- Email: [email protected]
Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus)
Biochemical and Biophysical Research Communications xxx (2016) 1e6
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus) Linyan Zhou a, 1, Feng Luo a, 1, Xuelian Fang a, Tapas Charkraborty b, Limin Wu a, Jing Wei a, Deshou Wang a, * a
Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, 400715, PR China South Ehime Fisheries Research Center, Ehime University, Ainan, 798-4206, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2016 Accepted 9 March 2016 Available online xxx
Previous studies indicated that maturation inducing hormone, 17a, 20b-Dihydroxy-4-pregnen-3-one (DHP), probably through nuclear progestin receptor (Pgr), might be involved in spermatogenesis and oogenesis in fish. To further elucidate DHP actions in teleostean ovarian differentiation, we analyzed the expression of pgr in the ovary of Nile tilapia (Oreochromis niloticus), and performed RU486 (a synthetic Pgr antagonist) treatment in XX fish from 5 days after hatching (dah) to 120dah. Tilapia Pgr was abundantly expressed in the follicular cells surrounding oocytes at 30 and 90dah. Continuous RU486 treatment led to the blockage of oogenesis and masculinization of somatic cells in XX fish. Termination of RU486 treatment and maintenance in normal condition resulted in testicular differentiation, and estrogen compensation in RU486-treated XX fish successfully restored oogenesis. In RU486-treated XX fish, transcript levels of female dominant genes were significantly reduced, while male-biased genes were evidently augmented. Meanwhile, both germ cell mitotic and meiotic markers were substantially reduced. Consistently, estrogen production levels were significantly declined in RU486-treated XX fish. Taken together, our data further proved that DHP, possibly through Pgr, might be essential in the ovarian differentiation and estrogen production in fish. © 2016 Elsevier Inc. All rights reserved.
Keywords: DHP Estrogen RU486 treatment Sex reversal Spermatogenesis
1. Introduction In teleosts, 17a, 20b-dihydroxypregn-4-en-3-one (17a, 20b-DP, DHP) and 17, 20b, 21-trihydroxy-4-pregnen-3-one (17, 20b, 21-P), have been identified as two major biologically active progestins to induce oocytes maturation and ovulation in female [1,2], as well as final sperm maturation and spermiation in male [3,4]. Moreover, DHP, mediated through progestin receptors (Pgr), stimulates early stage of spermatogenesis in Japanese eel (Anguilla japonica) [5], Atlantic salmon (Salmon salar) [6], zebrafish (Danio rerio) [7], cod (Gadus morhua) [8] and tilapia (Oreochromis niloticus) [9]. However, the role of DHP in early stage of ovarian differentiation remains elusive. Previous report showed, by in vitro culture of ovarian
* Corresponding author. School of Life Sciences, Southwest University, 400715, Beibei, Chongqing, PR China E-mail address: [email protected] (D. Wang). 1 The authors contribute to this work equally.
epithelial, that a low concentration of DHP was sufficient to promote DNA synthesis, formation of Synaptonemal-Complexes (SC) oocytes, and Spo11 expression in Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio) [10]. No further informations are available regarding the role of DHP in early stage of oogenesis in teleosts. Therefore, additional investigations are required to elucidate the mechanisms of DHP in germ cell meiotic initiation and ovarian differentiation. Estrogen is regarded as a natural inducer of ovarian differentiation in fish during female sex determination/differentiation [11]. Gonadal aromatase (encoded by the cyp19a1a gene), an enzyme catalyzing the conversion of androgens to estrogens, increased its expression specifically in the ovary from 5 days after hatching (dah) [12]. The treatment of XX tilapia fry with fadrozole (an aromatase inhibitor) or tamoxifen (an estrogen receptor antagonist) causes their complete sex reversal to functional males [13,14]. In fadrozole-treated XX tilapia gonads, germ cells meiotic initiation was delayed, and expression profiles of sycp3 were detected only
http://dx.doi.org/10.1016/j.bbrc.2016.03.045 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
2
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
Fig. 1. Pgr expression and effects of RU486/RU486 þ E2 treatment on ovarian differentiation. (AeD) Immunohistochemistry of Pgr in the ovary of 30 and 90dah. Positive staining was detected in the interstitial cells of the ovarian tissue at 30dah (A). The expression of Pgr was also detected in the follicular cells surrounding oocytes at 90dah (B). No immunostaining was detected in the sections incubated with normal rabbit serum as the first antibody (C, D). (EeP) Results of immunohistochemistry of three genes (Cyp19a1a, Cyp11b2, Dmrt1) expression in the gonad of different treatments and control fish at 120dah. Abundant expression of Cyp19ala was detected in interstitial cells of the gonads in XX control fish (E), while no positive signal of Cyp19ala expression was detected in the gonads of RU486-treated XX fish (H, K). Cyp11b2 and Dmrt1, which were strongly expressed in
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
3
from 90dah. Contrastingly, in estradiol-17b (E2)-treated XY gonads, meiotic initiation of germ cells and expression of sycp3 was preponed to 30dah. It indicated that E2 favors the ovarian fate probably via controlling the timing of meiotic initiation. Therefore, it is intriguing to investigate the crosstalk between DHP and E2 in early stage of ovarian differentiation. The Nile tilapia, a gonochoristic teleost fish with an XX/XY sexdetermining system, provides an excellent model for fish gametogenesis study. As a new player in oogenesis, the role of DHP in tilapia ovarian development during sexual differentiation remains largely unknown. To further elucidate the action of Pgr-mediated DHP functions in fish, XX fry were treated with a Pgr antagonist (RU486) from 5 to 120dah. Finally, E2 was added to rescue the RU486-disrupted ovarian differentiation.
(1:2500), Cyp11b2 (1:500) and Dmrt1 (1:100) genes overnight at 4 C, and rinsed with 0.01 M PBS three times for 5 min per wash. Subsequently, the tissue sections were incubated with a second antibody (goat anti-rabbit IgG) conjugated with horseradish peroxidase (Bio-Rad, USA) at 1:2000 for 30 min, and then rinsed with PBS three times for 5 min per wash. Immunoreactive signals were visualized using diaminobenzidine (Sigma, USA) as the substrate. Sections were counterstained with hematoxylin. For the negative control, the primary antibody was replaced with normal rabbit serum.
2. Materials and methods
Western blotting was performed to confirm the expression changes of Cyp19a1a, Cyp11b2 and Dmrt1 in both RU486-treated XX fish and control XX/XY fish at 120dah. Briefly, proteins were extracted with buffer containing 50 mM Tris-Cl (pH7.5), 140 mM NaCl, 1% Nonidet P-40, 2 mM EDTA and complete inhibitor cocktail. Then, 150 ng of the whole gonadal extract from each group was separated by SDS-PAGE, and transferred onto a PVDF membrane (Amersham, Sweden). The membranes were blocked with 5% low fat milk powder in TBST (20 mM Tris-HCL pH7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with primary antibody, and then with APlabeled secondary antibody. Finally, the immunoreactive signals were stained with NBT/BCIP substrates and visualized on Fusion FX7 (Vilber Lourmat, France).
2.1. Fish The tilapia for this study were reared in re-circulating aerated freshwater tanks at 26 C under constant photoperiod (12 h light: 12 h darkness). All genetic females (XX) and males (XY) were obtained by artificially fertilizing the eggs from normal females (XX) with sperm from either sex-reversed males (XX) or super males (YY), respectively. Super males (YY) were obtained by crossing normal XY males with an XY female, which was sex-reversed hormonally with E2 treatment. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Laboratory Animal Experimentation at Southwest University, China. 2.2. Treatment of XX tilapia by RU486 and E2 Previous studies revealed that the period around 5dah is critical for the initiation of sex determination/differentiation, in which undifferentiated gonads differentiate into testes or ovaries in tilapia [15]. All treatments were started from 5dah in aerated fresh water and sampled at 120dah. Briefly, fish were divided into five groups, i.e. I) continuous treatment with RU486 till 120dah, II) treatment with RU486 till 20dah, and then feeding with normal commercial diet before sampling, III) simultaneous treatment with RU486 and E2 to 120dah, IV) XX control group, V) XY control group. Fish were maintained in aerated fresh water and fed daily with commercial diet mixed with RU486 (500 mg/g, Sigma) with or without E2 (300 mg/g, Sigma). Meanwhile, the normal XX and XY fish fed with commercial diet were used as control. At 120dah, gonads from both control and treated groups were sampled for subsequent immunohistochemistry (IHC) and real-time PCR analyses.
2.4. Western blotting
2.5. Quantitative real-time PCR analysis Real-time PCR was carried out on an ABI-7500 real-time PCR machine according to the protocol of SYBR Premix Ex Taq II (Takara, Tokyo, Japan), using b-actin as internal control. Gonads from six control XX/XY and six RU486-treated XX fish were collected. Total RNAs were extracted from each sample and reverse transcribed using PrimeScript RT Master Mix Perfect Real Time Kit (Takara, Tokyo, Japan), according to the manufacturer's instructions. Gene expression of foxl2, cyp19a1a, cyp11b2, dmrt1, sycp3 and vasa were analyzed by real-time PCR using gene-specific primers (Supplementary Table S1). The relative abundance of mRNA transcripts was evaluated using the formula: R ¼ 2-△△Ct. Data are expressed as the mean ± SD. Statistical analyses of the results were conducted using one-way ANOVA (P < 0.05) in GraphPad Prism 4 software.
2.3. IHC
2.6. Measurement of E2 levels by enzyme immunology assay (EIA)
At 120dah, gonads from both treated and control groups fish were collected and fixed in Bouin's solution for IHC analysis. Briefly, specimens were embedded in paraffin and sectioned at 5 mm thickness. Paraffin sections were then deparaffinized and hydrated. After washing with 0.01 M PBS three times for 10 min per wash. The sections were then treated in a blocking solution (Roche, Germany), incubated with the polyclonal antibody anti-tilapia Cyp19a1a
For EIA, a minimum of 500 ml blood was drawn from the caudal vein of RU486-treated XX fish and control XX/XY fish at 120dah using a gauge needle and a disposable syringe. Blood samples were centrifuged at 10,000 g for 5 min at 4 C, and the supernatant was collected without disturbing the white buffy layer or the blood cells. Finally, E2 levels in the serum were measured using the E2 EIA Kit according to the manufacturer's instructions (Cayman, USA).
the Leydig cells (F) and the Sertoli cells (G) in XY control, were also detected in the RU486-treated XX fish (I, L, J, M). No positive expression of Cyp19a1a, Cyp11b2 and Dmrt1 were detected in XX gonads with RUXX þ E2 treatment (N, O, P). White arrowheads, black arrowheads and arrows indicate the positive immunostaining of Cyp19a1a, Cyp11b2 and Dmrt1, respectively. (Q) Western bloting analysis of three genes (Cyp19a1a, Cyp11b2, Dmrt1) expression in the gonad of continuous RU486 treatment and control fish. (RUXX, continuous RU486 treatment from 5dah to 120dah; RUXX20-120dah, RU486 treatment from 5dah to 20dah, then feeding with a normal diet until 120dah; RUXX þ E2, simultaneous treatment of RU486 and E2 from 5dah to 120dah; dah,day after hatching)
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
4
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
3. Results
3.2. Rescue of RU486-treated XX fish by estrogen
3.1. Expression of pgr and morphological changes of ovaries under RU486 treatment in XX fish
Previous reports revealed that estrogen is a prerequisite of ovarian differentiation. Therefore, simultaneous treatment of RU486 and E2 were carried out to explore whether estrogen could rescue the defects of RU486 treatment in XX gonad development. As expected, the compensation of estrogen in RU486-treated XX fish regained oogenesis suggesting reoccurrence of oocytes development. However, no positive expression of female-biased gene (Cyp19a1a) and male-biased gene (Cyp11b2 and Dmrt1) were detected in XX gonads with simultaneous treatment with E2 and RU486 (Fig. 1N, O, P).
The expression of Pgr in XX gonads during different developmental stages was investigated by IHC. Strong expression of Pgr was detected in interstitial cells at 30dah (Fig. 1A) and follicular cells at 90dah (Fig. 1B). Then, RU486 treatment was applied to study the role of Pgr-mediated DHP function in XX fish. By 120dah in normal XX tilapia, ovary was filled with oocytes surrounded by somatic cells (Fig. 1E). However, continuous treatment of XX fish with RU486 from 5 to 120dah led to blockage of meiotic entry and oogenesis, and resulted in complete loss of oocytes (Fig. 1HeJ). RU486-treated XX fish from 5 to 20dah and thereafter maintenance in normal condition up to 120dah led to sex reversal with spermatogenesis and differentiation of sperm at 120dah (Fig. 1KeM). It is well documented that aromatase, encoded by cyp19a1a gene, is the critical enzyme to catalyze the biosynthesis of estrogen in fish [16]. On the contrary, cyp11b2 is specifically expressed in the interstitial cells of testis and is required for the production of fish androgen 11-KT [17]. Meanwhile, dmrt1 has been proved to play a conserved role in fish testicular differentiation. Therefore, the expressions of Cyp19a1a, Cyp11b2 and Dmrt1 gene were examined in control and RU486-treated XX gonads by both IHC and western blotting. In control XX gonad, expression of Cyp19a1a could be detected in the interstitial cells at 120dah (Fig. 1E). However, it was severely repressed and disappeared in continuous RU486-treated XX gonads (Fig. 1H). On the other hand, abundant expression of Cyp11b2 and Dmrt1 were observed in RU486-treated XX gonads (Fig. 1I, J, Q). The rate of masculinization and sex reversal in continuous RU486-treated XX fish reached nearly 73.3% and 16.7% (Supplemental Table S2). Consistently, short-term RU486 treatment simultaneously caused the suppression of Cyp19a1a, and induction of Cyp11b2 and Dmrt1, which might have further accelerated the sex reversal process (Fig. 1K, L, M).
3.3. Changes of gene expression profiles Real-time PCR was carried out to further evaluate the molecular changes in RU486-treated XX fish, with or without E2, and control XX/XY fish. RU486 treatment resulted in significant reduction of female specific genes (foxl2 and cyp19a1a) and ectopic upregulation of male specific genes (dmrt1 and cyp11b2). Germ cell mitotic and meiotic marker genes (vasa and sycp3) were also severely inhibited. As aforementioned, compensation of E2 was able to rescue differentiation of oocytes in RU486-treated XX fish, therefore, germ cells marker genes of vasa and sycp3 were evidently increased. Consistent with IHC results, expression of female specific genes (foxl2 and cyp19a1a) were not stimulated in comparison to the control XX fish. Moreover, male dominant genes (dmrt1 and cyp11b2) were also repressed (Fig. 2). 3.4. Evaluation of E2 production level Estrogen has been proved to be essential for ovarian differentiation and maintenance. To explore the estrogen level change in XX fish treated with RU486 alone and combination of RU486 with E2, we collected blood samples from treated XX, control XX and control XY fish at 120dah, and measured the estrogen levels by EIA. No significant difference of estrogen production level was found
Fig. 2. Expression profiles of somatic cell and germ cell marker genes in the gonads of treated and control fish gonads at 120dah analyzed by real-time PCR. Data are expressed as mean ± SD (n ¼ 6), and different letters indicate significant difference (P < 0.05).
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
between XX fish treated with RU486 alone and normal XY fish, suggesting that RU486 treatment alone led to the significant decrease of estrogen in XX fish. However, estrogen level in XX fish treated with both RU486 and E2 was equivalent to control XX fish (Fig. 3).
4. Discussion So far, it has been proved that regulation of oogenesis in fish involves the functions of Follicle Stimulating Hormone (FSH) [18,19], estrogen and Retinoic Acid (RA) [20]. However, the regulatory mechanisms are far more complex and largely unknown. By using in vitro culture system, it was reported that fish progestin (DHP) is involved in proliferation of oogonia and meiotic initiation. In Japanese huchen, significant increase of DHP level was detected during the initiation of the first meiotic division, as indicated by many chromatin-nucleolus stage oocytes [10]. In sockeye salmon (Oncorhynchus nerka), the serum levels of DHP have been shown to increase from about 1 ng/ml to 2 ng/ml in immature females [21]. Using in vitro culture systems, it was shown that DHP was sufficient to promote DNA synthesis in ovarian germ cells. After preculture of the carp ovarian epithelium, these fragments were treated for 15 days with DHP, and BrdU incorporation in numerous ovarian germ cells in DHP-treated ovarian fragments was observed, indicating that DNA was synthesized in the germ cells. The percentage of oocytes displaying SC was significantly increased in the DHPtreated fragments in huchen. Moreover, the meiosis-specific marker Spo11 was expressed in germ cells in the ovarian fragments of DHP-treated carp. These data indicated that DHP might be involved in proliferation of oogonia and meiotic initiation [10]. However, the actions and mechanisms of Pgr-mediated DHP in early stage of ovarian differentiation in fish need to be further explored. In tilapia, proliferation of primordial germ cell (PGC) and initiation of meiosis occurred around 15 and 40dah, respectively. During early stage of ovarian differentiation in tilapia, transcript of cyp17a2, one of the critical steroidogenic enzymes for DHP production during oocytes maturation, starts to increase in the somatic cells from 20dah in XX gonad [22]. Therefore, these data suggest that DHP might be possibly synthesized around the timing of meiotic initiation during sexual differentiation of tilapia ovary. Meanwhile, abundant expression of DHP nuclear receptor (Pgr) in the interstitial cells and follicular cells during the early stages of ovarian differentiation, strongly suggested the possible role of DHP during the early stage of ovarian differentiation. It is well known that the functions of progesterone are mainly mediated by Pgr. RU486, an orally active compound for absorption used in FDA-approved medical treatment, binds to the progesterone receptor with two fold higher affinity than progesterone [23].
5
In this study, we carried out the RU486 treatment of XX fish from 5dah to investigate the potential role DHP in tilapia oogenesis. Our data demonstrated that RU486 treatment led to blockage of oogenesis, suggesting the essentiality of DHP in tilapia oogenesis. Meanwhile, continuous RU486 treatment in XX fish led to the suppression of female specific genes (foxl2 and cyp19ala) and ectopic increase of male specific gene (dmrt1 and cyp11b2), suggesting that blockage of Pgr-mediated DHP action led to the masculinization of somatic cells. Consistently, in comparison with normal XX fish, estrogen level in RU486-treated XX fish was dramatically reduced to the similar level to XY fish. Moreover, no meiotic germ cells were found in RU486-treated (from 5 to 120dah continuously) XX fish. Our data suggest that blockage of Pgrmediated progestin function in XX tilapia results in the repression of female specific genes and decrease of estrogen production, which in turn switches the direction of gonadal development towards male pathway. Therefore, estrogen and progestin might be acting synergistically to promote ovarian differentiation in early developmental stages in fish. However, upon the termination of RU486 treatment at 20dah resulted in the occurrence of spermatogenesis by 120dah. Our data indicated that short-term exposure to RU486 led to the defects of ovarian differentiation and masculinization of XX fish. RU486 treatment by 20dah caused the rapid up-regulation of Gonadal soma-derived factor (gsdf) and dmrt1 gene, two male specific genes in the Sertoli cells in testis, suggesting that the somatic cell environment has switched towards male properties in RU486-treated XX fish (unpublished data). The latest reports revealed that Gsdf and Dmrt1 are the key factors to initiate the male pathway and ensure the testicular differentiation, furthermore, the null mutant of gsdf and dmrt1 gene in XY fish results in the male to female sex reversal and ovarian development in medaka [24,25]. Moreover, over-expression of Gsdf in XX tilapia induced sex reversal from female to male and testicular development [26]. Therefore, ectopic expression of Gsdf and Dmrt1 in RU486-treated XX fish and recovery of endogenous steroids might be the reasons to cause the sex reversal by 120dah. Estrogens play an important role in ovarian differentiation and maintenance in fish. The treatment of tilapia XX fry with fadrozole causes complete sex reversal to functional males [27]. Exogenous/ endogenous estrogen was sufficient to induce the development of fertile ovary. To elucidate the possible interaction of estrogen and DHP in fish oogenesis, simultaneous treatments of exogenous E2 and RU486 in XX fish were carried out. Our data demonstrated that compensation of exogenous estrogen successfully rescued the progression of oogenesis and oocyte differentiation. Intriguingly, estrogen failed to induce complete female development when Pgrmediated DHP function was blocked due to the significant lower expression level of foxl2 and cyp19a1a gene in somatic cells. Our data further strengthened that both estrogen and DHP are required for proper female development. In our opinion, we suppose that occurrence of oogenesis induced by extra estrogen in RU486disrupted XX fish might have originated from the direct function of estrogen on germ cells, but not via endogenous estrogen production in somatic cells. However, the mechanisms need further investigations. In conclusion, for the first time, we have investigated the critical role of DHP in early stage oogenesis of XX tilapia by RU486 treatment. Our data further proves that both DHP and estrogen might be essential for ovarian differentiation. Conflict of interest
Fig. 3. Serum17b-estradiol (E2) level in the control group (XX/XY) and treated group (RUXX, RUXX þ E2) by EIA assay. Data are expressed as the mean ± SD (n ¼ 6), and different letters indicate significant difference(P < 0.05).
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
6
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
reported. Acknowledgments The authors cordially thank Prof. Yong Zhu from East Carolina University, North Carolina, for providing the zebrafish nuclear Pgr antibody. This work was supported by grants 31572597, 31201986, 91331119 and 31030063 from the National Natural Science Foundation of China; grant 20130182130003 from the Specialized Research Fund for the Doctoral Program of Higher Education of China; This work was also supported in part by Research Foundation of Talent Introduction of Southwest University, China (SWU111003) and Fundamental Research Funds for the Central Universities, China (XDJK2015A004 and XDJK2014B040) from Ministry of Education of China. The special fund of Chongqing key laboratory (CSTC). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.03.045. References [1] Y. Nagahama, M. Yoshikuni, M. Yamashita, T. Tokumoto, Y. Katsu, Regulation of oocyte growth and maturation in fish, Curr. Top. Dev. Biol. 30 (1995) 103e145. [2] Y. Nagahama, M. Yamashita, Regulation of oocyte maturation in fish, Dev. Growth Differ. 50 (Suppl 1) (2008) S195eS219. [3] H. Ueda, A. Kambegawa, Y. Nagahama, Involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon, Oncorhynchus rhodurus, and goldfish, Carassius auratus, Gen. Comp. Endocrinol. 59 (1985) 24e30. [4] R.W. Schulz, L.R. de Franca, J.J. Lareyre, F. Le Gac, H. Chiarini-Garcia, R.H. Nobrega, T. Miura, Spermatogenesis in fish, Gen. Comp. Endocrinol. 165 (2010) 390e411. [5] T. Miura, M. Higuchi, Y. Ozaki, T. Ohta, C. Miura, Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 7333e7338. [6] S.X. Chen, J. Bogerd, E. Andersson, F.F. Almeida, G.L. Taranger, R.W. Schulz, Cloning, pharmacological characterization, and expression analysis of Atlantic salmon (Salmo salar L.) nuclear progesterone receptor, Reproduction 141 (2011) 491e500. [7] S.X. Chen, J. Bogerd, A. Garcia-Lopez, H. de Jonge, P.P. de Waal, W.S. Hong, R.W. Schulz, Molecular cloning and functional characterization of a zebrafish nuclear progesterone receptor, Biol. Reprod. 82 (2010) 171e181. [8] S.X. Chen, F.F. Almeida, E. Andersson, G.L. Taranger, R. Schmidt, R.W. Schulz, J. Bogerd, Cloning, pharmacological characterization and expression analysis of Atlantic cod (Gadus morhua, L.) nuclear progesterone receptor, Gen. Comp. Endocrinol. 179 (2012) 71e77. [9] G. Liu, F. Luo, Q. Song, L. Wu, Y. Qiu, H. Shi, D. Wang, L. Zhou, Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus), J. Mol. Endocrinol. 53 (2014) 57e70. [10] C. Miura, T. Higashino, T. Miura, A progestin and an estrogen regulate early
stages of oogenesis in fish, Biol. Reprod. 77 (2007) 822e828. [11] Y. Nagahama, Molecular mechanisms of sex determination and gonadal sex differentiation in fish, Fish. Physiol. Biochem. 31 (2005) 105e109. [12] S. Ijiri, H. Kaneko, T. Kobayashi, D.S. Wang, F. Sakai, B. Paul-Prasanth, M. Nakamura, Y. Nagahama, Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus, Biol. Reprod. 78 (2008) 333e341. [13] T. Kobayashi, H. Kajiura-Kobayashi, Y. Nagahama, Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia, Cytogenet Genome Res. 101 (2003) 289e294. [14] D.S. Wang, T. Kobayashi, L.Y. Zhou, B. Paul-Prasanth, S. Ijiri, F. Sakai, K. Okubo, K. Morohashi, Y. Nagahama, Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1, Mol. Endocrinol. 21 (2007) 712e725. [15] W. Tao, J. Yuan, L. Zhou, L. Sun, Y. Sun, S. Yang, M. Li, S. Zeng, B. Huang, D. Wang, Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes, PLoS One 8 (2013) e63604. [16] Y. Guiguen, A. Fostier, F. Piferrer, C.F. Chang, Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish, Gen. Comp. Endocrinol. 165 (2010) 352e366. [17] M.A. Alam, R.K. Bhandari, Y. Kobayashi, S. Nakamura, K. Soyano, M. Nakamura, Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra, Mol. Reprod. Dev. 73 (2006) 206e214. [18] B. Levavi-Sivan, J. Bogerd, E.L. Mananos, A. Gomez, J.J. Lareyre, Perspectives on fish gonadotropins and their receptors, Gen. Comp. Endocrinol. 165 (2010) 412e437. [19] E. Lubzens, G. Young, J. Bobe, J. Cerda, Oogenesis in teleosts: How fish eggs are formed, Gen. Comp. Endocrinol. 165 (2010) 367e389. [20] R.J. Feng, L.L. Fang, Y.Y. Cheng, X. He, W.T. Jiang, R.R. Dong, H.J. Shi, D.N. Jiang, L.N. Sun, D.S. Wang, Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus), Sci. Rep. 5 (2015). [21] R.K. Bhandari, I. Ushikoshi, H. Fukuoka, N. Koide, K. Yamauchi, H. Ueda, Effects of Rhizopus extract administration on somatic growth and sexual maturation in lacustrine sockeye salmon Oncorhynchus nerka, Fish. Sci. 68 (2002) 776e782. [22] L.Y. Zhou, D.S. Wang, T. Kobayashi, A. Yano, B. Paul-Prasanth, A. Suzuki, F. Sakai, Y. Nagahama, A novel type of P450c17 lacking the lyase activity is responsible for C21-steroid biosynthesis in the fish ovary and head kidney, Endocrinology 148 (2007) 4282e4291. [23] O. Heikinheimo, R. Kekkonen, P. Lahteenmaki, The pharmacokinetics of mifepristone in humans reveal insights into differential mechanisms of antiprogestin action, Contraception 68 (2003) 421e426. [24] T. Imai, K. Saino, M. Matsuda, Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development, Biochem. Biophys. Res. Commun. 467 (2015) 109e114. [25] H. Masuyama, M. Yamada, Y. Kamei, T. Fujiwara-Ishikawa, T. Todo, Y. Nagahama, M. Matsuda, Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka, Chromosome Res. 20 (2012) 163e176. [26] H. Kaneko, S. Ijiri, T. Kobayashi, H. Izumi, Y. Kuramochi, D.S. Wang, S. Mizuno, Y. Nagahama, Gonadal soma-derived factor (gsdf), a TGF-beta superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus, Mol. Cell Endocrinol. 415 (2015) 87e99. [27] V. Gennotte, P. Mafwila Kinkela, B. Ulysse, D. Akian Djetouan, F. Bere Sompagnimdi, T. Tomson, C. Melard, C. Rougeot, Brief exposure of embryos to steroids or aromatase inhibitor induces sex reversal in Nile tilapia (Oreochromis niloticus), J. Exp. Zool. A Ecol. Genet. Physiol. 323 (2015) 31e38.
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus) Linyan Zhou a, 1, Feng Luo a, 1, Xuelian Fang a, Tapas Charkraborty b, Limin Wu a, Jing Wei a, Deshou Wang a, * a
Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, 400715, PR China South Ehime Fisheries Research Center, Ehime University, Ainan, 798-4206, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2016 Accepted 9 March 2016 Available online xxx
Previous studies indicated that maturation inducing hormone, 17a, 20b-Dihydroxy-4-pregnen-3-one (DHP), probably through nuclear progestin receptor (Pgr), might be involved in spermatogenesis and oogenesis in fish. To further elucidate DHP actions in teleostean ovarian differentiation, we analyzed the expression of pgr in the ovary of Nile tilapia (Oreochromis niloticus), and performed RU486 (a synthetic Pgr antagonist) treatment in XX fish from 5 days after hatching (dah) to 120dah. Tilapia Pgr was abundantly expressed in the follicular cells surrounding oocytes at 30 and 90dah. Continuous RU486 treatment led to the blockage of oogenesis and masculinization of somatic cells in XX fish. Termination of RU486 treatment and maintenance in normal condition resulted in testicular differentiation, and estrogen compensation in RU486-treated XX fish successfully restored oogenesis. In RU486-treated XX fish, transcript levels of female dominant genes were significantly reduced, while male-biased genes were evidently augmented. Meanwhile, both germ cell mitotic and meiotic markers were substantially reduced. Consistently, estrogen production levels were significantly declined in RU486-treated XX fish. Taken together, our data further proved that DHP, possibly through Pgr, might be essential in the ovarian differentiation and estrogen production in fish. © 2016 Elsevier Inc. All rights reserved.
Keywords: DHP Estrogen RU486 treatment Sex reversal Spermatogenesis
1. Introduction In teleosts, 17a, 20b-dihydroxypregn-4-en-3-one (17a, 20b-DP, DHP) and 17, 20b, 21-trihydroxy-4-pregnen-3-one (17, 20b, 21-P), have been identified as two major biologically active progestins to induce oocytes maturation and ovulation in female [1,2], as well as final sperm maturation and spermiation in male [3,4]. Moreover, DHP, mediated through progestin receptors (Pgr), stimulates early stage of spermatogenesis in Japanese eel (Anguilla japonica) [5], Atlantic salmon (Salmon salar) [6], zebrafish (Danio rerio) [7], cod (Gadus morhua) [8] and tilapia (Oreochromis niloticus) [9]. However, the role of DHP in early stage of ovarian differentiation remains elusive. Previous report showed, by in vitro culture of ovarian
* Corresponding author. School of Life Sciences, Southwest University, 400715, Beibei, Chongqing, PR China E-mail address: [email protected] (D. Wang). 1 The authors contribute to this work equally.
epithelial, that a low concentration of DHP was sufficient to promote DNA synthesis, formation of Synaptonemal-Complexes (SC) oocytes, and Spo11 expression in Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio) [10]. No further informations are available regarding the role of DHP in early stage of oogenesis in teleosts. Therefore, additional investigations are required to elucidate the mechanisms of DHP in germ cell meiotic initiation and ovarian differentiation. Estrogen is regarded as a natural inducer of ovarian differentiation in fish during female sex determination/differentiation [11]. Gonadal aromatase (encoded by the cyp19a1a gene), an enzyme catalyzing the conversion of androgens to estrogens, increased its expression specifically in the ovary from 5 days after hatching (dah) [12]. The treatment of XX tilapia fry with fadrozole (an aromatase inhibitor) or tamoxifen (an estrogen receptor antagonist) causes their complete sex reversal to functional males [13,14]. In fadrozole-treated XX tilapia gonads, germ cells meiotic initiation was delayed, and expression profiles of sycp3 were detected only
http://dx.doi.org/10.1016/j.bbrc.2016.03.045 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
2
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
Fig. 1. Pgr expression and effects of RU486/RU486 þ E2 treatment on ovarian differentiation. (AeD) Immunohistochemistry of Pgr in the ovary of 30 and 90dah. Positive staining was detected in the interstitial cells of the ovarian tissue at 30dah (A). The expression of Pgr was also detected in the follicular cells surrounding oocytes at 90dah (B). No immunostaining was detected in the sections incubated with normal rabbit serum as the first antibody (C, D). (EeP) Results of immunohistochemistry of three genes (Cyp19a1a, Cyp11b2, Dmrt1) expression in the gonad of different treatments and control fish at 120dah. Abundant expression of Cyp19ala was detected in interstitial cells of the gonads in XX control fish (E), while no positive signal of Cyp19ala expression was detected in the gonads of RU486-treated XX fish (H, K). Cyp11b2 and Dmrt1, which were strongly expressed in
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
3
from 90dah. Contrastingly, in estradiol-17b (E2)-treated XY gonads, meiotic initiation of germ cells and expression of sycp3 was preponed to 30dah. It indicated that E2 favors the ovarian fate probably via controlling the timing of meiotic initiation. Therefore, it is intriguing to investigate the crosstalk between DHP and E2 in early stage of ovarian differentiation. The Nile tilapia, a gonochoristic teleost fish with an XX/XY sexdetermining system, provides an excellent model for fish gametogenesis study. As a new player in oogenesis, the role of DHP in tilapia ovarian development during sexual differentiation remains largely unknown. To further elucidate the action of Pgr-mediated DHP functions in fish, XX fry were treated with a Pgr antagonist (RU486) from 5 to 120dah. Finally, E2 was added to rescue the RU486-disrupted ovarian differentiation.
(1:2500), Cyp11b2 (1:500) and Dmrt1 (1:100) genes overnight at 4 C, and rinsed with 0.01 M PBS three times for 5 min per wash. Subsequently, the tissue sections were incubated with a second antibody (goat anti-rabbit IgG) conjugated with horseradish peroxidase (Bio-Rad, USA) at 1:2000 for 30 min, and then rinsed with PBS three times for 5 min per wash. Immunoreactive signals were visualized using diaminobenzidine (Sigma, USA) as the substrate. Sections were counterstained with hematoxylin. For the negative control, the primary antibody was replaced with normal rabbit serum.
2. Materials and methods
Western blotting was performed to confirm the expression changes of Cyp19a1a, Cyp11b2 and Dmrt1 in both RU486-treated XX fish and control XX/XY fish at 120dah. Briefly, proteins were extracted with buffer containing 50 mM Tris-Cl (pH7.5), 140 mM NaCl, 1% Nonidet P-40, 2 mM EDTA and complete inhibitor cocktail. Then, 150 ng of the whole gonadal extract from each group was separated by SDS-PAGE, and transferred onto a PVDF membrane (Amersham, Sweden). The membranes were blocked with 5% low fat milk powder in TBST (20 mM Tris-HCL pH7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with primary antibody, and then with APlabeled secondary antibody. Finally, the immunoreactive signals were stained with NBT/BCIP substrates and visualized on Fusion FX7 (Vilber Lourmat, France).
2.1. Fish The tilapia for this study were reared in re-circulating aerated freshwater tanks at 26 C under constant photoperiod (12 h light: 12 h darkness). All genetic females (XX) and males (XY) were obtained by artificially fertilizing the eggs from normal females (XX) with sperm from either sex-reversed males (XX) or super males (YY), respectively. Super males (YY) were obtained by crossing normal XY males with an XY female, which was sex-reversed hormonally with E2 treatment. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Laboratory Animal Experimentation at Southwest University, China. 2.2. Treatment of XX tilapia by RU486 and E2 Previous studies revealed that the period around 5dah is critical for the initiation of sex determination/differentiation, in which undifferentiated gonads differentiate into testes or ovaries in tilapia [15]. All treatments were started from 5dah in aerated fresh water and sampled at 120dah. Briefly, fish were divided into five groups, i.e. I) continuous treatment with RU486 till 120dah, II) treatment with RU486 till 20dah, and then feeding with normal commercial diet before sampling, III) simultaneous treatment with RU486 and E2 to 120dah, IV) XX control group, V) XY control group. Fish were maintained in aerated fresh water and fed daily with commercial diet mixed with RU486 (500 mg/g, Sigma) with or without E2 (300 mg/g, Sigma). Meanwhile, the normal XX and XY fish fed with commercial diet were used as control. At 120dah, gonads from both control and treated groups were sampled for subsequent immunohistochemistry (IHC) and real-time PCR analyses.
2.4. Western blotting
2.5. Quantitative real-time PCR analysis Real-time PCR was carried out on an ABI-7500 real-time PCR machine according to the protocol of SYBR Premix Ex Taq II (Takara, Tokyo, Japan), using b-actin as internal control. Gonads from six control XX/XY and six RU486-treated XX fish were collected. Total RNAs were extracted from each sample and reverse transcribed using PrimeScript RT Master Mix Perfect Real Time Kit (Takara, Tokyo, Japan), according to the manufacturer's instructions. Gene expression of foxl2, cyp19a1a, cyp11b2, dmrt1, sycp3 and vasa were analyzed by real-time PCR using gene-specific primers (Supplementary Table S1). The relative abundance of mRNA transcripts was evaluated using the formula: R ¼ 2-△△Ct. Data are expressed as the mean ± SD. Statistical analyses of the results were conducted using one-way ANOVA (P < 0.05) in GraphPad Prism 4 software.
2.3. IHC
2.6. Measurement of E2 levels by enzyme immunology assay (EIA)
At 120dah, gonads from both treated and control groups fish were collected and fixed in Bouin's solution for IHC analysis. Briefly, specimens were embedded in paraffin and sectioned at 5 mm thickness. Paraffin sections were then deparaffinized and hydrated. After washing with 0.01 M PBS three times for 10 min per wash. The sections were then treated in a blocking solution (Roche, Germany), incubated with the polyclonal antibody anti-tilapia Cyp19a1a
For EIA, a minimum of 500 ml blood was drawn from the caudal vein of RU486-treated XX fish and control XX/XY fish at 120dah using a gauge needle and a disposable syringe. Blood samples were centrifuged at 10,000 g for 5 min at 4 C, and the supernatant was collected without disturbing the white buffy layer or the blood cells. Finally, E2 levels in the serum were measured using the E2 EIA Kit according to the manufacturer's instructions (Cayman, USA).
the Leydig cells (F) and the Sertoli cells (G) in XY control, were also detected in the RU486-treated XX fish (I, L, J, M). No positive expression of Cyp19a1a, Cyp11b2 and Dmrt1 were detected in XX gonads with RUXX þ E2 treatment (N, O, P). White arrowheads, black arrowheads and arrows indicate the positive immunostaining of Cyp19a1a, Cyp11b2 and Dmrt1, respectively. (Q) Western bloting analysis of three genes (Cyp19a1a, Cyp11b2, Dmrt1) expression in the gonad of continuous RU486 treatment and control fish. (RUXX, continuous RU486 treatment from 5dah to 120dah; RUXX20-120dah, RU486 treatment from 5dah to 20dah, then feeding with a normal diet until 120dah; RUXX þ E2, simultaneous treatment of RU486 and E2 from 5dah to 120dah; dah,day after hatching)
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
4
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
3. Results
3.2. Rescue of RU486-treated XX fish by estrogen
3.1. Expression of pgr and morphological changes of ovaries under RU486 treatment in XX fish
Previous reports revealed that estrogen is a prerequisite of ovarian differentiation. Therefore, simultaneous treatment of RU486 and E2 were carried out to explore whether estrogen could rescue the defects of RU486 treatment in XX gonad development. As expected, the compensation of estrogen in RU486-treated XX fish regained oogenesis suggesting reoccurrence of oocytes development. However, no positive expression of female-biased gene (Cyp19a1a) and male-biased gene (Cyp11b2 and Dmrt1) were detected in XX gonads with simultaneous treatment with E2 and RU486 (Fig. 1N, O, P).
The expression of Pgr in XX gonads during different developmental stages was investigated by IHC. Strong expression of Pgr was detected in interstitial cells at 30dah (Fig. 1A) and follicular cells at 90dah (Fig. 1B). Then, RU486 treatment was applied to study the role of Pgr-mediated DHP function in XX fish. By 120dah in normal XX tilapia, ovary was filled with oocytes surrounded by somatic cells (Fig. 1E). However, continuous treatment of XX fish with RU486 from 5 to 120dah led to blockage of meiotic entry and oogenesis, and resulted in complete loss of oocytes (Fig. 1HeJ). RU486-treated XX fish from 5 to 20dah and thereafter maintenance in normal condition up to 120dah led to sex reversal with spermatogenesis and differentiation of sperm at 120dah (Fig. 1KeM). It is well documented that aromatase, encoded by cyp19a1a gene, is the critical enzyme to catalyze the biosynthesis of estrogen in fish [16]. On the contrary, cyp11b2 is specifically expressed in the interstitial cells of testis and is required for the production of fish androgen 11-KT [17]. Meanwhile, dmrt1 has been proved to play a conserved role in fish testicular differentiation. Therefore, the expressions of Cyp19a1a, Cyp11b2 and Dmrt1 gene were examined in control and RU486-treated XX gonads by both IHC and western blotting. In control XX gonad, expression of Cyp19a1a could be detected in the interstitial cells at 120dah (Fig. 1E). However, it was severely repressed and disappeared in continuous RU486-treated XX gonads (Fig. 1H). On the other hand, abundant expression of Cyp11b2 and Dmrt1 were observed in RU486-treated XX gonads (Fig. 1I, J, Q). The rate of masculinization and sex reversal in continuous RU486-treated XX fish reached nearly 73.3% and 16.7% (Supplemental Table S2). Consistently, short-term RU486 treatment simultaneously caused the suppression of Cyp19a1a, and induction of Cyp11b2 and Dmrt1, which might have further accelerated the sex reversal process (Fig. 1K, L, M).
3.3. Changes of gene expression profiles Real-time PCR was carried out to further evaluate the molecular changes in RU486-treated XX fish, with or without E2, and control XX/XY fish. RU486 treatment resulted in significant reduction of female specific genes (foxl2 and cyp19a1a) and ectopic upregulation of male specific genes (dmrt1 and cyp11b2). Germ cell mitotic and meiotic marker genes (vasa and sycp3) were also severely inhibited. As aforementioned, compensation of E2 was able to rescue differentiation of oocytes in RU486-treated XX fish, therefore, germ cells marker genes of vasa and sycp3 were evidently increased. Consistent with IHC results, expression of female specific genes (foxl2 and cyp19a1a) were not stimulated in comparison to the control XX fish. Moreover, male dominant genes (dmrt1 and cyp11b2) were also repressed (Fig. 2). 3.4. Evaluation of E2 production level Estrogen has been proved to be essential for ovarian differentiation and maintenance. To explore the estrogen level change in XX fish treated with RU486 alone and combination of RU486 with E2, we collected blood samples from treated XX, control XX and control XY fish at 120dah, and measured the estrogen levels by EIA. No significant difference of estrogen production level was found
Fig. 2. Expression profiles of somatic cell and germ cell marker genes in the gonads of treated and control fish gonads at 120dah analyzed by real-time PCR. Data are expressed as mean ± SD (n ¼ 6), and different letters indicate significant difference (P < 0.05).
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
between XX fish treated with RU486 alone and normal XY fish, suggesting that RU486 treatment alone led to the significant decrease of estrogen in XX fish. However, estrogen level in XX fish treated with both RU486 and E2 was equivalent to control XX fish (Fig. 3).
4. Discussion So far, it has been proved that regulation of oogenesis in fish involves the functions of Follicle Stimulating Hormone (FSH) [18,19], estrogen and Retinoic Acid (RA) [20]. However, the regulatory mechanisms are far more complex and largely unknown. By using in vitro culture system, it was reported that fish progestin (DHP) is involved in proliferation of oogonia and meiotic initiation. In Japanese huchen, significant increase of DHP level was detected during the initiation of the first meiotic division, as indicated by many chromatin-nucleolus stage oocytes [10]. In sockeye salmon (Oncorhynchus nerka), the serum levels of DHP have been shown to increase from about 1 ng/ml to 2 ng/ml in immature females [21]. Using in vitro culture systems, it was shown that DHP was sufficient to promote DNA synthesis in ovarian germ cells. After preculture of the carp ovarian epithelium, these fragments were treated for 15 days with DHP, and BrdU incorporation in numerous ovarian germ cells in DHP-treated ovarian fragments was observed, indicating that DNA was synthesized in the germ cells. The percentage of oocytes displaying SC was significantly increased in the DHPtreated fragments in huchen. Moreover, the meiosis-specific marker Spo11 was expressed in germ cells in the ovarian fragments of DHP-treated carp. These data indicated that DHP might be involved in proliferation of oogonia and meiotic initiation [10]. However, the actions and mechanisms of Pgr-mediated DHP in early stage of ovarian differentiation in fish need to be further explored. In tilapia, proliferation of primordial germ cell (PGC) and initiation of meiosis occurred around 15 and 40dah, respectively. During early stage of ovarian differentiation in tilapia, transcript of cyp17a2, one of the critical steroidogenic enzymes for DHP production during oocytes maturation, starts to increase in the somatic cells from 20dah in XX gonad [22]. Therefore, these data suggest that DHP might be possibly synthesized around the timing of meiotic initiation during sexual differentiation of tilapia ovary. Meanwhile, abundant expression of DHP nuclear receptor (Pgr) in the interstitial cells and follicular cells during the early stages of ovarian differentiation, strongly suggested the possible role of DHP during the early stage of ovarian differentiation. It is well known that the functions of progesterone are mainly mediated by Pgr. RU486, an orally active compound for absorption used in FDA-approved medical treatment, binds to the progesterone receptor with two fold higher affinity than progesterone [23].
5
In this study, we carried out the RU486 treatment of XX fish from 5dah to investigate the potential role DHP in tilapia oogenesis. Our data demonstrated that RU486 treatment led to blockage of oogenesis, suggesting the essentiality of DHP in tilapia oogenesis. Meanwhile, continuous RU486 treatment in XX fish led to the suppression of female specific genes (foxl2 and cyp19ala) and ectopic increase of male specific gene (dmrt1 and cyp11b2), suggesting that blockage of Pgr-mediated DHP action led to the masculinization of somatic cells. Consistently, in comparison with normal XX fish, estrogen level in RU486-treated XX fish was dramatically reduced to the similar level to XY fish. Moreover, no meiotic germ cells were found in RU486-treated (from 5 to 120dah continuously) XX fish. Our data suggest that blockage of Pgrmediated progestin function in XX tilapia results in the repression of female specific genes and decrease of estrogen production, which in turn switches the direction of gonadal development towards male pathway. Therefore, estrogen and progestin might be acting synergistically to promote ovarian differentiation in early developmental stages in fish. However, upon the termination of RU486 treatment at 20dah resulted in the occurrence of spermatogenesis by 120dah. Our data indicated that short-term exposure to RU486 led to the defects of ovarian differentiation and masculinization of XX fish. RU486 treatment by 20dah caused the rapid up-regulation of Gonadal soma-derived factor (gsdf) and dmrt1 gene, two male specific genes in the Sertoli cells in testis, suggesting that the somatic cell environment has switched towards male properties in RU486-treated XX fish (unpublished data). The latest reports revealed that Gsdf and Dmrt1 are the key factors to initiate the male pathway and ensure the testicular differentiation, furthermore, the null mutant of gsdf and dmrt1 gene in XY fish results in the male to female sex reversal and ovarian development in medaka [24,25]. Moreover, over-expression of Gsdf in XX tilapia induced sex reversal from female to male and testicular development [26]. Therefore, ectopic expression of Gsdf and Dmrt1 in RU486-treated XX fish and recovery of endogenous steroids might be the reasons to cause the sex reversal by 120dah. Estrogens play an important role in ovarian differentiation and maintenance in fish. The treatment of tilapia XX fry with fadrozole causes complete sex reversal to functional males [27]. Exogenous/ endogenous estrogen was sufficient to induce the development of fertile ovary. To elucidate the possible interaction of estrogen and DHP in fish oogenesis, simultaneous treatments of exogenous E2 and RU486 in XX fish were carried out. Our data demonstrated that compensation of exogenous estrogen successfully rescued the progression of oogenesis and oocyte differentiation. Intriguingly, estrogen failed to induce complete female development when Pgrmediated DHP function was blocked due to the significant lower expression level of foxl2 and cyp19a1a gene in somatic cells. Our data further strengthened that both estrogen and DHP are required for proper female development. In our opinion, we suppose that occurrence of oogenesis induced by extra estrogen in RU486disrupted XX fish might have originated from the direct function of estrogen on germ cells, but not via endogenous estrogen production in somatic cells. However, the mechanisms need further investigations. In conclusion, for the first time, we have investigated the critical role of DHP in early stage oogenesis of XX tilapia by RU486 treatment. Our data further proves that both DHP and estrogen might be essential for ovarian differentiation. Conflict of interest
Fig. 3. Serum17b-estradiol (E2) level in the control group (XX/XY) and treated group (RUXX, RUXX þ E2) by EIA assay. Data are expressed as the mean ± SD (n ¼ 6), and different letters indicate significant difference(P < 0.05).
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045
6
L. Zhou et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
reported. Acknowledgments The authors cordially thank Prof. Yong Zhu from East Carolina University, North Carolina, for providing the zebrafish nuclear Pgr antibody. This work was supported by grants 31572597, 31201986, 91331119 and 31030063 from the National Natural Science Foundation of China; grant 20130182130003 from the Specialized Research Fund for the Doctoral Program of Higher Education of China; This work was also supported in part by Research Foundation of Talent Introduction of Southwest University, China (SWU111003) and Fundamental Research Funds for the Central Universities, China (XDJK2015A004 and XDJK2014B040) from Ministry of Education of China. The special fund of Chongqing key laboratory (CSTC). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.03.045. References [1] Y. Nagahama, M. Yoshikuni, M. Yamashita, T. Tokumoto, Y. Katsu, Regulation of oocyte growth and maturation in fish, Curr. Top. Dev. Biol. 30 (1995) 103e145. [2] Y. Nagahama, M. Yamashita, Regulation of oocyte maturation in fish, Dev. Growth Differ. 50 (Suppl 1) (2008) S195eS219. [3] H. Ueda, A. Kambegawa, Y. Nagahama, Involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon, Oncorhynchus rhodurus, and goldfish, Carassius auratus, Gen. Comp. Endocrinol. 59 (1985) 24e30. [4] R.W. Schulz, L.R. de Franca, J.J. Lareyre, F. Le Gac, H. Chiarini-Garcia, R.H. Nobrega, T. Miura, Spermatogenesis in fish, Gen. Comp. Endocrinol. 165 (2010) 390e411. [5] T. Miura, M. Higuchi, Y. Ozaki, T. Ohta, C. Miura, Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 7333e7338. [6] S.X. Chen, J. Bogerd, E. Andersson, F.F. Almeida, G.L. Taranger, R.W. Schulz, Cloning, pharmacological characterization, and expression analysis of Atlantic salmon (Salmo salar L.) nuclear progesterone receptor, Reproduction 141 (2011) 491e500. [7] S.X. Chen, J. Bogerd, A. Garcia-Lopez, H. de Jonge, P.P. de Waal, W.S. Hong, R.W. Schulz, Molecular cloning and functional characterization of a zebrafish nuclear progesterone receptor, Biol. Reprod. 82 (2010) 171e181. [8] S.X. Chen, F.F. Almeida, E. Andersson, G.L. Taranger, R. Schmidt, R.W. Schulz, J. Bogerd, Cloning, pharmacological characterization and expression analysis of Atlantic cod (Gadus morhua, L.) nuclear progesterone receptor, Gen. Comp. Endocrinol. 179 (2012) 71e77. [9] G. Liu, F. Luo, Q. Song, L. Wu, Y. Qiu, H. Shi, D. Wang, L. Zhou, Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus), J. Mol. Endocrinol. 53 (2014) 57e70. [10] C. Miura, T. Higashino, T. Miura, A progestin and an estrogen regulate early
stages of oogenesis in fish, Biol. Reprod. 77 (2007) 822e828. [11] Y. Nagahama, Molecular mechanisms of sex determination and gonadal sex differentiation in fish, Fish. Physiol. Biochem. 31 (2005) 105e109. [12] S. Ijiri, H. Kaneko, T. Kobayashi, D.S. Wang, F. Sakai, B. Paul-Prasanth, M. Nakamura, Y. Nagahama, Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus, Biol. Reprod. 78 (2008) 333e341. [13] T. Kobayashi, H. Kajiura-Kobayashi, Y. Nagahama, Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia, Cytogenet Genome Res. 101 (2003) 289e294. [14] D.S. Wang, T. Kobayashi, L.Y. Zhou, B. Paul-Prasanth, S. Ijiri, F. Sakai, K. Okubo, K. Morohashi, Y. Nagahama, Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1, Mol. Endocrinol. 21 (2007) 712e725. [15] W. Tao, J. Yuan, L. Zhou, L. Sun, Y. Sun, S. Yang, M. Li, S. Zeng, B. Huang, D. Wang, Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes, PLoS One 8 (2013) e63604. [16] Y. Guiguen, A. Fostier, F. Piferrer, C.F. Chang, Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish, Gen. Comp. Endocrinol. 165 (2010) 352e366. [17] M.A. Alam, R.K. Bhandari, Y. Kobayashi, S. Nakamura, K. Soyano, M. Nakamura, Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra, Mol. Reprod. Dev. 73 (2006) 206e214. [18] B. Levavi-Sivan, J. Bogerd, E.L. Mananos, A. Gomez, J.J. Lareyre, Perspectives on fish gonadotropins and their receptors, Gen. Comp. Endocrinol. 165 (2010) 412e437. [19] E. Lubzens, G. Young, J. Bobe, J. Cerda, Oogenesis in teleosts: How fish eggs are formed, Gen. Comp. Endocrinol. 165 (2010) 367e389. [20] R.J. Feng, L.L. Fang, Y.Y. Cheng, X. He, W.T. Jiang, R.R. Dong, H.J. Shi, D.N. Jiang, L.N. Sun, D.S. Wang, Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus), Sci. Rep. 5 (2015). [21] R.K. Bhandari, I. Ushikoshi, H. Fukuoka, N. Koide, K. Yamauchi, H. Ueda, Effects of Rhizopus extract administration on somatic growth and sexual maturation in lacustrine sockeye salmon Oncorhynchus nerka, Fish. Sci. 68 (2002) 776e782. [22] L.Y. Zhou, D.S. Wang, T. Kobayashi, A. Yano, B. Paul-Prasanth, A. Suzuki, F. Sakai, Y. Nagahama, A novel type of P450c17 lacking the lyase activity is responsible for C21-steroid biosynthesis in the fish ovary and head kidney, Endocrinology 148 (2007) 4282e4291. [23] O. Heikinheimo, R. Kekkonen, P. Lahteenmaki, The pharmacokinetics of mifepristone in humans reveal insights into differential mechanisms of antiprogestin action, Contraception 68 (2003) 421e426. [24] T. Imai, K. Saino, M. Matsuda, Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development, Biochem. Biophys. Res. Commun. 467 (2015) 109e114. [25] H. Masuyama, M. Yamada, Y. Kamei, T. Fujiwara-Ishikawa, T. Todo, Y. Nagahama, M. Matsuda, Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka, Chromosome Res. 20 (2012) 163e176. [26] H. Kaneko, S. Ijiri, T. Kobayashi, H. Izumi, Y. Kuramochi, D.S. Wang, S. Mizuno, Y. Nagahama, Gonadal soma-derived factor (gsdf), a TGF-beta superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus, Mol. Cell Endocrinol. 415 (2015) 87e99. [27] V. Gennotte, P. Mafwila Kinkela, B. Ulysse, D. Akian Djetouan, F. Bere Sompagnimdi, T. Tomson, C. Melard, C. Rougeot, Brief exposure of embryos to steroids or aromatase inhibitor induces sex reversal in Nile tilapia (Oreochromis niloticus), J. Exp. Zool. A Ecol. Genet. Physiol. 323 (2015) 31e38.
Please cite this article in press as: L. Zhou, et al., Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus), Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.045