The potential roles of androgen and estrogen receptors during reproductive cycle in Pampus argenteus
Zhongsu Wan, Ming Ding, Xinru Zhang, Chang Li, Jiajie Zou, Danli Wang, Xubo Wang, Chunyang Guo
TL;DR
This study explores how androgen and estrogen receptors function in the reproductive cycle of Pampus argenteus fish, revealing their roles in gonadal development.
Contribution
The study identifies tissue- and stage-specific expression patterns of androgen and estrogen receptors in P. argenteus, linking them to gonadal development and hormone responsiveness.
Findings
Androgen and estrogen receptors show distinct tissue-specific expression in P. argenteus.
Hormone treatments influence receptor expression differently in ovarian and testicular tissues.
Receptor expression patterns correlate with specific stages of gonadal development.
Abstract
Steroid hormones are indispensable for regulating the typical reproductive processes during the development of gonads. This study aimed to clarify the expression patterns of androgen receptors (Ars) and estrogen receptors (Ers) in Pampus argenteus to better understand gonadal development and improve artificial breeding. In this study, the androgen receptors (arα and arβ) and estrogen receptors (esr1, esr2a, and esr2β) of P. argenteus were cloned and the expression profiles were detected by qPCR. The results showed that the highest expression of arα was found in hypothalamus, while arβ was mainly found in midbrain of male fish. The esr1, esr2β and esr2α showed the highest expression level in pituitary, ovary, and liver of female fish, respectively. During the gonadal development, arα and arβ reached their highest expression in stage IV, while esr1, esr2a, and esr2β showed the strongest…
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Fig 10- —KC Wong Magna Fund in Ningbo University
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Taxonomy
TopicsReproductive biology and impacts on aquatic species · Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities · Hypothalamic control of reproductive hormones
Introduction
Androgen display unique physiological functions by binding to androgen receptor (Ar). Ars belong to the nuclear receptor superfamily of steroid receptors and act as ligand-dependent transcriptional regulators. The Ar protein comprises four functional domains: the N-terminal activation domain (NTD), the DNA-binding domain (DBD), the labile hinge region, and the C-terminal ligand binding domain (LBD). Two types of Ars have been identified in some teleost species [1]. The two Ar subtypes, Arα and Arβ, were successfully cloned in the Atlantic croaker (Micropogonias undulatus) [2], rainbowfish (Melanotaenia fluviatilis) [3], and olive flounder (Paralichthys olivaceus) [4]. In contrast, only a single Ar form has been identified in red seabream (Chrysophrys major), and its structural features closely resemble those of Arβ [5]. The two Ar subtypes exhibit distinct tissue expression patterns. For example, in rainbow trout Arα was more widely expressed than Arβ [6,7]. In Atlantic croaker Arα was primarily expressed in the brain, whereas Arβ was detected in both the brain and gonads of both sexes [2]. Teleost species that possess only a single type of Ar also display distinct tissue-specific expression patterns.
Estrogen is a key steroid hormone that regulates oocyte maturation in females and controls spermatogonia survival and spermatogenesis in males [8–10], and it exerts these essential physiological functions, which are comparable in importance to those of androgens, through binding to Ers. The Er protein consist of six functional domains (A-F): the A/B region, which contains the N-terminal transcriptional activation domain; the C region, known as the DNA-binding domain (DBD), the D region, or hinge region; the E region, which corresponds to the ligand-binding domain (LBD); and the F region located at the C-terminal. According to previous studies, other variants that can only be found in mammals as two subtypes of Erα and Erβ [11, 12] have been found in teleost fish. For example, four Er subtypes (erα1, erα2, erβ1 and erβ2) have been identified in rainbow trout (Oncorhynchus mykiss) [13], whereas only three (erα, erβ1 and erβ2) have been identified in zebrafish (Danio rerio) [14]. Ers exhibit multiple subtypeswith distinct tissue distribution patterns. In teleost such as rainbow trout and gold fish, Erα was mainly distributed in the heart, gonads, liver and other tissues, whereas Erβ was mainly detected in the gonad, kidney, pituitary and other tissues [12,13]. These diverse distribution patterns suggest that different Er subtypes play distinct roles in gonadal development of teleost fish.
The sex steroid hormones present in teleost fish include androgens (e.g., testosterone, androstenedione), estrogens (e.g., estradiol, estriol, progesterone), and progestin (e.g., progesterone) [15,16]. These hormones primarily regulate the initiation, development, maturation and maintenance of the gonads, stimulate the onset of reproductive behavior, and ensure the normal physiological functioning of the organisms [17]. Androgens play distinct roles at different developmental stages in teleost fish. For example, androgens regulate spermatogenesis in males and suppress the development of immature oocytes and embryos in females [18], while in males, testosterone (T) promotes gonadal development and induces sexual differentiation or transformation [19]. These hormones act through two main pathways: one is that hormones are transferred to the nucleus by binding to nuclear hormone receptors and then combined with the corresponding response elements to enable gene expression, the other is that hormones activate second messengers or ion channels by binding to cell surface receptors, and then induce expression of corresponding genes. Thus, the whole process is that androgens binding to the Ars, which in turn produces the appropriate biological response via different pathways. Estrogen, as a key sex steroid hormone, promotes ovarian development and the maturation of secondary sexual characteristics in teleost fish. In addition, it plays important roles in the central nervous system [20] and the immune system [21]. These hormones primarily exert their effects through Ers, which mediate two types of responses: membrane receptor-mediated non-genomic effects and nuclear receptor-mediated genomic effects [22–24].The non-genomic effects are mediated by GPR30, a member of the G protein-coupled receptors (GPCRs) family [25], whereas genomic effects represent the classical signaling pathway, in which estrogen binds directly to nuclear receptors Erα and Erβ, leading to the activation of target gene transcription and subsequent physiological effects.
Pampus argenteus is a commercially important fish in China, recognized by its light blue back, silver-white abdomen, and silvery body. In recent years, both the abundance and quality of wild populations have sharply declined due to overfishing and ongoing deterioration of the marine environment [26,27]. This decline underscores the urgent need for efficient artificial breeding techniques to ensure sustainable aquaculture of this species [28,29]. Artificial breeding of P. argenteus is challenging because of its delicate body and sensitivity to stress. Moreover, females mature later than males, leading to premature sperm release by males and delayed ovulation in females. This asynchrony significantly reduces fertilization and hatching rates, representing a major bottleneck for aquaculture productivity. Sex steroid hormones, including androgens and estrogens, play central roles in regulating gonadal development and reproductive function in teleosts. Their actions are mediated through Ars and Ers, which control hormone signaling and gene expression in a tissue- and stage-specific manner. Although these receptors have been studied in other teleosts, their distribution and dynamic expression patterns during the gonadal reproductive cycle of P. argenteus remain poorly understood. To address this knowledge gap, the present study aims to investigate the tissue-specific expression of Ars and Ers and their temporal expression patterns during the reproductive cycle of P. argenteus. Elucidating these patterns will provide a molecular basis for understanding gonadal development and offer insights to overcome asynchronous maturation in cultured populations, ultimately contributing to the improvement of artificial breeding strategies.
2. Materials and methods
2.1 Animal and sample collection
All fish experiments were conducted in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Animal Care and Use Committee of Ningbo University (#2015051701). The MS-222 (Tricaine methanesulfonate, Sigma-Aldrich Co. LLC.) was used to anesthetize the fish. P. argenteus were rared at Xiangshan Bay, Zhejiang, China. The year-round sampling of cultured P. argenteus were performed. One-year-old P. argenteus were reared in a 45 m^2^ cement tank with a water depth of 1.5 m. The tank was supplied with seawater that had undergone secondary filtration. Throughout the culture period, natural water temperatures ranged from 9 to 33 ℃, salinity remained between 23 and 26‰, and pH values ranged from 7.7 to 8.3. Dissolved oxygen was maintained at 5–7 mg/L. The fish were fed Yubao commercial feed(Hayashikane Sangyo Co., Ltd., Japan) twice daily. Each sampled fish was assigned an identification number. After one side of its gonad was used for histological examination to determine the developmental stage, the other side of the gonad was frozen at −80℃ according to the corresponding ID number and used for qPCR analysis. Based on histological characteristics, six distinct stages were identified in both the ovary and the testis.The pituitaries of P. argenteus during stages I-VI of gonadal development were also collected. Furthermore, the liver, kidney, gill, laterral, heart, pituitary, forebrain, hypothalamus, midbrain, spleen, intestine, muscleand gonad tissues were collected.
2.2 Gonadal histology
The fish gonads were fixed with 4% paraformaldehyde solution at 4℃ for 12 hours. The fixed gonads were dehydrated in methanol and stored at −20℃. The dehydrated gonads were transferred from methanol to ethanol and then embedded in paraffin wax following dealing with xylene and paraffin. Fixed tissues were embedded in paraffin and sectioned at 5 µm thickness. The sections were deparaffinized in xylene for 10 min, followed by rehydration through a graded ethanol series (100%, 95%, 80%, and 70%, 5 min each) and rinsed in distilled water. Subsequently, the sections were stained with hematoxylin for 5 min, rinsed under running tap water for 30 min, and then immersed in eosin for 1 min. Finally, the sections were dehydrated through ascending ethanol series, cleared in xylene, and mounted with a coverslip using a synthetic mounting medium for microscopic examination. All sampled fish were classified according to their gonadal development stages through histological analysis.
2.3 Sequence alignment and phylogenetic analysis
For phylogenic analysis, a subset of arα, arβ and esr1, esr2α, esr2β sequences from three different species were retrieved from GenBank. The deduced amino acid sequence of P. argenteus Ars and Ers were subjected to multiple sequence alignment using MUSCLE. The phylogenetic tree was constructed using the neighbor-joining method on MEGA 7. The number at each node represents the bootstrap probability (% from 1000 replicates). The accession numbers of the sequences obtained in the analysis are shown in the Supplementary S1 Table.
2.4 Total RNA extraction and cDNA synthesis
After determining the gonadal status of all fish through histology, we used six different stages of pituitaries and gonads to ascertain gene expression patterns. Total RNA was extracted following the TransZol Up Kit (Transgene, ET111) manufacturer’s protocol. The quantity of total RNA was measured using a NanoDrop™ 1000 spectrometer (Thermo Fisher Scientific). First-strand cDNA was synthesized from 1 μg of total RNA using the HiFiScript gDNA Removal RT MasterMix Kit (CWBIO) according to the manufacturer’s protocol.
2.5 Quantitative real-time PCR
Gene quantification of standards, samples, and controls was conducted simultaneously by qPCR (Quantagene q225 Real-Time PCR System) with the MagicSYBR mixture (CWBIO). beta-actin (β-actin; GenBank accession no. KF982333) was used as an internal control to normalize the gene expression level. Specific primers for esr1, esr2a, esr2β, arα, and arβ were used, and all primers are listed in Table 1. The specificity of PCR was confirmed through a single melting curve of unknown samples and standards. No signal was detected in non-template controls by qPCR. The data were calibrated according to the 2^-ΔΔCt^ method. The reaction efficiency of different genes was assessed through the analysis of serially diluted cDNA templates. Based on linear regression analysis, the accuracy of real-time PCR assays was high (R^2^ > 0.99) across all dilutions. The relative expression values of target genes in all samples were normalized to β-actin, with the highest value of the target genes set to 100%.
Table 1: Oligonucleotides for specific primer.
2.6 Sex hormone treatment
For sex hormone treatment, β-estradiol (E2), methyltestosterone (MT) and anastrozole (an aromatase inhibitor, AI) were used. Different optimal concentrations of sex hormones including 50 ng/ml of E2 (GC11282, GLPBIO), 120 ng/ml of MT (GC19961, GLPBIO), and 1000 ng/ml of AI (GC10256, GLPBIO) were used to gonadal tissue culture. E2 + AI, 50 ng/mL of β-estradiol and 1000 ng/mL of anastrozole; E2 + MT, 50 ng/mL of β-estradiol and 120 ng/mL of methyltestosterone solution. The gonadal tissue culture followed the methods described in previous studies [30,31]. Testes or ovaries were dissected into pieces less than 1 mm thick. The gonadal tissues, with six replicates per group, were incubated in the presence or absence of sex hormones for 7 days at 26 ℃. The basal culture medium consisted of Leibovitz’s L-15 Medium (11415064, Gibco) supplemented with 10% fetal bovine serum and 2% penicillin-streptomycin solution (P1400, Solarbio). After tissue culture, the gonad tissues were collected and stored at −80℃ immediately. Gonadal tissues were used for gene expression analysis.
2.7 Data analysis
Data were analyzed by one-way ANOVA using SPSS 26.0 software. The data were presented as the mean ± SD (standard deviation). The values were subjected to analysis by one-way ANOVA, followed by Duncan test, with P < 0.05 indicating a significant difference.
3. Results
3.1 Phylogenetic analysis of Ars and Ers
As shown in Fig 1A, among the Ars, P. argenteus Arβ exhibited high homology with Micropterus salmoides, while Arα showed high homology with Epinephelus coioides (Fig 1A). Among Ers, P. argenteus Esr2a represented the highest similarity with E. coioides, and so did the Esr1. The Esr2β of P. argenteus showed close relationship with that of E. coioides, Acanthopagrus schlegelii, and Dicentrarchus labrax (Fig 1B).
Comparison of the P. argenteus Ars and Ers protein sequences with other animals.(A) Phylogenetic relationship of androgen receptor (Arα/Arβ) proteins from P. argenteus with other animals. (B) Phylogenetic relationship of estrogen receptor (Esr1/Esr2a/Esr2β) proteins from P. argenteus with other animals.
3.2 Tissue distributions of Ars and Ers
The distributions of various Ars were different in females and males. The arα was the mainly expreesed in ovaries (P < 0.05), and the expression of arα in liver was significantly higher than that in other tissues except ovaries (Fig 2A). In males, arα was predominantly expressed in the brain, with the highest expression in the hypothalamus, followed by the forebrain, pituitary, and midbrain, while its expression in other tissues was significantly lower (Fig 2B). In female, the expression of arβ was the highest in gills and the lowest in midbrain (Fig 2C), while in male arβ presented the strongest expression in midbrain (Fig 2D).
The tissue distribution expression of arα and arβ in female and male fish.(A and B) The tissue distribution expression of arα in female and male fish, respectively (n = 3). (C and D) The tissue distribution expression of arβ in female and male fish, respectively (n = 3). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05). “N.D.” indicate not detect by qPCR.
The distributions of different Ers in female and male tissues also varied. In females, esr1 was mainly expressed in the pituitary and ovary (Fig 3A), whereas in males, esr1 was strongly expressed in the brain, with the highest level in the hypothalamus, followed by the pituitary, midbrain, and forebrain (Fig 3B). Regarding esr2α, its expression was significantly higher in the ovary than in other tissues in females (Fig 3C). In males, the expression pattern of esr2α was similar to that of esr1, showing high expression in the brain (Fig 3D). The expression of esr2β in females was highest in the liver, followed by the pituitary and ovary (Fig 3E), whereas in males, esr2β was also mainly expressed in the brain, with the highest level in the hypothalamus, followed by the pituitary, midbrain, and forebrain (Fig 3F).
The tissue distribution expression of esr1, esr2a and esr2β in female and male fish.(A and B) The tissue distribution expression of esr1 in female and male fish, respectively (n = 3). (C and D) The tissue distribution expression of esr2a in female and male fish, respectively (n = 3). (E and F) The tissue distribution expression of esr2β in female and male fish, respectively (n = 3). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
3.3 Gonadal developmental stage
Based on the histological characteristics, six developmental stages were identified in both ovary and testis (Fig 4). During ovarian development, oogonia and primary oocytes were observed in stage I (Fig 4A). The diameter of primary oocytes increased progressively from stage II to stage III (Fig 4B and 4C). In stage IV, vitellogenic oocytes appeared (Fig 4D). During stage V, when P. argenteus entered breeding season, mature vitellogenic oocytes were present in the ovary (Fig 4E). After the breeding season, regressed oocytes were observed in stage VI (Fig 4F). For the testicular characteristics, spermatogonia type A cells were observed in stage I (Fig 4G). At the stage II, spermatogonia type A and type B, as well as primary spermatocyte, were present (Fig 4H). Subsequently, secondary spermatocytes were observed in stage III (Fig 4I).The sperm was observed at the stage IV (Fig 4J), and the testis was filled with sperm during the breeding season (Fig 4K). After the breeding season, fewer sperm and spermatogonia were found in the testis (Fig 4L).
Profiles of gonadal development in P. argenteus.The ovarian development in different stages, including stage I (A), stage II (B), stage III (C), stage IV (D), stage V (E), and stage VI (F). The testicular development in different stages, including stage I (G), stage II (H), stage III (I), stage IV (J), stage V (K), and stage VI (L). OG, oogonia; PO, primary oocyte; VO, vitellogenic oocyte; Fc, follicle cells; RO, regressed oocyte. SGA, spermatogonia type A; SGB, spermatogonia type B; PSC, primary spermatocyte; SSC, secondary spermatocyte; SP, sperm.
3.4 Expression of Ars and Ers genes during gonadal development
In the ovary, arα was strongly expressed in stage IV, followed by stages Ⅲ and Ⅴ (Fig 5A). In the testis, arα showed its highest expression in stage I, followed by stage II, and was lowest in stage VI in the testis during testicular development (Fig 5B). The highest expression level of arβ was observed in stage IV ovaries, while its expression in stage I was also significantly higher than in other stages (Fig 5C). In the testis, arβ showed its highest values in stage II testis (Fig 5D), suggesting that this receptor may play a role in promoting early testis development. In the ovary, esr1 was predominantly expressed in stage V, suggesting its involvement in the increased estrogen levels during oocyte maturation and ovulation. In stage VI, after ovulation, the expression of esr1 decreased significantly compared with stage V but remained higher than in other stages (Fig 6A). In the testis, the esr1 showed the highest expression in stage IV and the lowest in stage VI (Fig 6B). In females, esr2α showed the highest expression in stage V ovaries, followed by a significant decrease in stage VI (Fig 6C). The expression pattern of esr2α during ovarian development is similar to that of esr1, suggesting that they may have comparable roles in ovarian development of P. argenteus. During testicular development, esr2α exhibited a similar pattern to esr1, with the highest expression observed in stage IV testes (Fig 6D). In female, esr2β showed the highest expression in stage V ovaries, and its expression in stages I and IV was significantly higher than in other stages (Fig 6E). In males, esr2β expression was highest in stage I, followed by stage II, which may be involved in promoting spermatogonia proliferation (Fig 6F).
The expression of arα and arβ in gonad during gonadal development.(A) The expression of arα during ovarian developmental stages (n = 3). (B) The expression of arα during testicular developmental stages (n = 3). (C) The expression of arβ during ovarian developmental stages (n = 3). (D) The expression of arβ during testicular developmental stages (n = 3). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
The expression of esr1, esr2a and esr2β in gonad during gonadal development.(A) The expression of esr1 during ovarian developmental stages (n = 3). (B) The expression of esr1 during testicular developmental stages (n = 3). (C) The expression of esr2a during ovarian developmental stages (n = 3). (D) The expression of esr2a during testicular developmental stages (n = 3). (E) The expression of esr2β during ovarian developmental stages (n = 3). (F) The expression of esr2β during testicular developmental stages (n = 3). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
3.5 Expression of Ars and Ers genes in pituitary during gonadal development
Expressions of ars and ers in the pituitary at different stages of gonad development were detected. The results showed that in the pituitary, arα exhibited the highest in stage IV ovaries, followed by stage III and V (Fig 7A), In contrast, no significant differences were observed in arα expression in the pituitary during testicular development (Fig 7B). During both ovarian and testicular development, esr2β expression was not detected in the pituitary. In the pituitary, the expression of esr1 showed no significant changes during ovarian development (Fig 8A), while it reached its highest level in stage VI during testicular development (Fig 8B). The expression of esr2a in the pituitary was highest in stage IV during ovarian development (Fig 8C), and in stage III during testicular development (Fig 8D). Regarding esr2β, its expression in the pituitary was significantly higher at stage III during ovarian development (Fig 8E) and at stage VI during testicular development (Fig 8F).
The expression of arα in pituitary during gonadal development.(A) The expression of arα in pituitary during ovarian development (n = 3). (B) The expression of arβ in pituitary during testicular development (n = 3). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
The expression of esr1, esr2a and esr2β in pituitary during gonadal development.(A) The expression of esr1 in pituitary during ovarian development (n = 3). (B) The expression of esr1 in pituitary during testicular development (n = 3). (C) The expression of esr2a in pituitary during ovarian development (n = 3). (D) The expression of esr2a in pituitary during testicular development (n = 3). (E) The expression of esr2β in pituitary during ovarian development (n = 3). (F) The expression of esr2β in pituitary during testicular development (n = 3). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
3.6 The influence of different hormones on gonadal tissue culture
The results of the hormone treatments indicated that arα expression was significantly elevated in the E2 group in both ovarian (Fig 9A) and testicular tissue culture (Fig 9B). Similarly, arβ expression was significantly elevated in the E2 group in the ovarian tissue culture (Fig 9C). In the testicular tissue culture, arβ was significantly expressed in the MT group, E2 group, and E2 + MT groups (Fig 9D). In ovarian tissue culture ers and ars were highly expressed in the E2 group (Fig 10A, 10C and 10E). In the testicular tissue culture, MT treatment significantly increased the expression levels of esr1, esr2a and esr2β (Fig 10B, 10D and 10F). Taken together, these findings sugget that E2 and MT may induce the expressions of ars and ers in ovarian and testicular tissue culture, respectively.
Effects of different treatments on ars expression in gonadal tissue.The expression of arα was analyzed by qPCR (n = 6 for each value) after different hormones treatment in ovarin tissue (A) and testicular tissue (B). The expression of arβ was analyzed by qPCR (n = 6 for each value) after different hormones treatment in ovarin tissue (C) and testicular tissue (D). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
Effects of different treatments on ers expression in gonadal tissue.The expression of esr1 was analyzed by qPCR (n = 6 for each value) after different hormones treatment in ovarin tissue (A) and testicular tissue (B). The expression of esr2a was analyzed by qPCR (n = 6 for each value) after different hormones treatment in ovarin tissue (C) and testicular tissue (D). The expression of esr2a was analyzed by qPCR (n = 6 for each value) after different hormones treatment in ovarin tissue (E) and testicular tissue (F). Superscript letters indicate one-way ANOVA, followed by Duncan’s test (P < 0.05).
4. Discussion
4.1 The distribution of Ars and Ers in teleost
In this study, the cDNA sequences of androgen receptors (arα and arβ) and estrogen receptors (esr1, esr2a, and esr2β) of P. argenteus were successfully cloned and compared with other teleost species. Phylogenetic analysis showed that these receptors are closely related to their counterparts in several teleosts, suggesting conservation of their molecular features. These results confirm the identity of the cloned sequences and provide a basis for further functional studies on the roles of Ars and Ers in regulating gonadal development and reproduction in P. argenteus.
Distributions of Ars and Ers in tissue are different due to the diversity of their subtypes and function mechanism. Androgens synthesized by the testis, adrenal glands, brain or other parts of male fish (Acconcia et al., 2017) bind to Ars, and thus play different roles, including promoting spermatogenesis in the testis, regulating reproductive behaviors via the brain, and modulating steroidogenesis in endocrine organs such as the adrenal glands. The expression of arα was mainly detected in the hypothalamus of males and in the ovary of females. Studies on Ctenosciaena gracilicirrhus [2] (Sperry et al., 2000) reported similar conclusions, suggesting that arα, through its binding to sex steroid hormones, regulates hormone secretion in the hypothalamus and supports normal gonadal development in males, while also promoting gonadal growth, oocyte maturation, and ovulation in females. The expression of arβ was highest in the midbrain of male P. argenteus, suggesting that this receptor may regulate midbrain hormone secretion and participate in testicular development, consistent with findings reported in European seabass [15]. Estrogen receptors are widely distributed in the ovaries and testis of teleost fish [32,33], and play important roles in regulating the hypothalamic-pituitary-gonadal reproductive axis, with esr1 recognized as the most functionally important subtype in teleosts [34]. In females, the highest expression of esr1, esr2a and esr2β were detected in pituitary, ovary and liver, respectively, consistent with findings reported in Scophthalmus maximus [35]and Sininiperca chuatsi [36]. In male, these Ers were mainly expressed in the brain, with the highest levels observed in the hypothalamus. Studies on Ers of P. argenteus are consistent with findings in Oncorhynchus mykiss [37], Dicentrarchus labrax [14], D. rerio [38], Oryzias latipes [39], and Lateolabrax japonicus [40], suggesting that in females, esr1 regulates gonadotropin release in pituitary, whereas esr2a may promote ovulation and ovarian development. Taken together, these results indicate that the tissue-specific distributions of Ars and Ers in P. argenteus are closely linked to their distinct physiological roles.
4.2 The potential roles of Ars and Ers in gonadal development
In fish, on the basis of experiment results, Ars plays an important role in ovary as well as testis [41]. In the testis of P. argenteus, arα expression was highest at stage I, whereas arβ was detected only from stages I to II, reaching its peak at stage II during testicular development, consistent with observations in Rattus norvegicus [42] and A. schlegelii [43]. The highest expression levels of both arα and arβ occured in stage IV of the P. argenteus ovary. The current results on Ars expression in ovaries are similar to that on stone flounder (Kareius bicoloratus) [42] (He et al., 2003). The expression patterns of Ers differ from those of ARs in P. argenteus. In males, the highest expression levels for esr2β were found in stage I, while that for esr1 and esr2a reached their maximum in stage IV. In females, the highest levels of the three Ers were all detected in stage V, and some similar patterns were observed in Spinibarbus denticulatus [44], E. coioides [45], and D. rerio [46]. The arβ similar to arα primarily promotes the formation of primary spermatocytes in males [47]. The receptors esr1, esr2a, and esr2β all facilitate ovulation and the development of stage V oocytes in females, as reported in tilapia (Oreochromis mossambicus) [48] and medaka (O. latipes) [49]. Meanwhile, esr1 and esr2a contribute to sperm synthesis in males, with esr2a exhibiting a similar function in tilapia [48]. Like arα, esr2β also regulates sperm formation in males. Overall, in the gonads of P. argenteus, ARs mainly promote oocyte formation and spermatogenesis, whereas ERs primarily regulate ovulation and sperm synthesis, in addition to controlling the activities of oocytes and spermatogonia.
4.3 The potential roles of Ars and Ers in pituitary
Sex steroids are key regulators of the hypothalamic–pituitary–gonadal (HPG) axis, and the pituitary serves as a primary target through which these hormones modulate gonadotropin synthesis and release [50]. In teleosts, androgen and estrogen receptors expressed in the pituitary mediate feedback mechanisms that coordinate reproductive timing, gametogenesis, and sexual maturation [51]. Building upon these established roles, we examined the expression patterns of Ars and Ers across different gonadal developmental stages in P. argenteus. Our results showed that arα expression reached a peak at ovarian stage IV, in contrast to patterns described in Carassius auratus [52] and Astatotilapia burtoni [7,53], suggesting a species-specific feature of androgen signaling in P. argenteus. In females, esr2α and esr2β peaked at stage V, consistent with the involvement of ERβ-type receptors in late oocyte development [14]. In males, esr1 and esr2β exhibited peak expression at stages V and I, respectively, whereas esr2α reached its maximum at stage IV, aligning with previous findings that ER isoforms show stage-dependent expression dynamics during spermatogenesis in teleosts [14]. Overall, these results indicate clear sex- and stage-specific expression patterns of Ar and Er isoforms in the pituitary, suggesting their coordinated roles in regulating gametogenesis.
4.4 Regulation of sex steroids on Ars and Ers
Our hormone treatment experiments revealed strikingly distinct and tissue-specific responses of Ars and Ers in ovarian and testicular tissue cultures of P. argenteus. In ovarian tissues, E2 treatment markedly upregulated arα and arβ, suggesting that estrogens may promote ovarian maturation not only through classical estrogenic pathways but also by modulating androgen signaling. Consistently, Ers were highly expressed under E2 stimulation, indicating a coordinated activation of estrogen and androgen pathways to drive oocyte development and ovulation. By contrast, in testicular tissues, MT treatment predominantly enhanced the expression of Ar and Er subtypes, highlighting the pivotal role of androgens in promoting testicular differentiation and spermatogenesis. Interestingly, combined E2 + MT treatment also increased arβ expression in testes, suggesting potential crosstalk between androgenic and estrogenic signaling during early testis development. These observations indicate that the regulation of Ars and Ers by sex steroids is highly context-dependent, with receptor- and tissue-specific sensitivity to hormonal cues. Our results are in line with previous studies in teleosts. In orange-spotted grouper, MT-induced masculinization was accompanied by differential regulation of Er subtypes: Erα decreased during ovarian regression, while Erβ1 and Erβ2 increased, supporting early ovarian development and MT-induced testicular differentiation [54]. In Blackhead seabream, Erβ2 was identified as a key regulator during sex reversal [55]. Extending these insights to P. argenteus, our data suggest that E2 primarily activates Ar and Er signaling in ovaries, whereas MT predominantly stimulates Ar and Er in testes, reflecting sex- and tissue-specific responsiveness to steroid hormones.
5. Conclusion
In P. argenteus, Ars and Ers show clear tissue specificity, sex-biased distribution, and stage-dependent expression during gonadal development. Ars predominantly function in oocyte growth and early spermatogenesis, while Ers mainly regulate oocyte maturation, ovulation, and late-stage sperm formation. Pituitary expression patterns further indicate essential endocrine feedback during the reproductive cycle. Hormone-induction experiments reveal strong tissue-specific regulation, with the ovary highly sensitive to estrogen and the testis strongly responsive to androgen. Together, these findings demonstrate the complementary and coordinated roles of Ars and Ers in reproductive regulation and provide a theoretical basis for improving artificial breeding of P. argenteus.
Supporting information
S1 TableGenBank accession numbers of androgen and estrogen receptors used in this study.(DOCX)
S1 FileRaw qPCR data for Figs 2–3 and 5–10.(ZIP)
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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