RESEARCH ARTICLE Molecular Reproduction & Development 82:694–708 (2015)

Differential Expression Patterns of Three Aromatase Genes and of Four Estrogen Receptors Genes in the Testes of Trout (Oncorhynchus mykiss) CHRISTELLE DELALANDE,1,2,3 ANNE-SOPHIE GOUPIL,4 JEAN-JACQUES LAREYRE4,

AND

FLORENCE LE GAC4*

1

Normandie Univ, France UNICAEN, EA 2608, France 3 INRA USC 1377, 14032 CAEN cedex 5, France 4
nomique des Poissons, SFR BIOSIT, Biogenouest, 35042 Rennes, France INRA, UR1037 Laboratoire de Physiologie et Ge 2

SUMMARY Estrogens are implicated in male gonad function, although their physiological roles remain uncertain. In the present study, we take advantage of the original model of spatio-temporal organization of trout spermatogenesis to revisit the synthesis and action sites of estrogens in fish testis. Within this system, somatic cell and germ cell development are synchronized due to a strict seasonal spermatogenetic cycle and the cystic organization of gonads. We evaluated the expression patterns and regulation of three aromatase isoforms (cyp19a,cyp19b-I, and cyp19b-II) and four estrogen receptors (esr1a, esr1b, esr2a, and esr2b) by quantitative reversetranscriptase PCR during testicular maturation and in isolated germ cell populations. Our data demonstrated a reciprocal relationship between cyp19a and cyp19b (I and II) expression during testicular development (cyp19a decreased while cyp19b increased with maturation). Furthermore, cyp19b is significantly expressed in late germ cells. At the protein level, aromatase was immunohistochemically identified in interstitial tissue and in germ cells. Remarkable elevation of esr1a and esr2a was observed during the final stage of spermiation, while esr1b was expressed in an early stage of spermatogenetic development. Estrogen implants reduced testicular cyp19a transcript abundance while up-regulating cyp19b levels, whereas androgens upregulated testicular esr1a, esr2a, and esr2b. Together, the distinct spatio-temporal expression profiles and regulation of aromatases and estrogen receptors suggest that estrogens have discrete physiological functions during an early step of spermatogenesis and in the final stages of germ cell maturation and/or excretion. Mol. Reprod. Dev. 82: 694
708, 2015. ß 2015 Wiley Periodicals, Inc. Received 16 June 2014; Accepted 24 May 2015



Corresponding author: INRA, UR1037 Laboratoire de Physiologie et Genomique des Poissons SFR BIOSIT, Biogenouest 35042 Rennes, France. E-mail: [email protected]

Part of this work was presented at the 7th International Symposium on Fish Endocrinology, Buenos Aires, Argentina. Grant sponsor: INRA PHASE; Grant sponsor: French National Research Agency; Grant number: ANR-06-GANI-014

Published online 16 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22509

INTRODUCTION Spermatogenesis is a tightly regulated and complex process resulting in the production of spermatozoa from spermatogonia. It begins with the proliferation of spermatogonia by mitosis, leading to the formation of primary spermatocytes. These spermatocytes then undergo meiosis to

ß 2015 WILEY PERIODICALS, INC.

Abbreviations: 11KT, 11-ketotestosterone; cyp19, cytochrome P450 aromatase gene; E2, estradiol; esr, estrogen receptor gene

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become haploid round spermatids, which will be transformed into mature spermatozoa by differentiation (spermiogenesis). The basic features and the regulatory mechanisms of spermatogenesis are well-conserved throughout vertebrate evolution. For example, spermatogenesis is under the control of the gonadotropins luteinizing hormone and follicle-stimulating hormone, which respectively control the production of androgens by Leydig cells and the physiological function of Sertoli cells. Other endocrine, paracrine, and autocrine factors also help regulate the production of male gametes in mammals (reviewed in McLachlan et al., 2002; Huleihel and Lunenfeld, 2004) and in fish (Schulz et al., 2010). Estrogens are classically known to play a major role in female reproduction, but there is now compelling evidence that they may also be involved in the development and control of male reproductive functions. Studies in mammals, in particular, were the first to link estrogen activity with male reproduction: low fertility in patients with congenital estrogen deficiency (reviewed in Jones et al., 2006) or estrogen resistance (Smith et al., 1994), or infertility in mouse models that lack aromatase (Ar knockouts) or estrogen receptors (Esr1 and Esr2 knockouts) (Eddy et al., 1996; Robertson et al., 1999; Antal et al., 2008). Specifically, impaired sperm motility and the arrest of mature germ cell production are observed in aromatase-deficient men and mice (reviewed in Rochira et al., 2005). Conversely, exposure of adult male rats to estradiol (E2) or to a high phytoestrogen diet disrupts spermatogenesis through a failure in spermiation and an increase in germ cell apoptosis, respectively (D’Souza et al., 2005; Assinder et al., 2007). Over-expression of the aromatase gene in mice leads to infertility in all or half of the males in a litter, depending on when overexpression occurs

e.g., during the fetal life or at puberty, respectively (reviewed in Li and Rahman, 2008). Together, these studies suggest that a regulated level of estrogen is essential for normal testicular physiology and reproduction. A number of studies in various mammalian models have focused on the specific question of testicular functional modulation by estrogens, but the way E2 regulates spermatogenesis remains largely misunderstood (reviewed in Carreau and Hess, 2010). Notably, very little is known about the localization and the hormonal regulation of estrogen production and estrogen receptor-expressing cells in different vertebrate models, or about the role of E2 at specific steps during male gonad development. Seasonal-breeding fish, such as the rainbow trout (Onchorynchus mykiss), are useful models to investigate those specific relationships in the testis because their seasonal spermatogenetic cycle plus the cystic organization of the testis help to synchronize the developmental state of somatic cells. Furthermore, endocrine data for several fish species support a potential role for estrogens in the male (Vizziano et al., 2008; Schulz et al., 2010). In particular, E2 was reported to play a physiological role during the mitotic phase of spermatogonia (Miura et al., 1999; Amer et al., 2001). Conversely, deleterious effects of estrogen treatment on spermatogenesis were also demonstrated in fish (de Waal et al., 2009), and exposure to xenobiotics with

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estrogen-mimetic actions or anti-estrogen potential can be detrimental to male reproduction, testicular differentiation, or testis function (reviewed in Cheshenko et al., 2008; Segner et al., 2013). These data suggest that fish are an important model for analyzing the role of endogenous estrogens during the male reproductive cycle. Estrogen production and target-cell signalling are regulated by the aromatase and estrogen receptors, respectively. In teleostean species, functional aromatase enzyme can be produced from two paralogous genes, cyp19a1a (generally named cyp19a) and cyp19a1b (generally named cyp19b) (Tchoudakova and Callard, 1998; Zhao et al., 2001)

although cyp19a is predominantly expressed in the ovary and the cyp19b form in the brain. More recently, a third gene encoding a variant of the brain aromatase (cyp19b-II) has been identified in the rainbow trout (Dalla Valle et al., 2005). Four genes have been described for nuclear estrogen receptors: esr1a, esr1b, esr2a, and esr2b (Nagler et al., 2007). We analyzed the spatio-temporal expression pattern of these genes during the annual cycle of testicular development and in isolated populations of germ cells to evaluate the relationship of estrogens in the hormonal control of rainbow trout spermatogenesis. As these genes are potentially under the control of sex steroids (reviwed in Bourguiba et al., 2003; Nelson and Habibi, 2013), the regulation of transcription of these genes by estrogens and androgens was also examined in vivo.

RESULTS Expression of cyp19 Genes and Aromatase Protein in the Testis Follows the Stage of Testicular Pubertal Development and the Germ Cell Type In teleosts, two paralogs (cyp19a1a and cyp19a1b) were described that encode for aromatase A (abundant in the ovary) and aromatase B (abundant in the brain). Both forms are expressed in rainbow trout prepubertal testes (Stage I), although cyp19a transcript abundance may be higher than cyp19b (about four- to sixfold). Aromatase transcript abundance in the testis, however, is low: cyp19a transcript was 50-fold more abundant in the vitellogenic ovary than in the prepubertal testis, while cyp19b transcript was 100-fold more abundant in the brain than in the prepubertal testis. We also considered the expression of two recently proposed trout genes encoding aromatase B, cyp19a1b-I and cyp19a1b-II (Dalla Valle et al., 2005). Although we could not find these cyp19a1b-II genes in the trout genome, we tested primers designed to discriminate between cyp19b-I and cyp19b-II transcripts, based on sequences reported by Dalla Valle et al. (2005), and found that cyp 19b-II transcript is present in the brain and in testis, with cyp19b-II transcript 10- to 30-fold less abundant than cyp19b-I in the brain (Supplemental Fig. 1). Expression profiling during testis development revealed a decrease in the relative expression of cyp19a mRNA during organ maturation, reaching an undetectable level in the fully

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mature gonad (Stage VIII) (Fig. 1A). In contrast, the relative abundance of both cyp19b-I and cyp19b-II increased in later stages of testis development, when post-meiotic cells (spermatocytes and spermatids) accumulate (Fig. 1B). As important changes occur in germ cell composition that can strongly affect the ‘‘relative abundance’’ of a transcript during gonad development, we also present the ‘‘total amounts’’ of cyp19a and cyp19b transcripts per pair of gonads (Fig. 1C). Again, cyp19b transcripts increased significantly from Stage III to V
VI whereas cyp19a decreased, indicating its overall down-regulation during the reproductive cycle despite increasing gonad size. Note that when using primers that do not distinguish between cyp19b-I and cyp19b-II (Table 1), total cyp19b has a similar transcript profile as each variant alone (Supplemental Fig. 2) We next investigated the involvement of the germinal component in aromatase expression by evaluating gene expression in stage-specific germ-cell populations separated by centrifugation and enriched in spermatogonia, spermatocytes, or spermatids, as described in the Material and Methods section. cyp19a gene expression was absent from the germ cell fractions. In contrast, cyp19b-I and cyp19b-II were expressed at low levels in early germ cells; indeed, the relative transcript abundance of cyp19b-I increased in the fractions containing spermatocytes and spermatids/spermatozoa (Fig. 2A), implicating that the increase in testicular cyp19b expression between Stages II and V
VI was, at least in part, linked to aromatase expression in the meiotic/ post meiotic germ cells. Again, total cyp19b has similar transcript profiles as each variant separately (Supplemental Fig. 3). The cyp19b product obtained from the meiotic/postmeiotic germ cell fraction was sequenced and confirmed to correspond to an aromatase B transcript. In addition, the enrichment of the different cellular germ cell fractions was confirmed by the differential expression profiles of vasa and rsph3, which are respectively expressed in spermatogonia and in meiotic/post-meiotic germ cells (Fig. 2B,C). The cellular localization of the aromatase protein was further investigated using an anti-mouse aromatase antibody that had been generated against a peptide homologous to the trout sequence, which labelled interstitial cells and post-meiotic germ cells of the rat testes (Fig. 3B), as previously reported (Levallet et al., 1998). Specific immunostaining was observed in the somatic interstitial compartment in immature Stage I and in Stage IV
V of trout testis development (Fig. 3C,D). Labelling was also detected in germ-cell cysts containing spermatogonia, spermatocytes, or spermatids (Fig. 3E,F). No staining in trout testis was observed without primary antibody (Fig. 3A). The faint germ-line staining pattern obtained with this anti-mouse antibody was similar to that reported using anti-aromatase generated by Forlano et al., (2001) (Supplemental Fig. 4).

Expression of the Genes Encoding for Nuclear Estrogen Receptors by Testicular Stage of Maturation and Germ Cell Type

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Figure 1. Expression of cyp19a, cyp19b-I, and cyp19b-II during testicular developmental stages. Gene expression was determined by quantitative reverse-transcriptase PCR and normalized to rs15 gene expression. A,B: The relative abundance of the transcripts (A) cyp19a and (B) cyp19b-I and cyp19b-II throughout the male reproductive cycle. C: The total abundance of cyp19 transcripts for two gonads. Roman numerals (I-VIII) indicate testicular development stages. Results are means  standard error of the mean (n ¼ 4
5). Values are statistically different when P < 0.05, according to the following symbols:  , I6¼VIII; #, II6¼VIII; §, I6¼V
VI; and , II6¼V
VI. In the total abundance graph (C), P(Stage I versus Stage V-VI) < 0.001 and > esr1b), with their individual expression profiles changing significantly over the reproductive cycle. esr1b expression was low compared to others esr isoforms; in particular, esr1b transcript abundance was 30- to 100-fold lower than esr1a transcript (Fig. 4A). esr1a, esr2a, and ers2b transcripts decreased from Stages I to V
VI, as germ cells accumulate in the seminiferous tubules; this decrease was statistically significant only for esr2b (Fig. 4A). In contrast, the relative abundance of esr1b transcript accumulated between Stages I and IIIa, when differentiating and proliferating type B spermatogonia accumulate in the tubules and when the first primary spermatocytes appear (see Supplemental Table 1). Later during development, dramatic increases in esr1a and esr2a mRNA level were observed at Stage VIII, when only haploid cells are present and spermatozoa have been released into the lumen of seminiferous tubules (Fig. 4A,B) (62- and 17-fold changes in mean quantities, respectively). All of these receptor transcripts were detected in the germ cell fractions. esr1a and maybe esr2a abundance increased in meiotic/post-meiotic cells. The relative abundances of esr transcripts in the germ cell fractions were lower than total testicular tissue (relative expressions esr1a: isolated ‘‘Spc þ Spt’’ ¼ 0.012  0.009 compared to testis in Stage VIII ¼ 0.076  0.038; esr2a: isolated ‘‘Spc þ Spt’’ ¼ 0.002  0.001 compared to testis in Stage VIII ¼ 0.022  0.011), suggesting their preferential expression in somatic cells (Fig. 4C).

Effects of Hormonal Treatment on Aromatase Transcripts Supplementation by E2 implants at two doses caused a significant decrease in cyp19a mRNA levels (Fig. 5A). In contrast, a stimulatory effect was observed on cyp19b mRNA levels following the highest E2 dose (Fig. 5B). On the other hand, the aromatisable androgen, testosterone, but not 11-ketotestoterone (11KT), also inhibited cyp19a expression, whereas neither androgen treatment affected the expression of cyp19b gene (Fig. 6A,B).

Effects of Hormonal Treatment on Estrogen Receptor Transcripts E2 treatment induced an elevation in esr1b transcripts at the highest dose, but had no significant effect on the levels of esr1a, esr2a, or esr2b mRNA (Fig. 7). Interestingly, testosterone and 11KT elevated esr1a and esr2a mRNA levels (Figs. 8A and 8C), while a stimulatory effect on esr2b mRNA level was only observed after 14 days of testosterone treatment (Fig. 8D). Conversely, 11KT had no effect on esr1b or esr2b gene transcript levels (Figs. 8B and 8D).

DISCUSSION The mammalian testis is able to produce estrogens, and that estrogen-receptor signalling is involved in normal

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testicular function. The present study explored the possible analogous roles of estrogen in testicular physiology in fish, reporting on the expression pattern of genes related to estrogen synthesis and signalling pathways

cyp19a and cyp19b, esr1 and esr2

together with their regulation by estrogens and androgens.

Reciprocal Transcription Profiles of Aromatase A and B Genes During Testis Maturation In teleost fish, functional aromatase enzyme can be produced from both cyp19a and cyp19b genes (Tchoudakova and Callard, 1998; Zhao et al., 2001). Teleosts have further evolved a brain-specific cyp19b, probably for the specialized function of estrogen synthesis in the brain (Chiang et al., 2001; Diotel et al., 2010). Although cyp19a is predominantly expressed in the ovary while cyp19b expression is enriched in the brain, transcripts of both forms were found in each of these tissues (Dalla Valle et al., 2002). The present study highlights the transcription of the two aromatases (A and B) in the trout testis. When analysing each cyp19b-I and cyp19b-II variant, first described by Dalla Valle et al. (2005), we found that cyp19b-II was less-abundant in the brain compared to cyp19b-I, although both forms are expressed at a low level in the immature trout testis. These data are in accordance with previous reports for Nile tilapia (Chang et al., 2005), sea bass (Blazquez and Piferrer, 2004), Atlantic salmon (Von Schalburg et al., 2013), Atlantic halibut (Matsuoka et al., 2006), and zebrafish (Sawyer et al., 2006; Hinfray et al., 2013). Taking advantage of the highly cyclic spermatogenetic process in trout, we found that cyp19 expression is generally low in the testis compared to the brain or ovaries, but is enriched at particular stages of gonadal development. cyp19a transcript was highest in early stages of testis development, but rapidly decreased during testicular maturation and spermatogenetic development; conversely, cyp19b-I and cyp19b-II transcripts increased during maturation. Thus, cyp19a and cyp19b isoforms appear to be expressed in specific cell types or co-localize to similar specialized cells in the testis that can differentially regulated cyp19 gene expression.

Cyp19a is Likely of Somatic Origin While cyp19b-I Increases in Post-Meiotic Germ Cells Our study indicates that the cyp19a and cyp19b gene expression is differentially distributed among testicular cell types. Expression profiles indicate that cyp19a mRNA is most abundant in early stages of development, but rapidly decrease from Stage I to VI as the proportion of germ cells rapidly increases in seminiferous tubules. The dilution of somatic mRNA by germ-cell mRNA at advanced stages of testis growth and differentiation may explain this drop in the relative expression of cyp19a mRNA, as previously found for other genes (Rolland et al., 2009). Furthermore, cyp19a transcript was not detectable in isolated germ cell fractions. Together these observations suggest that the expression of cyp19a is restricted to the somatic cells.

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Figure 2. Expression of cyp19a, cyp19b-I, and cyp19b-II in fractions enriched for isolated germ cells. Gene expression was determined by quantitative reverse-transcriptase PCR and normalized to rs15 gene expression. Relative abundance of (A) cyp19a, cyp19b-I, and cyp19bII; (B) vasa; and (C) rsph3 transcripts. cyp19a, cyp19b-I, and cyp19b-II values are shown normalized to Stage I abundance. vasa and rsph3 values are shown in comparison to the fraction of type A spermatogonia. ND, not detected. Sg A, type A spermatogonia (n ¼ 3
6); Sg A þ B, type A and B spermatogonia (n ¼ 4
5); Sg B þ Sc, type B spermatogonia and spermatocytes (n ¼ 2
6); Sc þ St, spermatocytes and spermatids (n ¼ 3
6). Results are means  standard errors of the mean. Values are statistically different when P < 0.05, according to the following symbols:  , Sc þ St 6¼ other germ cells fractions in panel A;  , Sc þ St 6¼ Sg A and 6¼Sg A þ B in panel C.

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cyp19b expression, on the other hand, rapidly increases as meiotic and post-meiotic germ cells accumulate in the gonad. This isoform is detected in isolated germ-cell populations, with cyp19b-I specifically enriched in spermatocytes and spermatids at both the transcript and protein level. This is consistent with its immunodection in isolated germ cell fractions (this study) and in the spermatozoa of the spawning trout (Kotula-Balak et al., 2008). Furthermore, some label could be observed in pre- and post-meiotic germ cells when using a specific anti-salmon aromatase B, graciously provided by Dr. Von Schalburg

although the staining was faint (data not shown). Besides, aromatase B was recently immuno-detected in the spermatogonia of immature salmon (von Schalburg et al., 2013) and in most germ cells in the zebrafish testis (Hinfray et al., 2013). We therefore propose that the cyp19b expression in post-meiotic cells could at least partially account for the increasing expression of cyp19b at late stages of trout testis maturation. From our data, we hypothesize that the expression of cyp19a is predominant in interstitial cells while the expression of cyp19b occurs in germ cells. We cannot, however, exclude that cyp19b is also expressed in the somatic component of the trout testis, as suggested in salmon (von Schalburg et al., 2013). Based on specific immunodetection of the different forms of aromatase in the zebrafish, Hinfray et al. (2013) proposed that Leydig cells express aromatase A (Cyp19a) while testicular germ cells express aromatase B (Cyp19b), which is in line with our hypothesis in trout. Yet these authors also detected Cyp19a in germ cells, an observation that differs from our quantitative reverse-transcriptase PCR findings on isolated germ cells; this point will need further clarification. Further studies are also planned to elucidate the functionality of aromatase B in trout germ cells. Mammalian somatic cells and germ cells clearly express aromatase (reviewed in Carreau and Hess, 2010). In the adult rat, Cyp19 gene expression peaks in pachytene spermatocytes whereas aromatase activity is maximal or maximum? in haploid cells (Levallet et al., 1998; Silandre et al., 2007). Consistent with this observation, our profiling data suggest that aromatase B in post-meiotic germ cells could be of functional importance for the late stages of testicular maturation in trout. Indeed, significant expression and activity of aromatase A in the late-vitellogenic oocyte was previously described in trout (Gohin et al., 2011), which supports the participation of the germinal compartment in fish gonadal E2 synthesis. We also found that in vivo E2 supplementation negatively regulated cyp19a mRNA levels in prepubertal testes. This is in agreement with the inhibitory effect of E2 on cyp19a mRNA in previtellogenic ovarian follicles described in trout (Nakamura et al., 2009) and on ovarian cyp19a gene expression in zebrafish (Hinfray et al., 2006). In contrast, E2 treatment increased cyp19b mRNA levels in our experiment, which is in line with previous studies reporting that brain cyp19b was up-regulated by exposure to estrogen or aromatisable androgen in goldfish, zebrafish, or male Japanese medaka brains (Gelinas et al., 1998; Sawyer et al., 2006; Zhang et al., 2008). In fact, high

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Figure 3. Immunolocalisation of aromatase in rainbow trout testes. Slices were incubated with a polyclonal rabbit anti-mouse aromatase antibody (B
F) followed by a biotinylated secondary antibody, streptavidin peroxidase, and development with 3, 30 -diaminobenzidine (DAB). A: Negative control, without primary antibody. B: Positive control, from adult rat testis. C: Stage I of spermatogenesis, with positive-stained interstitial cells indicated by arrows. Insert, higher magnification showing positive immunostaining of interstitial cell. D
F: Stage IV of spermatogenesis, with positive labelling observed in interstitial cells (arrow, D) and in cysts containing spermatogonia (arrow) and spermatocytes or spermatids (arrow head) (E
F). Scale bars, 100 mm (A
C); 200 mm (D), and (E
F).

expression of cyp19b in brain radial glial cells of zebrafish could result from an auto-regulatory loop involving an estrogen response element located on the cyp19b proximal promoter (Diotel et al., 2010). Yet, our data contradict the absence of an E2 effect on cyp19 transcripts or aromatase proteins reported for zebrafish testis (Sawyer et al., 2006; Hinfray et al., 2013). This inconsistency may reflect important differences in experimental design (e.g., reproductively mature zebrafish males were exposed to 10
100 nM waterborne E2 versus immature trout were exposed via E2 implants). In particular, the physiological age of the male may be essential to observe the impact of E2 exposure on cyp19a, which was unambiguous in the prepubertal trout testis. The mechanism underlying this effect is not known. In conclusion, the teleost cyp19 genes that encode for aromatases A and B demonstrate distinct spatio-temporal expression profiles during the testicular cycle, and are differentially regulated by sex steroids. This alterative usage is analogous to the mammalian testicular regulation of a single aromatase that encodes distinct transcripts with distinct

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regulations, which results from the use of alternative promoters and untranslated first exons (Bulun et al., 2003). The physiological significance of these different aromatase transcripts in vertebrate reproductive tract is still open to debate.

Remarkable Elevation of esr1a and esr2a During Final Testis Maturation and Expression of esr1b in an Early Stage of Germ Cell Differentiation Juvenile rainbow trout gonads express the four forms of estrogen receptors: esr1a, esr1b, esr2a, and esr2b (Nagler et al., 2007); our study described the expression profile of each isoform in the testis, from pre-pubertal spermatogenesis to final maturation, and in response to sex steroids. The four esr variants exhibit distinct, although partially overlapping, expression patterns. The unique period suggest that each has a non-redundant function in the teleost testis. Most interestingly esr1a and esr2a presented similar profiles with a remarkable peak of expression during final testicular maturation and spawning (Stage VIII). These two forms

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Figure 4. Expression of esr1a, esr1b, esr2a, and esr2b during testicular development, in whole testis or in enriched fractions of isolated germ cells. Gene expression was determined by quantitative reverse-transcriptase PCR and normalized to rs15 gene expression. A: Relative abundance throughout the male reproductive cycle. B: Abundance for two gonads. Roman numerals (I
VIII) indicate testicular development stages. Results are means  standard errors of the mean (n ¼ 4
5). Note the different scale for esr1a and esr1b. Values are statistically different when P < 0.05, according to the following symbols:  , IIIb 6¼ VIII (esr1a in panels A and B, P < 0.01); #, V
VI 6¼ VIII (esr1a and esr2a in panel A, P < 0.01); ^, IIIa 6¼ V
VI; X, IIIa 6¼ I; &, I 6¼ IIIb; §, I 6¼ V
VI (esr2b in panel A and esr1b in panel B, P < 0.01); , II 6¼ V
VI. C: Relative abundance in germ cells.  , Sc þ St 6¼ other germ cells fractions in panel C. Sg A, type A spermatogonia (n ¼ 3
6); Sg A þ B, type A and B spermatogonia (n ¼ 4
5); Sg B þ Sc, type B spermatogonia and spermatocytes (n ¼ 2
6); Sc þ St, spermatocytes and spermatids (n ¼ 3
6).

were also detected in germ cell fractions, which is in line with the detection by immunohistology of Esr1 in the interstitial and endothelial cells but also in the germ line in trout testis (Massart et al., 2014). The elevation of esr1a and esr2a expression at the end of the trout reproductive cycle is consistent with the recent finding that cod esr1a and esr2a are also elevated in the testis before the spawning period (Nagasawa et al., 2014). Moreover, this profile

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corresponds to the typical transcription profile of a group of mRNAs that we previously described in a large-scale analysis of the testicular transcriptome during the trout reproductive cycle (cluster ‘‘C’’ in Rolland et al., 2009). Several of the transcripts that were preferentially or specifically expressed at the spawning stage encode for proteins involved in water transport and in Hþ, Kþ, and Ca2þ regulation, and therefore are probably important for spermatozoa

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Figure 5. Effects of E2 administration on (A) cyp19a and (B) cyp19b mRNA levels. Gene expression was determined by quantitative reverse-transcriptase PCR, and was normalized to rs15 gene expression. The E2 implants were present for 7 days (7D) or 14 days (14D). Results are means  standard errors of the mean (n ¼ 4
5 per treatment). Values are statistically different when P < 0.05, according to the following symbols:  , P < 0.05;  , P < 0.01.

final maturation and for the constitution of semen fluid in teleosts (Rolland et al., 2009; unpublished data by Bella€ıche and Le Gac). In such contexts, it is interesting to note that Esr1-knockout mice are infertile, and present seminiferous tubule degeneration due to an inhibition of normal fluid reabsorption in the efferent ducts leading to an accumulation of fluid in the lumen (Eddy et al., 1996, for review Joseph et al., 2011). The physiological functions of ESR2 in vertebrate gonad are still controversial. One study revealed that Esr2-null mutant males mice are sterile, despite the fact the testis and epididymis histology of these knockout males were normal (Antal et al., 2008). In cod, esr1a, esr2a, and esr2b transcripts were enriched within interstitial fibroblasts composed of immature and mature Leydig cells, suggesting a direct function of estrogen in testicular somatic cell (Nagasawa et al., 2014). Yet, we found that esr1a and esr2a are expressed in differentiated trout germ cells, which opens the question of a direct function for Esr2 in the final maturation of germ cells.

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esr1b expression was generally very low in trout testis, but exhibited an interesting increase at Stages II and IIIa, during which rapid proliferation of differentiated type B spermatogonia begins. Although esr1b transcript was detected in germ cell fractions, its relative levels were around 40-fold lower compared to the Stage-IIIa gonad. Therefore, germinal esr1b might not significantly contribute to the regulation of germ cell proliferation. We found little significant regulation of estrogen receptor expression in trout testes after individuals were exposed to E2 implants, although esr1b transcription did change following the highest dose of E2. Within in the same experiments, known target genes of Esr

such as vitellogenin and choriogenin L

were strongly up regulated by the E2 treatment (see Schulz et al., 2010), confirming that the E2 implants were active. While autoregulation of Esr1 by estrogens is fairly ubiquitous across species and tissues, regulation of Esr2 varies significantly among species/studies or tissues (review by Nelson and Habibi, 2013). Indeed, contradictory data were obtained regarding zebrafish gonads: Exposure to 17a-ethinylestradiol (EE2) had no effect on esr expression (Zhang et al., 2008), whereas E2 modestly increased the expression of esr1 in adult testis (but not of esr2a or esr2b, and not in the brain or ovary) (Chandrasekar et al., 2010). We also observed a significant stimulatory effect of E2 on esr1b transcript, but only in the immature trout testis. Taking into account that esr1a, esr2a, and esr2b have previously been reported as target genes for E2 in other organs of the trout and zebrafish (Pakdel et al., 1991; Flouriot et al., 1996; Menuet et al., 2004; Boyce-Derricott et al., 2009), our results reveal that the control of esr transcription by E2 depends on the organ/ cellular context or on the developmental stage of the testis In contrast, esr1a and esr2a mRNA levels were positively regulated by 11KT and testosterone, which is consistent with our observations that esr1a and esr2a are overexpressed in Stage VIII of testicular maturation, when circulating androgen levels during the trout reproductive cycle are greatest (Rolland et al., 2013). Although we presume that the cyp19a response to E2 and esr1 and esr2 responses to 11KT and testosterone occurred via the intra-testicular action of supplemented steroids, we cannot exclude that the effects we observed after in vivo hormone supplementation partially result from alteration in the circulating levels of gonadotropin or other endocrine factors. Indeed, positive and negative effects on basal gonadotropin secretion by testosterone and E2 during the early recrudescence phase of the gonadal cycle have been reported (Dickey and Swanson, 1998; Khan et al., 1999), and at least part of the inhibitory effects of in vivo E2 treatment on spermatogenesis in zebrafish was exerted via feedback inhibition of gonadotropin release (De Waal et al., 2009).

The Physiological Role of the E2 Pathway Would Depend on the Stage of Testis Development The role of E2-Esr signalling in the fish testis remains poorly understood, and its study is made more complex by the expression of numerous aromatase and estrogen

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Figure 6. Effects of testosterone (T) and 11 KT administration on (A) cyp19a and (B) cyp19b mRNA levels. Gene expression was determined by quantitative reverse-transcriptase PCR, and was normalized to rs15 gene expression. The androgen implants were present for 7 days (7D) or 14 days (14D). Results are means  standard error of the mean (n ¼ 4
6 per treatment). Values are statistically different when P < 0.05, according to the following symbol:  , P < 0.05.

receptor variants. The differential expression patterns of cyp19a and cyp19b (aromatase A and B, respectively) plus the four esr genes

as well as their differential sensitivities to steroid exposure

indicate that the corresponding proteins may have different physiological functions during testis development. It must also be mentioned that two transitory increases in male trout blood E2 levels are observed during the reproductive cycle: one in early stages of active spermatogenesis (II
III), when spermatogonia start to differentiate and proliferate, and one in Stage VII, at the end of spermiogenesis and before sperm release (unpublished results). Our data demonstrate tight regulation of estrogen production and Esr signalling during testicular spermatogenic cycling, and thereby clearly highlight the importance of this pathway in fish testis physiology. Our results allow us to speculate on the physiological implications of testicular aromatase and E2 receptors during maturation in trout. In early stages of testis development, the expression of

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cyp19a may provide the major source of intratesticular aromatase activity. Intratesticular E2 production and esr1b expression at Stages II to IIIa could be involved in the proliferation of spermatogonia that occurs at these stages. This last hypothesis is supported by the fact that E2 stimulates the proliferation of spermatogonia in fish (Miura et al., 1999; Amer et al., 2001). On the other hand, the increase in cyp19b abundance towards the end of the spermatogenetic cycle is consistent with the second, transitory elevation of E2 plasma levels that we observe during late stages of the reproductive cycle. Interestingly, a role for estrogen at this stage of trout testicular development was suggested before: 17b-estradiol treatment inhibited the production of progestin (17,20 bP), which is known to regulate sperm maturation or release (Vizziano et al., 1996), whereas the inhibition of aromatase stimulated 17,20bP production and advanced the release of sperm (Afonso et al., 2000). Finally, as discussed above, the remarkable increase of esr1a and esr2a that we

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Figure 7. Effects of E2 administration on (A) esr1a, (B) esr1b, (C) esr2a, and (D) esr2b mRNA levels. Gene expression was determined by quantitative reverse-transcriptase PCR, and was normalized to rs15 gene expression. The E2 implants were present for 7 days (7D) or 14 days (14D). Results are means  standard error of the mean (n ¼ 4
5 per treatment). Values are statistically different when P < 0.05, according to the following symbol:  , P < 0.05.

observed could contribute to the final maturation of male gametes and/or to the semen fluid composition of teleosts.

MATERIALS AND METHODS Animals, Hormonal Treatment, and Testis Samples Ethics statement. Male Rainbow trout (Onchorhynchus mykiss) were obtained from the INRA experimental fish farm (Drennec, France), where they are maintained under natural conditions of light intensity and photoperiod. Experimental animal research reported here was performed in conformity with the principles for the use and care of laboratory animals, in compliance with French and European regulations on animal welfare. Experimenters were authorized by the French ‘‘Direction des Services
te
rinaires’’ to conduct or supervise experimentations Ve on live animals. Androgen treatment. Prepubertal males (150  17 g) were supplemented with either testosterone (0.2 mg) or Mol. Reprod. Dev. 82:694–708 (2015)

11KT (0.25 mg) implants (Innovative Research of America) for 7 or 14 days. These treatments resulted in a significant increase in corresponding blood-plasma androgen concentrations. Mean values of 28.2  2.5 ng/ml were measured in animals treated for 7 days, and 13.9  3 ng/ml in animals treated for 14 days with testosterone, whereas levels of 1.00  0.35 ng/ml were found in control animals. Similarly, the mean circulating levels of 11KT were measured in animals treated for 7 days with this hormone and in control animals (42.8  7.6 ng/ml and 1.3  0.3 ng/ml, respectively) (Rolland et al., 2013).

E2 treatment. Prepubertal males (236  35 g) were treated with 0.05 mg or 0.25 mg E2 (Innovative Research of America) for 7 or 14 days. Exposure to E2 implants resulted in a large increase in blood plasma concentration, from 43  5 pg/ml to 4030  398 and 7100  1100 pg/ml on Day 7 for doses of 0.05 and 0.25 mg, respectively, and from 26  11 pg/ml to 6948  785 pg/ml on Day 14 for the 0.25 mg dose. Testicular development stages. Testes samples analysed included: testes in early stages, containing slowly 703

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TABLE 1. Primer Sequences and Real-Time PCR Product Sizes Gene target

Accession number

Primers

Fragment size

cyp19a

BX871650 BX876154

50 -CTGACGTAGAACTACAGCTCCT 50 -ACATCATCAGACAGTGCCCGG

169 bp

cyp19b-I

AJ311937

50 -ACTAGCGTGTCTGGAGTCCT 50 -CCTGGTATGTGTGAGGCGTT

86 bp

cyp19b-II

AJ311938

50 -CGGTGGCAAGTAGCAATCCT 50 -CGCTCCAGATGAACCGAGAG

194 bp

cyp19b

AJ311937 AJ311938

50 -GAGGAAGGCACTGGAAGATGAC 50 -GCTGGAAGAAACGACTGGGC

164 bp

esr1a

AJ242740 AJ242741

50 -CTGAGGAGACACGCGGAGGT 50 -TTGGGCAGGGGCTGTGTAGT

80 bp

esr1b

DQ177438

50 -AATTTTTGGGGGTCGGGTGT 50 -CGCTGAAGAGGTTTGGGAGT

87 bp

esr2a

DQ177439

50 -GGCACGACTTCAACCCCTTG 50 -GAGGGACTGGTGGGTGGATG

95 bp

esr2b

DQ248229

50 -CCACGGACCCCTAGCCTTCT 50 -TGTTTTGCTGGCCATGGTTG

128 bp

rs15

NM_001165174

50 -CTGGGGGAGTTCTCTATCACCT 50 -GGATGAAACGGGAAGAATGTGT

82 bp

Histological analysis. Gonads fixed in Bouin’s solution were dehydrated, embedded in paraffin, cut into 5-mm thick sections, and then stained with Regaud’s haematoxylin-orange G-aniline blue. The stage of the gonads was determined according to the classification described by Gomez et al. (1999). No remarkable change in gonad weight or cellular composition was observed after 7 or 14 days of treatment with androgen or estrogen supplementation (data not shown).

cell sorting by centrifugal elutriation (JE5 Beckman Instruments). Cell separation was performed at constant rotation (2000 rpm) and increasing flow rates in L15 media with 0.5% BSA, as described in Bellaiche et al. (2014). Germcell fractions were pelleted, transferred to Trizol reagent, and stored at
808C for RNA extraction or fixed in Bouin’s solution for histological examination. Centrifugal elutriation resulted in the following populations: Spg-A, populations containing 70
90% of undifferentiated type A spermatogonia; A þ B, populations with a mixture of type A and B spermatogonia; B þ Spc, populations containing 70% type B spermatogonia, with the spermatocyte-enriched population contained 70% primary spermatocytes; and Spc þ Spt, the post-meiotic-enriched fraction contained essentially secondary spermatocytes, spermatids, and contaminating spermatozoa.

Germ Cell Isolation

Gene Expression Studies

Populations of germ cells at different stages of differentiation were obtained from immature males or maturing males, as previously described in Rolland et al. (2009) and Bellaiche et al. (2014). In brief, testes were minced, submitted to enzymatic digestion in collagenase, followed by mechanical dispersion using a Dounce homogenizer. The resulting cell suspensions were filtered through nylon gauze (150-mm, 50-mm, and 32-mm pore sizes). The cells were resuspended in L15 medium with 1% bovine serum albumin (BSA), loaded into 45% percoll, and centrifuged for 20 min at 500g and then 20 min at 100g to remove cell clusters, erythrocytes, and most spermatozoa, if present. The upper floating layers were recovered and submitted to

Total RNA from testis was isolated using Trizol reagent (Invitrogen, Fischer Scientific, Illkirch, France), quantified on a NanoDrop ND-1000 (Thermo Scientific, Fischer Scientific, Illkirch, France), and visualized on a Bionanalyser 2100 (Agilent, Les Ulis, France). Two micrograms of total RNA treated with DNase was reverse transcribed using res les Bains, random primers (Promega, Charbonnie France) and MMLV reverse transcriptase (Promega). Target cDNA was amplified from 1/10 or 1/20 volumes of the 1 reverse transcription reaction, 1X Fast SYBR Green Master Mix (Applied Biosystems, Fischer Scientific, Illkirch, France), and 600 nM of gene-specific forward and reverse primers (Eurogentec, Seraing, Belgique) (Table 1) in a

dividing type A spermatogonia (Stage I) or growing numbers of actively dividing type B spermatogonia (Stage II); maturing testes containing increasing numbers of meiotic spermatocytes (Stages IIIa and IIIb) and post-meiotic spermatids (Stage V); and spawning testes containing essentially mature spermatozoa (Stage VIII).

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Figure 8. Effects of testosterone (T) and 11 KT administration on (A) esr1a, (B) esr1b, (C) esr2a, and (D) esr2b mRNA levels. Gene expression was determined by quantitative reverse-transcriptase PCR, and was normalized to rs15 gene expression. The androgen implants were present for 7 days (7D) or 14 days (14D). Results are means  standard error of the mean (n ¼ 4
6 per treatment). Values are statistically different when P < 0.05, according to the following symbols:  , P < 0.05;  , P < 0.01.

StepOne Plus Thermocycler (Applied biosystems). All samples were measured in duplicate. The end of the amplification was followed by a melt curve to verify the amplification of a single product. PCR efficiency was determined for each set of primers and for each run using a standard composed of a dilution series of reverse-

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transcription products. Negative controls for each sample were generated from pools of RNA samples in each treatment group in the absence of reverse transcriptase. The relative abundance of specific mRNA was obtained by the formula 2(delta Ct) (relative gene expression) or by the determination of the ratios between the mean quantity of

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the gene of interest divided by the mean quantity of the reference gene rs15 (relative gene expression) compared to values from Stage I, type A spermatogonia (Spg-A), or control samples (Rolland et al., 2013). In studies concerning the stages of spermatogenesis, the total abundance of specific mRNA in the two gonads was determined by taking into account the quantity of total RNA extracted from the two gonads and normalized to 1 kg of fish body weight: 2(delta Ct)  cDNA dilution factor  tot. extracted RNA (mg)  2 testes weight  1.

Aromatase Immunohistochemistry Testes were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde, and then processed for immunohistochemistry studies. After rehydration of the 5mm slices, immunohistochemistry was performed at room temperature with the UltraVision Detection System AntiPolyvalent HRP/DAB (Thermo Scientific) and the In Situ Pro VS robot (Intavis, Koln, Germany). After one wash for 7 min in PBS containing 0.05% Tween20 (PBST), samples were incubated for 15 min in Hydrogen Peroxide Block, followed by another washing in PBST for 7 min. Sections were then saturated with a PBST solution containing 1% BSA for 10 min, incubated for 2 h with polyclonal rabbit antimouse aromatase antibody (1:250, a generous gift from Dr. Carretero, Laboratory of neuroendocrinology, University of Salamenca, spain), 10 min with biotinylated goat anti-polyvalent secondary antibody, and finally 10 min with peroxidase-labelled streptavidin; each of these incubation steps was separated by four washes in PBST for 7 min. The addition of three, 3’-diaminobenzidine (DAB) was performed manually. The slices were mounted with Mowiol (Calbiochem, VWR international, Fontenay Sous Bois, France), allowed to polymerize, and then examined by microscopy (Leica). Images were acquired with an Olympus camera. Negative controls included sections incubated with the saturating solution instead of the primary antibody.

Statistical Analyses Data are expressed as means  standard errors of the mean of n individuals. The statistical differences between stages were analyzed by one-way ANOVA followed by the Kruskal-Wallis test (comparison of at least three groups of data). Differences were considered significant when P < 0.05. The statistical difference between each treatment and its corresponding control was analyzed by the Mann
Whitney U test (comparison of two groups of data). Differences were considered significant when P < 0.05, and were noted for P < 0.01 or P < 0.001.

ACKNOWLEDGMENTS We are grateful to Chantal Cauty and Claude Sevellec from the histology service in LPGP for their help in immunohistochemistry. We acknowledge Jose Carretero for anti-

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mouse aromatase antibody, Andrew Bass for anti-teleost aromatase proteins, and Kris Von Schalburg for anti-atlantic salmon aromatase A and aromatase B antibodies.

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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.

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