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Biology of Reproduction 65, 60-65 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Estrogen Receptor-{alpha} Protein Localization in the Testis of the Rainbow Trout (Oncorhynchus mykiss) During Different Stages of the Reproductive Cycle1

Jerry Boumaa, and James J. Nagler2,a

a Center for Reproductive Biology, Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844-3051

ABSTRACT

Estrogen receptor-alpha (ER-{alpha}) is important for male reproduction in mammals; however, no information is available on ER-{alpha} protein distribution in the testes of fishes. The cellular localization of the rainbow trout (Oncorhynchus mykiss) ER-{alpha} (rtER-{alpha}) protein, throughout the annual reproductive cycle was determined in this study. An antibody was designed based on a 15-amino acid sequence from the D-domain of the rtER-{alpha}, and its specificity was confirmed using Western blot analysis. Immunohistochemical analysis revealed rtER-{alpha} protein to be present only in the testicular interstitium, at every stage of the annual reproductive cycle. The localization of rtER-{alpha} protein in the interstitial fibroblasts, the Leydig cell precursor in the rainbow trout, suggests a role for estrogens in the differentiation of these precursor cells into mature Leydig cells. This is the first study to report the cellular localization of an estrogen receptor protein in the testis of any fish species.

estradiol receptor, interstitial cells, seasonal reproduction, spermatogenesis, testes

INTRODUCTION

In vertebrates the genomic actions of the sex steroid 17ß-estradiol (E2) are mediated through nuclear estrogen receptors (ERs) [1–3]. The two best-known ERs are estrogen receptor-alpha (ER-{alpha}) and estrogen receptor-beta (ER-ß). Studies using ER-{alpha} knockout (ERKO) mice have shown the importance of ER-{alpha} in male reproduction, as these male ERKO mice are completely infertile [4, 5]. The absence of ER-{alpha} in the mouse testis resulted in a decrease in testis weight, and by Day 150 the testis was completely atrophied due to reduced fluid reabsorption in the efferent ducts [5, 6]. In addition, the testis of these male ERKO mice exhibited a decrease in sperm count and sperm motility, and hyperplasia of the Leydig cells due to a slight elevation of LH levels [6]. In mammals, the histological localization of ER-{alpha} is restricted to fetal and adult Leydig cells [7], while ER-ß is present in gonocytes, A type spermatogonia, late spermatocytes, round spermatids, Sertoli cells, and fetal Leydig cells [8]. There is no information on the localization of ER proteins in the gonads of any nonmammalian vertebrate.

Studies have shown that fishes have high affinity binding sites for E2 in their gonads [9, 10], and the mRNAs for both ER-{alpha} and ER-ß have been identified in the testis of a variety of fish species [11–15]. In one species, the Atlantic croaker (Micropogonias undulatus), three different ER mRNAs (ER-{alpha}, -ß, -{gamma}) have been discovered, all of them present in the testis [16]. In the rainbow trout (Oncorhynchus mykiss) ER-{alpha} has been characterized [17] and the transcript detected in the testis [13]. Recently, an ER-ß has been reported in the rainbow trout (GenBank accession no. AJ289883); however, there is no information on tissue distribution of the mRNA or protein. Although ER transcripts have been detected and characterized in the fish testis, the cellular localization of ER proteins within the fish testis is still unknown. Furthermore, there is no information on the physiological role of estrogens or ERs in the testes of any fish species.

This study was designed to determine the cellular localization of ER-{alpha} protein in the testis of the rainbow trout by immunohistochemistry and examine the pattern of expression during the reproductive cycle.

MATERIALS AND METHODS

Fish and Tissues

Male rainbow trout (O. mykiss, House Creek strain) were sampled from a population kept at the University of Idaho's Aquaculture Experiment Station in Hagerman, ID. The fish were held in an outdoor raceway under ambient conditions of light and temperature. Ten male trout were sedated using MS-222 and killed by a blow to the head on five separate occasions between August 1999 and May 2000 (10 x 5 = 50 fish total), to provide testis samples throughout an annual reproductive cycle. During each sampling, both testes from each fish were weighed to determine the gonadosomatic index (GSI = [gonad weight/body weight - gonad weight] x 100). Small pieces (approximately 0.5 cm2) of the middle portion of one testis were fixed in Histochoice MB (Amresco, Solon, OH). After 2 wk of fixation at 4°C, the tissue was stored in 70% ethanol at room temperature.

Histology

Pieces of testicular tissue were dehydrated through a graded series of ethanol, cleared with xylene, and embedded in paraffin. For histological analysis, two serial sections (3–4 µm thick) were cut and stained with hematoxylin and eosin. The reproductive stage of the testis of each fish was determined according to the classification scheme of Schulz [18] for rainbow trout using GSI and histological criteria.

Antibody Production and Characterization

A polyclonal antibody, based on a 15-amino acid peptide (Lys-Asp-Lys-Arg-Tyr-Cys-Gly-Pro-Ala-Gly-Asp-Arg-Glu-Lys-Pro) from the D-domain region of the rainbow trout ER-{alpha} sequence [17], conjugated to keyhole limpet hemocyanin, was produced in New Zealand white rabbits (Research Genetics Inc., Huntsville, AL). Both the postimmune and the preimmune rabbit sera were purified on a HiTrap Protein G affinity column (Pharmacia Biotech, Piscataway, NJ) according to manufacturer's instructions.

To determine the specificity of the rainbow trout ER-{alpha} (rtER-{alpha}) antibody, Western blot analysis was conducted with recombinant rtER-{alpha} C–D domain and rtER-{alpha} E–F domain fusion protein fragments produced in bacteria. The production and purification of these recombinant fusion protein fragments have been described in Pakdel et al. [19]. Briefly, two regions (232 amino acids of the C–D and E–F domains, respectively) of the rtER-{alpha} cDNA were subcloned into a bacterial expression plasmid pGEX-3X. Bacteria (Escherichia coli DH5{alpha} strain) were transformed with either the rtER-{alpha} C–D or rtER-{alpha} E–F plasmid (kindly provided by F. Pakdel, University of Rennes, France). Production of the protein fragments was induced by 0.3 mM isopropylthio-ß-D-galactoside (IPTG). Bacterial cell extracts were prepared by centrifuging and resuspending the pellet in 50 µl SDS-PAGE loading buffer (0.0625 M Tris-HCl, 2% SDS, 10% glycerol, 40 mM dithiothreitol, pH 6.8). The bacterial proteins were denatured by boiling for 5 min, after which the samples were stored at -20°C. The suspensions containing the rtER-{alpha} C–D and rtER-{alpha} E–F proteins were briefly centrifuged (8000 rpm for 10 sec), and the supernatant was separated on duplicate 10% SDS-PAGE gels for 2.5 h at 100 V (Mini-Protean; Bio-Rad, Hercules, CA). For immunodetection the proteins from one gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) for 1 h at 100 V. The membrane was subsequently incubated in 10% Blot Qualified BSA (Promega, Madison, WI) overnight at 50°C. The following day, diluted rtER-{alpha} antibody (1:3000) was applied to the PVDF membrane for 60 min, rinsed three times with a Tris-buffered saline solution (10 mM Tris-HCl, 0.15 M NaCl, pH 8.0) containing 0.05% Tween 20 (TBST) for 10 min each time, and incubated for 30 min with an alkaline phosphatase-conjugated secondary anti-rabbit IgG antibody (1:5000 dilution; Promega). Unbound secondary antibody was removed by rinsing the membrane three times for 10 min each time with TBST. Specific signals were detected using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate color development substrate (Promega). To verify that the proteins had separated properly, the duplicate gel was fixed for 30 min in 50% methanol and 10% acetic acid, stained for 25 min in 0.3% Coomassie brilliant blue R (Sigma, St. Louis, MO) in 50% methanol and 15% acetic acid, and destained overnight in 10% methanol and 10% acetic acid. Protein markers (#161-0314 and #161-0372; Bio-Rad) were used to determine the size of the protein fragments.

Immunohistochemistry

For immunohistochemical analysis, three different fish (two serial sections per fish) from each stage of the reproductive cycle were analyzed for the presence of rtER-{alpha} protein. Tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Next, the sections were placed in a 0.01 M sodium citrate (pH 6.0) solution and boiled for at least 5 min in a microwave oven. The slides were cooled to room temperature and rinsed for 15 min in PBS. Nonspecific binding sites were blocked by incubation in 10% normal goat serum (Vector Labs, Burlingame, CA) for 15 min. The tissue sections were then incubated with the rtER-{alpha} antibody (1:300 for stage I tissue, 1:200 for all other stages) overnight at 4°C.

The next day the slides were rinsed in PBS three times, 2 min each time, and a biotinylated goat-anti-rabbit antibody (1:500; Vector Labs) was added to the sections. After 60 min, unbound antibody was removed by rinsing in PBS three times, 2 min each time. Immunodetection was performed using streptavidin-peroxidase (20-min incubation) and an AEC chromogen substrate reagent set (Zymed Laboratories Inc., San Francisco, CA). As controls, the sections were incubated with preimmune sera, and sections were treated without rtER-{alpha} antibody. The tissue sections were examined using a DAS Mikroskop Leica DMR light microscope equipped with a SPOT (Diagnostic Instruments Inc., Wetzlar, Germany) digital camera. All micrographs were taken at 400x magnification and stored on TDK certified plus CD•R recordable compact discs.

RESULTS

Antibody Characterization

Western blot analysis, using the rtER-{alpha} antibody, detected a major band of the predicted size of 53 kDa in IPTG-induced bacteria transformed with the rtER-{alpha} C–D protein fragment (Fig. 1A). The 53-kDa band, representing the rtER-{alpha} C–D protein, was detected only in bacterial protein cell extracts from IPTG-induced bacteria and not in noninduced bacteria or in bacteria with the IPTG-induced rtER-{alpha} E–F fragment (Fig. 1A). A minor band of approximately 45 kDa was also detected in bacteria in which the rtER-{alpha} C–D fragment was induced. No bands were observed when the membrane was incubated with the rabbit preimmune serum (data not shown). Staining of the duplicate SDS-PAGE gel with Coomassie blue shows the separation of the bacterial proteins and induction of the rtER-{alpha} C–D protein fragment (Fig. 1B).



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  		FIG. 1. Western blot analysis of rtER-{alpha} C–D domain (rtER-{alpha} C–D) and E–F domain (rtER-{alpha} E–F) protein fragments. A) Immunodetection of IPTG-induced rtER-{alpha} C–D proteins (lane 1), uninduced rtER-{alpha} C–D proteins (lane 2), and IPTG-induced rtER-{alpha} E–F proteins (lane 3) by the rtER-{alpha} antibody (1:3000). B) SDS-PAGE gel loaded with crude bacterial cell extract containing rtER-{alpha} C–D protein fragment induced by IPTG (lane 1), and uninduced cell extract (lane 2). Arrow depicts the band representing the rtER-{alpha} C–D protein fragment. The molecular weights (in kDa) of the protein markers used are indicated on the left in each case

Histology

Representative stages of the histology of the male rainbow trout testis throughout the reproductive cycle are shown in Figure 2. At stage I the male gonad consists of numerous small cysts, called spermatogenic cysts, containing spermatogonia (Fig. 2A). The interstitial space between these cysts is composed of myoid cells that are present in all the stages of the reproductive cycle and fibroblasts. Stages II through IV correspond to the process of spermatogenesis during which the cysts greatly increase in size and the space between the cysts becomes very narrow (Fig. 2, B–D). Spermatogonia are present in stages II–IV, but their number gradually decreases due to their development into spermatocytes (stage II). The spermatocytes in turn undergo meiosis to produce spermatids (stage III), and eventually spermatozoa (stage IV).



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  		FIG. 2. Histological sections of six representative stages of the reproductive cycle from the rainbow trout testis, according to Schulz [18]. A) Stage I testis showing numerous cysts containing spermatogonia (black arrow). B) Stage II testis representing the start of spermatogenesis, with the cysts increasing in size due the appearance of spermatocytes (black arrowhead) in addition to the presence of spermatogonia (black arrow). C) Stage III testis, in which the cysts contain spermatogonia (black arrow), spermatocytes (black arrowhead), and spermatids (black asterisk). D) Stage IV testis representing the last stage of spermatogenesis with the presence of spermatozoa (white arrowhead). E) Stage V testis showing the start of spermiation. The tubules are filled with spermatozoa (white arrowhead), and the interstitium becomes very narrow (white arrow). F) Stage VI testis that shows a regressing testis at the end of the reproductive cycle. The black arrow points to spermatogonia, whereas the white arrowhead indicates spermatozoa that remained in the tubules. Reabsorption of spermatozoa is denoted by the black arrowhead. Bar = 5 µm

Spermiation, defined as the release of sperm cells from the spermatogenic cysts into tubules leading to the sperm duct, starts in stage V (Fig. 2E) and continues in stage Va, during which most of the spermiating males are found. The interstitial spaces now consist primarily of fully differentiated Leydig cells. Stage VI are regressing testes that contain tubules filled with spermatozoa, and developing spermatogenic cysts similar to stage I together with degenerating tubules. In these testes spermatozoa can be seen that are actively being phagocytosed by Sertoli cells and macrophages (Fig. 2F).

Immunohistochemistry

The rtER-{alpha} protein was detected in the interstitium of the rainbow trout testis, at all the stages of the reproductive cycle (Fig. 3). Intense staining for rtER-{alpha} protein was observed throughout the interstitial space at stage I, in both myoid cells and fibroblasts (Fig. 3A). During spermatogenesis (stages II–IV) rtER-{alpha} protein was detected in the interstitial cells between the cysts (Fig. 3, B and C) that at these stages have both characteristics of Leydig cells and fibroblasts and have been referred to as intermediate cells [20]. At stages V and Va (spermiation) rtER-{alpha} protein staining was found in the interstitium, both in the myoid cells and the Leydig cells (Fig. 3D). Staining for rtER-{alpha} protein in the regressing testis (stage VI) is in the interstitium between the tubules but appears faint and diffuse around and sometimes even within the cysts and the regressed tubules (Fig. 3E). The rtER-{alpha} protein was also found associated with blood vessels within the testes during the entire reproductive cycle (see Fig. 3, A and B). Both controls, incubation with preimmune rabbit serum and exclusion of rtER-{alpha} antibody, were completely negative for any staining as shown in Figure 3, F and G, respectively.



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  		FIG. 3. Immunohistochemical localization of the rtER-{alpha} protein in the testis of the rainbow trout. A) Stage I testis showing positive staining in the interstitial cells (black arrow), including endothelial cells (black arrowhead). B) Start of spermatogenesis (stage II) with the interstitial cells (black arrow) being positive for rtER-{alpha} protein. Black arrowhead indicates positive staining in the endothelial cells. C) Stage IV showing the end of spermatogenesis, again rtER-{alpha} protein being present only in the interstitium (black arrow). D) Start of spermiation (stage V). The interstitial space contains Leydig cells that are positive for rtER-{alpha} protein (black arrow). E) Stage VI showing the regressing testis. Staining for rtER-{alpha} protein around the tubules containing spermatozoa is similar to stage V. F) Negative control showing incubation with purified rabbit preimmune serum on a stage I testis section. G) Negative control showing a stage IV testis section in the absence of primary antibody. Bar = 5 µm

DISCUSSION

This study is the first to report the cellular localization of ER-{alpha} protein in the testis of a fish, the rainbow trout. Using immunohistochemistry the rtER-{alpha} protein was found in the interstitial cells, between the cysts in the testis. The ER-{alpha} protein was detected in interstitial cells at every stage of the annual reproductive cycle in this species.

The localization of rtER-{alpha} protein in the interstitial cells and blood vessels of the rainbow trout testis corresponds to the situation in mammals, in which ER-{alpha} is also present in Leydig cells and endothelial cells of the blood vessels [7, 8, 21]. In mammals, fetal Leydig cells are responsible for masculinization of the male reproductive tract during fetal life, whereas adult Leydig cells appearing during puberty produce testosterone that is required for spermatogenesis [22]. The adult Leydig cells are derived from mesenchymal cells that are localized in the peritubular region [23]. Contrary to mammals, the rainbow trout has an annual reproductive cycle that can be divided into two main processes: spermatogenesis (stages II–IV) and spermiation (stages V–Va). According to Loir [20], it is not until the process of spermiation commences that fully differentiated Leydig cells are present in the interstitium, when they become the primary cell type. These Leydig cells are presumably derived from fibroblasts [20]. In this study, rtER-{alpha} protein is not only present in the fully differentiated Leydig cells during spermiation, but also in myoid cells and fibroblasts at the start of the reproductive cycle, and intermediate cells during spermatogenesis. In addition, it has been shown that rainbow trout testicular fragments obtained from stage II (start of spermatogenesis) and stage V/Va (spermiation) testes are able to synthesize E2 [24]. The localization of rtER-{alpha} protein in the precursor Leydig cells during the early stages of the reproductive cycle suggest a role for E2 in the differentiation of precursor cells into fully mature Leydig cells. This idea is supported by studies in mammals [25, 26].

There is some evidence that exogenous treatment with estrogen or estrogenic compounds can affect the testes of rainbow trout, presumably acting through ERs. Studies have found that when E2 was added to the diet the process of spermatogenesis was inhibited and the testis regressed in adult rainbow trout [27]. These effects of E2 were believed to be due to alterations of the somatic cells within the testis (Sertoli cells and Leydig cells) as the testis was unresponsive to circulating gonadotropins. The presence of rtER-{alpha} proteins within the Leydig cells of the rainbow trout testis could therefore explain these results as they provide the binding sites for E2. In addition, studies have shown that a growing number of environmental contaminants called xenoestrogens are able to mimic or block the action of endogenous E2 [28]. Testicular growth was inhibited when rainbow trout were exposed to various xenoestrogens [29]. These negative, inhibitory effects of exogenous estrogens may therefore be mediated through the rtER-{alpha} that is found to be present throughout the annual reproductive cycle. It has also been well documented that genetically male fish embryos are sensitive to estrogens and xenoestrogens, and that exposure to these sex steroids during the critical period of gonadal differentiation can result in partial or complete sex reversal [30–32]. This suggests that ERs are also important in the differentiating testis of embryonic fishes.

Recently, Pakdel et al. [33] found two isoforms of rtER-{alpha}, generated from the same gene, in the liver of the rainbow trout. These isoforms are identical in sequence except for the N-terminus (A/B-domain), one isoform being 45 amino acids longer than the other. The antibody used in this study (designed against a region in the D-domain) would thus detect both of these rtER-{alpha} isoforms. However, it is unknown whether both of these rtER-{alpha} isoforms are present in rainbow trout gonads. Preliminary studies in this laboratory have identified another ER mRNA in the rainbow trout testis, which, based on structural analysis, is also classified as an ER-{alpha} [13]. This probably resulted from a genome duplication event thought to have occurred during the evolution of fishes [34] that resulted in two copies of this gene. The nucleotide sequence of this other rtER-{alpha} shows little homology in the A/B domains, D-domain, and F-domain compared to the previously identified rtER-{alpha} [17] and is unique (unpublished results). Although the native protein has not been demonstrated in the rainbow trout testis, we have compared the deduced amino acid sequence of this ER-{alpha} with the other rtER-{alpha}s [17, 33]. All of the 15 amino acids in the peptide used to produce the antisera in this study are different between the two rtER-{alpha}s. Therefore, it is unlikely that the rtER-{alpha} antibody used would detect this other, distinctive ER-{alpha}, provided the protein is present in rainbow trout testes.

Western blot analysis confirmed that the antibody used in this study is specific for the D-domain of rtER-{alpha}, relative to other domains of the rtER-{alpha} (Fig. 1A, lane 1 vs. 3). In addition to the 53-kDa rtER-{alpha} C–D protein, a smaller protein band of approximately 45 kDa was observed in the rtER-{alpha} C–D protein induced cell extracts. This is most likely the result of degradation or incomplete production of the rtER-{alpha} C–D recombinant protein, as this band was not observed in the rtER-{alpha} C–D-noninduced or the rtER-{alpha} E–F-induced bacterial cell extracts.

In conclusion, an immunohistochemical method was used with an rtER-{alpha} antibody that specifically detected rtER-{alpha} protein in the interstitial cells of the rainbow trout testis. The localization of rtER-{alpha} protein in the interstitium of the testis was observed throughout every stage of the annual reproductive cycle. The presence of rtER-{alpha} protein in interstitial fibroblasts, thought to be precursors of Leydig cells, suggests a role for E2 in the differentiation of mature Leydig cells in the testis of this fish.

ACKNOWLEDGMENTS

The authors thank Ms. My Vo and Dr. K.H. Kim (Washington State University, WA) for their help with the immunohistochemistry. Dr. V.P. Eroschenko's (University of Idaho, ID) help with the histology and review of this manuscript is greatly appreciated. The rtER-{alpha} C–D and E–F plasmids were kindly provided by Dr. F. Pakdel (University of Rennes, France).

FOOTNOTES

First decision: 6 December 2000.

1 Supported by the NSF-Idaho EPSCoR Program and by the National Science Foundation Cooperative Agreement number EPS-9720634. Back

2 Correspondence. FAX: 208 885 7905; jamesn{at}uidaho.edu Back

Accepted: February 9, 2001.

Received: November 7, 2000.

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J. Aravindakshan, V. Paquet, M. Gregory, J. Dufresne, M. Fournier, D. J. Marcogliese, and D. G. Cyr
Consequences of Xenoestrogen Exposure on Male Reproductive Function in Spottail Shiners (Notropis hudsonius)
Toxicol. Sci., March 1, 2004; 78(1): 156 - 165.
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