differences in ovary structure were found among normal females in the control group, the intersex group, and normal females in LZ treatments. No degenerating oocytes were observed in the intersex gonads at 105 DPH.

Discussion

In the present study, masculinization of undifferentiated yellow catfish was induced by administration of the aromatase inhibitor (AI) letrozol (LZ). In Japanese flounder and zebrafish, treatments of fadrozole, another AI, during sex differentiation also caused sex inversion of genetic females to normal phenotypic males (Kitano et al., 2000; Uchida et al., 2004). Treatment with an Al also induces suppression of P450 gene expression in the gonads of genetic female flounder (Kitano et al., 2000). Therefore, these results indicate that gonadal masculinizaton of genetic all-females occurred by depletion of aromatase activity and, in turn, of estrogen synthesis. Additionally, degenerating oocytes were found, as well as vacuolate tissue likely formed by apoptosis of the oocytes, through histological examination in the present study (Fig. 5c). In zebrafish, which undergo a juvenile hermaphroditism stage, the disappearance of oocytes and the decomposition of the ovarian cavity caused by apoptosis during sex differentiation are male-specific events (Uchida et al., 2002). Moreover, oocyte apoptosis, depletion of P450arom activity, and differentiation of spematogonia are found to be caused by treatments with Al during gonadal sex-inversion (Uchida et al., 2004). Taken together, these observations suggest that estrogens play a vital role in ovarian differentiation, and that ovarian apoptosis is involved in the process of masculinization caused by AI treatment.

The large amount of spermatozoa and enlarged lobule lumens in the testes treated with LZ (Fig. 5a) compared with that of the control, which mainly comprise spermatogonia and spermatocytes (Fig. 5b), indicates that the LZ treatment may have the potential of stimulating or inducing spermatogenesis and promoting fertility. On the other hand, OUT results, via histological examination, demonstrate indirectly that estrogen or androgen-to-estrogen ratio plays a significant part in spermatogenesis regulation. Afonso et al. (2000) report that male coho salmon (Oncorhynchus ku-sutch) treated with fadrozole spermiate earlier than the control group, through the inhibition of 17 [beta]-estradiol (E2) and the increase of 17, 20[beta]-dihydroxy-4-pregnen-3-one plasma levels, suggesting that low estrogen level is a prerequisite in spermiation of coho salmon. It also has been proposed that a small concentration of estrogen is necessary for normal spermatogenesis (Miura et al., 1999). Our results are consistent with these reports to some extent. Although E2 levels were not determined in the present study, the fact that we did not observe a complete masculinization via LZ treatments suggests that E2 synthesis may not have been completely inhibited even at the highest dose. In the lizard Podarcis sicula, Cardone et al. (2002) suggest that the failure of spermatogenesis in autumn may be due to high estrogen levels, and that Al treatment induces an increased diameter of tubules and the stimulation of the seminiferous epithelium. Thus a relatively high androgen-to-estrogen ratio is proposed to be beneficial to testicular development and spermatogenesis. However, the side effects remain to be elucidated. This information will not only contribute to our understanding of the involvement of aromatase activity and estrogens (or androgen-to-estrogen ratio) in spermatogenesis, but could also be applied in artificial reproduction. Further research is necessary to characterize the biochemical and physiological parameters, so as to clarify the involvement of estrogen or the androgen-to-estrogen ratio during the early development of fish testis.

Finally, the changes of sex proportions in the low LZ treatments (L20 and L50) from 60 DPH to 105 DPH observed in the present study suggest that female germ cells of sex-differentiated yellow catfish still retain a certain level of bipotentiality (Fig. 4). Several previous studies report that the sexual bipotentiality of germ cells lasts into adulthood even in gonochoristic fish species (Pandian and Sheela, 1995; Baroiller et al., 1999; Strilssmann and Nakamura, 2002). Nakamura et al. (2003) found that both germ cells and somatic cells in developed tilapia ovaries still exhibit sexual bipotentiality long after the period of sex differentiation, and artificial sex inversion after sex differentiation is only possible through Al treatments but not with 17[alpha]-methyltestosterone. The information on the bipotentiality of germ cells after sex differentiation is interesting and remarkable, because in yellow catfish sex inversion may be accomplished through Al exposure after sex differentiation even during the sexual maturation period.

Acknowledgments

We thank Mr. Fan Qizhi and Mr. Wang Xiaojun for preparing the experiment and providing fish care. We are indebted to Joy Bauman and Laura Tiu for English revision.

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Received 7 March 2013; accepted 8 August 2013.

* To whom correspondence should be addressed. E-mail: kugeshenl@163.com

ZHI-GANG SHEN, QI-XUE FAN *, WEI YANG, YUN-LONG ZHANG, PEI-PEI HU AND CONG-XIN XIE

College of Fishery, Huazhong Agricultural University, Wuhan, Hubei, 430070, People's Republic of China
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Author:Shen, Zhi-Gang; Fan, Qi-Xue; Yang, Wei; Zhang, Yun-Long; Hu, Pei-Pie; Xie, Cong-Xin
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:9CHIN
Date:Sep 1, 2013
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