Abstract
Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.
| Original language | American English |
|---|---|
| Pages (from-to) | 94-105 |
| Number of pages | 12 |
| Journal | Theriogenology |
| Volume | 205 |
| DOIs | |
| State | Published - 15 Jul 2023 |
Bibliographical note
Funding Information:The seasonal reproductive stages in male birds are controlled by the hypothalamus-pituitary-gonad axis (HPG axis), which regulates the release of sex steroids from testes through a feedback mechanism [1]. Anterior pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play a primary role in the control of testicular spermatogenic, endocrine, and exocrine functions [2,3]. A direct action of FSH on testicular Sertoli cells and LH on Leydig cells is supported by the presence of receptors (FSHR and LHR) on these cells [4]. Moreover, the important role of prolactin (PRL), produced by pituitary lactotrophs, in the control of seasonality was postulated. The PRL receptors (PRLR) are expressed in the testes [5,6], and the direct action of PRL on testicular tissue was suggested [7]. It is generally known that gonadal steroids produced by the testes are crucial for the maintenance of male fertility. In the steroidogenic pathway, the initial rate-limiting and regulating step is the transport of cholesterol across the mitochondrial membrane realized by steroidogenic acute regulatory protein (StAR), then cholesterol is converted into pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the product of the CYP11A1 gene) [8]. In the next steps of steroid biosynthesis, a series of enzyme-catalyzed reactions occur with participation of 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and aromatase cytochrome P450 (P450arom; the product of the CYP19A1 gene), which converts androgen to estrogen [9]. It has been established that androgens and estrogen mediate their biological effects through binding to the intracellular androgen receptor (AR) and estrogen receptors (ERs), respectively [10–13]. Estrogen's action is displayed particularly by ER alpha (ERα) and ER beta (ERβ). The main sex steroids, testosterone (T) and estradiol (E2), act both as endocrine and local factor within the testis [14].In the present study, the ability of the testis to synthesize T is manifested not only in the breeding season, but also at the PoB and NB stages, with an increase to the maximum at the PR stage, followed by a decrease at the PoB and NB stages. The highest level of intra-testicular T was recorded when the massive spermatozoa production occurs, and it is important for maintenance of secondary sexual characteristics [6]. Androgens play an important role in preventing cell apoptosis in androgen-dependent tissue [36] and regulating the expression of testicular cell adhesion molecules [37]. A decrease in intra-testicular T concentration at the PoB stage noted in present study is probably the hormonal signal inducing seasonal germ cell depletion. In seasonal birds, like the quail (Coturnix coturnix) [16], gonadal steroid hormones are synthesized not only in Leydig and Sertoli cells but also in germ cells within the testis. Thus, if spermatozoa are an important source of androgens, a decrease in intra-testicular T concentration at the PoB and NB stages may be partially attributed to a low value of spermatogenic index in ganders [6] and a drastic reduction in the ST diameter [26]. Furthermore, the present results showed that the E2 production is a prominent feature of gander testes steroidogenic function at the PoB and NB stages. These results are in agreement with previous reports demonstrating seasonal fluctuations in the intra-testicular E2 concentration observed in ganders by Leska et al. [13]. A complex study provided information regarding the possible roles of E2 in the germ cells and in sperm differentiation and maturation but also indicate that this outcome may be dose dependent [9]. An elevated intra-testicular E2 level beyond a given threshold, would exert negative feedback on LH, which in turn leads to reduced plasma T concentration. This view finds support in the study of Weil et al. [20], who observed that a decline in the fertility of aging roosters was associated with an increase in plasma and intra-testicular E2 levels and a decrease in plasma T and LH concentrations. Moreover, the inhibitory effect of a high level of E2 on spermatogenesis might be the induction of the impairment of Sertoli cell function, which leads to disturbed communication with germ cells and their apoptosis. Our recent study in Zatorska ganders revealed that reduction in testis size at the NB stage, compared to reproductively active stages, was associated with a decrease in proliferation and an increase in apoptosis of germinal cells [26]. It may be suggested that estrogen interacts in these processes and plays an autocrine or paracrine role in the differentiation of spermatogenic cells during the annual period in domestic ganders.In the present research, the expression of FSHR mRNA, PGR mRNA, and AR mRNA remained unaffected by the annual stages, which suggests similar tissue sensitivity to hormonal action. Thus, in ganders, annual period variation in plasma and intra-testicular levels of P4 and the intra-testicular level of T are not reflected in changes in the availability of their cognate receptors. On the other hand, regulation by posttranscriptional and/or posttranslational modification of transcript or protein should also be considered. Our results were partially opposite to a previous study by Leska et al. [12] in which testicular AR mRNA expression increased during the transition from the breeding to the non-breeding phase and remained at a similar level in the sexual reactivation phase. It is noteworthy that the annual period variations in the expression of LHR and PRLR mRNA were partially similar with upregulation at the PrB and PoB stages. It is proposed that PRL facilitates signalling between the pituitary gonadotrophs and the testes in mammals [38]. PRL has been shown to increase sensitivity to LH in Leydig cells in rat testis by increasing the abundance of their respective receptors [39]. In our study, the pattern of PRL transcript abundance resembled the pattern of PRL plasma concentration reported in our previous study [6,25], with the highest level of plasma PRL in ganders occurring in the second half of the breeding-laying period. It may be suggested that PRL autoregulates the expression of its own receptor. The physiological mechanism adjusting the number of receptors based on the activating signal could be proposed for ganders' testis during the annual period. Present results are partially in agreement with an existing theory, suggesting that PRL exerts both pro- and anti-gonadal effects depending on the plasma level and expression of the receptor. In prepubertal roosters, PRL in low and medium levels potentiates testis development, but at high concentrations, it contributes to testicular regression. Upregulation of PRLR signalling occurs through downregulation of mRNA expression of LHR and T synthesis enzymes [7]. Furthermore, increasing the expression levels of serotonin-VIP-PRL axis molecules inhibits testis function and, consequently, reproductive performance in aging roosters [24]. Herein, we found that mRNA of ERα and ERβ were expressed in a similar manner in testicular tissues, suggesting that estrogen could function via both receptor subtypes with its overlapping roles in ganders, as was suggested by Leska et al. [13]. Importantly, the action of E2 may have differential effects when activating ERα versus ERβ in mammals. In free-ranging sand rats [40], a proliferative effect of E2 on Sertoli and germ cells, mediated by ERβ and GPER1 (G-protein-coupled estrogen receptor), occurred during the breeding season, whereas an inhibitory effect, mediated by ERα is present during resting season. There is also evidence that changes in immunohistochemical localization of ERα in the testis may contribute to a decrease in reproductive function and a decline of fertility in aging roosters [41]. Furthermore, our results demonstrating the annual fluctuation in ERs’ transcript abundance, with a higher level at the PoB and NB stages, partially support the findings by Leska et al. [13], who detected a higher expression of genes and proteins of ERs during the nonbreeding stage. Thus, increased testicular sensitivity to E2 at these stages suggests the importance of estrogenic action in assuring the proper functional changes during the remodelling processes of this tissue in ganders.This work was financially supported by the Ministry of Education and Science for the University of Agriculture in Krakow, Poland (subvention number: SUB-020013-D015 and SUB-020002-D015).
Funding Information:
This work was financially supported by the Ministry of Education and Science for the University of Agriculture in Krakow, Poland (subvention number: SUB- 020013-D015 and SUB-020002-D015 ).
Publisher Copyright:
© 2023 Elsevier Inc.
Keywords
- Aromatase
- Ganders
- Seasonal breeding
- Steroidogenesis
- Steroidogenic enzymes
- Testes