Introduction
The hypothalamus–hypophysis–gonad axis orchestrates the seasonality of reproduction via intracrine, paracrine, and endocrine signalling (Norris, Reference Norris2018). In female lizards, as in other vertebrates, sex steroids are considered the major hormones involved in the reproductive events (development, gametogenesis, mating behaviour, and courtship among others; Edwards, Reference Edwards2005; Sánchez and Smitz, Reference Sánchez and Smitz2012; Ramírez-Pinilla et al., Reference Ramírez-Pinilla, de Pérez, Alvarado-Ramírez, Rheubert, Siegel and Trauth2015). Generally, estradiol (E2) and progesterone (P4) have a dynamic behaviour depending on the stage of the reproductive cycle in vertebrate females (Amey and Whittier, Reference Amey and Whittier2000; Rhen et al., Reference Rhen, Sakata, Zeller and Crews2000; Edwards and Jones, Reference Edwards and Jones2001; Wack et al., Reference Wack, Fox, Hellgren and Lovern2008; Kummrow et al., Reference Kummrow, Smith, Crawshaw and Mastromonaco2010).
Although genomic (receptor-mediated) and non-genomic signalling is recognized for sex steroid actions in other vertebrates, the second has been scantly studied in Squamata (Contrò et al., Reference Contrò, Basile and Proia2015; Yatsu et al., Reference Yatsu, Katsu, Kohno, Mizutani, Ogino, Ohta, Myburgh, van Wyk, Guillette, Miyagawa and Iguchi2016; Yaşar et al., Reference Yaşar, Ayaz, User, Güpür and Muyan2017). In genomic signal transduction, sex steroids exert their effects by activating nuclear receptors that bind to their promoter regions and modulate gene expression (Sato et al., Reference Sato, Miyagawa, Iguchi, Takei, Ando and Tsutsui2016; Garg et al., Reference Garg, Ng, Baig, Driggers and Segars2017). In general, sex steroid receptors are conformed by a N-terminal region that stimulates gene transcription (domain A/B), a DNA binding domain (C), a nuclear localization signal (D), and a ligand-binding region (E) (Medina-Laver et al., Reference Medina-Laver, Rodríguez-Varela, Salsano, Labarta and Domínguez2021). These receptors have shown high-functional plasticity due to their number of combinations in modulators, levels of activity, and responses (Jacobsen and Horwitz, Reference Jacobsen and Horwitz2012).
In female lizards, E2 promotes follicular development and the synthesis of vitellogenin (VTG) in the liver. This hormone increases with the onset of vitellogenesis and ovulation in various species (Jones, Reference Jones, Norris and Lopez2011; Ramírez-Pinilla et al., Reference Ramírez-Pinilla, de Pérez, Alvarado-Ramírez, Rheubert, Siegel and Trauth2015; Barbosa-Moyano et al., Reference Barbosa-Moyano, Rodríguez-Chaparro, Santos and Ramírez-Pinilla2020). The ovary is the principal source of E2 to exert its multiple effects via receptors in different tissues (Contrò et al., Reference Contrò, Basile and Proia2015). The estrogen receptor (ER) has two isoforms: the alfa (ERα) and beta (ERβ). These receptors were reported in many lizard species such as Bradypodion pumilum, Plestiodon finitimus, Gekko japinicus (Yatsu et al., Reference Yatsu, Katsu, Kohno, Mizutani, Ogino, Ohta, Myburgh, van Wyk, Guillette, Miyagawa and Iguchi2016), and Hemodactylus flaviviridis (Tripathy and Rai, Reference Tripathy and Rai2017). At least one receptor has been detected in Podarcis sicula (Verderame and Limatola, Reference Verderame and Limatola2010), Anolis carolinensis (Beck and Wade, Reference Beck and Wade2009), and Calotes versicolor (Inamdar et al., Reference Inamdar, Khodnapur, Nindi, Dasari and Seshagiri2015). In P. sicula, ERα is involved in vitellogenesis (Verderame and Limatola, Reference Verderame and Limatola2010), while in H. flaviviridis, it enhances aromatase activity via gonadotropin hormones (Tripathy and Rai, Reference Tripathy and Rai2017).
P4 is another hormone with pleiotropic effects in females (Cox, Reference Cox2020). This hormone also modulates vitellogenesis and ovulation (Crews, Reference Crews2005; Kabelik et al., Reference Kabelik, Weiss and Moore2008; Al-Amri et al., Reference Al-Amri, Mahmoud, Waring, Alkindi, Khan and Bakheit2012). However, its main roles are pregnancy maintenance and parturition in viviparous species due to its influence on uterine epithelium proliferation, uterine contractility inhibition, and downregulation of follicular development by modification of gonadotropin secretion and antiestrogenic capacity (Bonnet et al., Reference Bonnet, Naulleau, Bradshaw and Shine2001; Girling et al., Reference Girling, Jones and Swain2002; Martínez-Torres et al., Reference Martínez-Torres, Elena Hernández-Caballero, Alvarez-Rodriguez, Alba Luis-Díaz and Ortíz-López2003; Custodia-Lora et al., Reference Custodia-Lora, Novillo and Callard2004; Holmes and Cree, Reference Holmes and Cree2006; Adams et al., Reference Adams, Biazik, Stewart, Murphy and Thompson2007). The principal source of P4 is the corpus luteum (Hosie et al., 2003; Martínez-Torres et al., Reference Martínez-Torres, Elena Hernández-Caballero, Alvarez-Rodriguez, Alba Luis-Díaz and Ortíz-López2003, 2010, 2014). This progestogen shows low concentrations during follicular development, a peak before ovulation and the highest concentrations near oviposition or during embryo development (Jones, Reference Jones, Norris and Lopez2011; Ramírez-Pinilla et al., Reference Ramírez-Pinilla, de Pérez, Alvarado-Ramírez, Rheubert, Siegel and Trauth2015; Barbosa-Moyano et al., Reference Barbosa-Moyano, Rodríguez-Chaparro, Santos and Ramírez-Pinilla2020).
Two forms of progesterone receptor (PRA and PRB) have been reported and are associated with the stage of the reproductive cycle in reptiles (Custodia-Lora and Callard, Reference Custodia-Lora and Callard2002; Biazik et al., Reference Biazik, Parker, Murphy and Thompson2012; Hammouche et al., Reference Hammouche, Remana and Exbrayat2012; Motta et al., Reference Motta, Tammaro, Di Lorenzo, Panzuto, Verderame, Migliaccio and Simoniello2020). The difference between PRA and PRB resides in an extra-domain with activating function (AF) in the PRB (Garg et al., Reference Garg, Ng, Baig, Driggers and Segars2017). In the lizard Uromastix acanthinura, PRA and PRB are positively influenced by E2, which denotes heterogeneity in responses among species (Hammouche et al., Reference Hammouche, Remana and Exbrayat2012). In the turtle Chrysemys picta, RPA expression inhibits vitellogenin synthesis (Duggan and Callard, Reference Duggan and Callard2003), whereas RPB promotes it (Giannoukos and Callard, Reference Giannoukos and Callard1995). In Squamata, the diminution of uterine contractions may be regulated via RPA by limiting the activity of proteins involved in cellular adhesion (Custodia-Lora and Callard, Reference Custodia-Lora and Callard2002; Lovern, Reference Lovern, Norris and Lopez2011; Hoss et al., Reference Hoss, Garcia, Earley and Clark2014; Blackburn, Reference Blackburn and Skinner2018; Refsnider et al., Reference Refsnider, Clifton and Vazquez2019).
Recent research on lizard reproductive biology has evaluated changes in sex steroid concentration and the molecular components involved in these processes (Motta et al., Reference Motta, Tammaro, de Stasio, Borrelli and Filosa2004, Reference Motta, Tammaro, Di Lorenzo, Panzuto, Verderame, Migliaccio and Simoniello2020; Tammaro et al., Reference Tammaro, Simoniello, Filosa and Motta2008; Tripathy and Rai, Reference Tripathy and Rai2017). However, most of the studies involve oviparous species. Here we propose the spiny collared lizard (Sceloporus torquatus), a viviparous lizard with distribution in the central region of México (Guillette Jr. and Méndez-de la Cruz, Reference Guillette and la Cruz1993), to provide more information on the reproductive biology of lizards. The females begin vitellogenesis in summer, ovulate in autumn, are pregnant until the following spring, and give birth in April–June (Guillette and Méndez-de la Cruz, Reference Guillette and la Cruz1993; Ortiz et al., Reference Ortiz, de Oca, Ugarte, de Oca and Ugarte2001; Cruz-Cano et al., Reference Cruz-Cano, Sánchez-Rivera, Álvarez-Rodríguez, Dávila-Govantes, Cárdenas-León and Martínez-Torres2021). This work aimed to characterize the presence of estrogen receptors (α and β) and PR in the ovarian follicles (previtellogenic, vitellogenic and preovulatory) of the lizard S. torquatus and relate them to the concentration of these sex steroids.
Materials and methods
Animals
Fifteen sexually mature females (snout–vent length > 70 mm) of S. torquatus were collected in the Sierra de Guadalupe State Park (19°37′N, 99°12′W), State of México. The collecting scientific license was granted by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT; SGPA/DGVS/00579/17 and SGPA/DGVS/02921/19). The collection was carried out during follicular development (June to November).
Sample collection
Immediately after the capture, we collected a 100-µl blood sample by cardiac puncture with a heparinized syringe to measure sex steroid concentrations. The samples were taken to the Reproductive Biology Laboratory at 4ºC and were centrifuged to store the plasma until hormonal quantification. We anaesthetized females and performed a paramedian celiotomy to extract the ovarian follicles (Cruz-Cano et al., Reference Cruz-Cano, Sánchez-Rivera, Álvarez-Rodríguez, Dávila-Govantes, Cárdenas-León and Martínez-Torres2021). Then, the tissues were fixed with Bouin solution, and processed for routine histological technique and paraffin embedded.
As soon as the lizards fully recovered, we returned them to the site where they were collected.
E2 and P4 quantification
Hormone quantification was performed according to Cruz-Cano et al. (Reference Cruz-Cano, Sánchez-Rivera, Álvarez-Rodríguez, Dávila-Govantes, Cárdenas-León and Martínez-Torres2021). We used ASCENT software to obtain the values of the sex steroids. E2 was measured using a commercial immunoenzyme assay kit (Abcam ab108667, USA). The sensitivity was 8.68 pg/ml, according to the manufacturer’s protocol. The inter-assay and intra-assay coefficients of variation were 5.9% and 7.2%, respectively. P4 was quantified with a commercial radioimmunoassay kit (PROGEST-RIA, CISBIO). We used I125-labelled P4 as a reactive tracer. The sensitivity of the assay was 0.05 ng/ml. The inter-assay and intra-assay coefficients of variation were 4.6% and 7.5%, respectively.
Immunohistochemistry (IHC)
We obtained 5-µm sections from follicles of at least five lizards per stage (previtellogenesis, vitellogenesis, and preovulatory). The slides were deparaffinized in xylol, rehydrated through graded alcohols, and rinsed with distilled water. Immunohistochemistry was performed using the Vectastain Universal Quick Kit PK-8800 (Vector Laboratories Inc., Burlingame, CA, USA). Antigen retrieval was carried out with 0.1 M citrate buffer (pH 6.0) using heat treatment in a water bath set at 100ºC for 20 min. Quenching of endogenous peroxidase was made with 0.3% H2O2 for 30 min and washed in phosphate-buffered saline (PBS) 0.1 M (pH 7.4). Background-staining was blocked with preincubation with a solution containing horse serum (1 drop) and bovine serum albumin (BSA) 5% in 2 ml of PBS for 30 min and rinsed with PBS. Sections were incubated overnight in a humidified chamber at room temperature with the polyclonal primary antibodies (rabbit anti-ERα, Thermo Fisher PA5-16440; anti-Erβ, Thermo Fisher 51-7700; or anti-PR, Thermo Fisher PA5-94983, diluted 1:100). We incubated tissues with a biotinylated Pan-Specific Universal antibody for 15 min and washed them twice with PBS. Then, the slides were incubated with streptavidin/peroxidase complex for 10 min. We used 0.03%, diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories) in PBS and 0.1% H2O2 to reveal the peroxidase activity. The sections were mounted in ClearVue (Thermo Fisher). The specificity and effectiveness of the IHC were assessed with two controls, a negative by omitting the primary antibody and a positive with mouse ovaries.
Statistical analysis
The values of E2 and P4 were assessed for normality with the Shapiro–Wilk test on raw data. We checked the homogeneity of the variances with Levene’s test. To determine the changes in the sex steroid concentrations among stages, we performed an analysis of variance (ANOVA) followed by a Holms–Sidak multiple comparison method. All the statistical analyses were performed with the significance level α = 0.05 using the SPSS program for macOS (Version 22).
Results
Ovarian follicles morphology
The morphology of the ovarian follicles presented changes associated with the stage of the reproductive cycle. The granulosa layer in the previtellogenic phase was composed of small, intermediate, and pyriform cells (Figure 1A). When the vitellogenesis began, the regression of the pyriform and intermediate cells reduced the height of the granulosa layer (Figure 1B). When the follicles reached the preovulatory stage, the small cells flattened, and the zona pellucida thickened (Figure 1C).
Sex steroid concentrations
Sex steroid concentrations changed depending on the stage of follicular development (Table 1). E2 concentrations were low in the previtellogenic (254.61 ± 26.88 pg/ml) and vitellogenic stage (241.84 ± 40.71) and significantly higher in the preovulatory phase (625.95 ± 54.80) (ANOVA F(2,12) = 26.522, P < 0.001). For P4, there were no statistical differences among the stages studied (P > 0.05 in all cases) and ranged from 1464 to 1790 pg/ml.
* P < 0.01 vs previtellogenic and vitellogenic phase.
Sex steroid receptors
The previtellogenic follicles from June to September and November showed an intense mark of ERα in pyriform cells (Figure 2A) and granular structures in the cytoplasm of pyriform cells and the cortex (Figure 2B). However, these follicles showed signs of atresia, such as thickening of the theca, abundant small cells, and absence of a clearly defined zona pellucida.
Therefore, we studied the previtellogenic follicles obtained in October that showed a weak mark in the cytoplasm of the granulosa and ooplasm with a strong localization in some granules close to the cortex and some pyriform cells granules (Figure 3A). In addition, the ERβ was strong in the oocyte cortex, erythrocytes, and moderate in granulosa and theca (Figure 3D). For PR, the stain was intense in the pyriform cells and moderate in the oocyte cortex and some small cells (Figure 3G). With the beginning of vitellogenesis, the ERα staining was strong in the erythrocytes located in the blood vessels and weak in the oocyte cortex (Figure 3B). Conversely, ERβ was moderate in theca, granulosa cells, and oocyte’s cortex (Figure 3E). The PR mark was strong during pyriform cells regression, and moderate in the oocyte cortex and cytoplasm of small cells (Figure 3H).
In the preovulatory stage, there is an evident reduction in the immunostaining of the three receptors. The ERα was intense in the cortex and some regions of the theca (Figure 3C). There was no ERβ expression in this stage (Figure 3F). Finally, the presence of PR was moderate in yolk (Figure 3I).
Discussion
Sex steroid receptors are involved in multiple events in the reproductive biology of female vertebrates. They have different patterns of expression depending on the tissue and the reproductive status of the organism (Hammes and Levin, Reference Hammes and Levin2007; Jacobsen and Horwitz, Reference Jacobsen and Horwitz2012; Baker, Reference Baker2019; Fuentes and Silveyra, Reference Fuentes and Silveyra2019). The interaction of these hormones with their receptors is necessary to activate the expression of one or multiple genes that act on the phenotype (Cox, Reference Cox2020). As they have cytoplasmic and nuclear localization in the cells of many tissues (Dressing et al., Reference Dressing, Goldberg, Charles, Schwertfeger and Lange2011), they show high-functional plasticity due to their large number of combinations in modulators, levels of activity, and responses (Jacobsen and Horwitz, Reference Jacobsen and Horwitz2012). Numerous studies have reported their participation in the reproductive biology of female vertebrates (Hamilton et al., Reference Hamilton, Hewitt, Arao, Korach, Forrest and Tsai2017; Baker, Reference Baker2019; Fuentes and Silveyra, Reference Fuentes and Silveyra2019). However, there is limited information concerning their presence in lizards. In this study, we observed immunolocalization of ERα, Erβ, and PR in previtellogenic, vitellogenic, and preovulatory follicles of S. torquatus.
The ovarian follicles showed changes in their morphology as reported for other lizards (Uribe et al., Reference Uribe, Omana, Quintero and Guillette1995; Raucci and di Fiore, Reference Raucci and di Fiore2010; Motta et al., Reference Motta, Tammaro, Di Lorenzo, Panzuto, Verderame, Migliaccio and Simoniello2020). The more evident changes were the modification of the granulosa layer and the deposit of yolk inside the oocyte. The expression of steroid receptors changed according to the stage of follicular development and the cellular types registered. E2 concentrations were significantly higher in the preovulatory stage. In addition, P4 showed similar levels among the different phases of follicular development. The same behaviour is reported for Sceloporus virgatus (Weiss et al., Reference Weiss, Jennings and Moore2002), Phrynosoma cornutum (Wack et al., Reference Wack, Fox, Hellgren and Lovern2008), Chameleo calyptratus (Kummrow et al., Reference Kummrow, Smith, Crawshaw and Mastromonaco2010), Salvator merianae (Zena et al., Reference Zena, Dillon, Hunt, Navas, Bícego and Buck2019) and Mabuya sp. (Barbosa-Moyano et al., Reference Barbosa-Moyano, Rodríguez-Chaparro, Santos and Ramírez-Pinilla2020). ER, and PR presence was not related to the sex steroid concentrations, as reported in the lizard U. acanthinura (Hammouche et al., Reference Hammouche, Gernigon and Exbrayat2017).
We found that the ER labelling was mainly expressed in the granulosa layer and oocyte cortex. In the previtellogenic phase, ERα in the small cells could be associated with the proliferative activity to differentiate in a polymorphic granulosa (Kocanova et al., Reference Kocanova, Mazaheri, Caze-Subra and Bystricky2010; Hamilton et al., Reference Hamilton, Hewitt, Arao, Korach, Forrest and Tsai2017). Also, its presence in the cytoplasm of the pyriform cells may indicate their role in the estrogen stimulation synthesis and secretion in previtellogenic follicles (Hammouche et al., Reference Hammouche, Gernigon and Exbrayat2017; Fuentes and Silveyra, Reference Fuentes and Silveyra2019), inducing aromatase activity at the beginning of the reproductive season (Tripathy and Rai, Reference Tripathy and Rai2017). This steroidogenic capacity has been observed in the granulosa cells of previtellogenic and early vitellogenic follicles in S. torquatus (unpublished data).
For ERβ, its hepatic expression remained constant in the reproductive cycle of the lizard P. sicula (Verderame and Limatola, Reference Verderame and Limatola2010) and changed in the ovaries of H. flaviviridis (Tripathy and Rai, Reference Tripathy and Rai2017). Both receptors’ presence may be involved with the regression of pyriform cells in S. torquatus during vitellogenesis, as we observed an increased expression in atretic follicles.
Although previous studies have reported the presence of sex steroid receptors in diverse tissues on lizards (Young et al., Reference Young, Lopreato, Horan and Crews1994; Beck and Wade, Reference Beck and Wade2009; Verderame and Limatola, Reference Verderame and Limatola2010; Hammouche et al., Reference Hammouche, Remana and Exbrayat2012; Inamdar et al., Reference Inamdar, Khodnapur, Nindi, Dasari and Seshagiri2015; Yatsu et al., Reference Yatsu, Katsu, Kohno, Mizutani, Ogino, Ohta, Myburgh, van Wyk, Guillette, Miyagawa and Iguchi2016), there is limited information available on this group. as their expression was mainly cytosolic, further studies must consider signalling via non-genomic mechanisms. ER presence in the cytosol is associated with an unbound state that allows the translocation between the nucleus and cytoplasm (Kocanova et al., Reference Kocanova, Mazaheri, Caze-Subra and Bystricky2010).
The expression of PR in S. torquatus showed a different spatial-temporal pattern expression. However, identifying the behaviour of each isoform could shed light on its role in the phases of follicular development. In reptiles, both isoforms have been reported and are associated with the stage of the reproductive cycle (Custodia-Lora and Callard, Reference Custodia-Lora and Callard2002; Biazik et al., Reference Biazik, Parker, Murphy and Thompson2012; Hammouche et al., Reference Hammouche, Remana and Exbrayat2012; Motta et al., Reference Motta, Tammaro, Di Lorenzo, Panzuto, Verderame, Migliaccio and Simoniello2020). During follicular development, PR could stimulate the proliferation in the germinal beds, recruitment, and transition to the primary oocyte (Custodia-Lora and Callard, Reference Custodia-Lora and Callard2002; Jones, Reference Jones, Norris and Lopez2011; Holding et al., Reference Holding, Frazier, Dorr, Pollock, Muelleman, Branske, Henningsen, Eikenaar, Escallón, Montgomery, Moore and Taylor2014; Ramírez-Pinilla et al., Reference Ramírez-Pinilla, de Pérez, Alvarado-Ramírez, Rheubert, Siegel and Trauth2015; Duarte-Méndez et al., Reference Duarte-Méndez, Quintero-Silva and Ramírez-Pinilla2018; Motta et al., Reference Motta, Tammaro, Di Lorenzo, Panzuto, Verderame, Migliaccio and Simoniello2020). As the follicular development progressed, we observed an increase in immunolabelling intensity. A similar effect was observed in Uromastix acanthinura, where E2 positively influenced PRA and PRB (Shao et al., Reference Shao, Markström, Friberg, Johansson and Billig2003; Hammouche et al., Reference Hammouche, Remana and Exbrayat2012). In the turtle Chrysems picta, PRA expression inhibits VTG synthesis (Duggan and Callard, Reference Duggan and Callard2003) whereas PRB promotes it (Giannoukos and Callard, Reference Giannoukos and Callard1995). In the preovulatory stage, PR presence can regulate ovulation and caspase 3 activity (Shao et al., Reference Shao, Markström, Friberg, Johansson and Billig2003; Hammouche et al., Reference Hammouche, Gernigon and Exbrayat2017). Therefore, the differences in the events may be mediated by each receptor in the different species.
Our observations indicate that granulosa cells can have different roles in oocyte maturation, depending on the sensitivity to diverse intraovarian factors. The immunolabelling of the sex steroid receptors studied in the granulosa cells and, particularly the pyriform cells, have similar behaviour as other receptors influencing gonadotropin secretion of bradykinin, c-kit, and stem cell factor (Singh et al., Reference Singh, Krishna, Sridaran and Tsutsui2008; Raucci and di Fiore, Reference Raucci and di Fiore2011). The above confirms their support role in transferring diverse components to the oocyte after their breakdown (Neaves, Reference Neaves1971; Taddei, Reference Taddei1972; Filosa and Taddei, Reference Filosa and Taddei1976; Andreuccetti, Reference Andreuccetti1992; Motta et al., Reference Motta, Scanderbeg and Filosa1995; Aldokhi et al., Reference Aldokhi, Alwasel and Harrath2019).
In conclusion, our study revealed that the immunolabelling of ER and PR was more pronounced in the pyriform cells and oocyte cortex. These observations suggest that in females of S. torquatus, steroid receptors participate in the regulation of follicular development as in other vertebrates. Those receptors have a spatio-temporal expression depending on the stage of the reproductive cycle.
Acknowledgements
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) through a scholarship awarded to N. B. Cruz-Cano (CVU 621940). We are grateful to the Sierra de Guadalupe State Park staff, Juan Gerardo Valverde (regional delegate of Tultitlán) and Mario Alberto Serrano Pérez (Protection and Surveillance Assistant). Also, we thank the field assistance of Romeo Eduardo Loya-Zurita, Yabín Josué Castro-Camacho, Alejandro Cadena Velázquez, Julián Torres Gloria and Rodrigo Dávila-Govantes. For the manuscript review to David Osvaldo Cruz-Cano. We express our gratitude to the anonymous reviewers.
Financial support
This work was supported by the CONACYT through a scholarship awarded to N. B. Cruz-Cano (CVU 621940).
Competing interest
None
Ethical standard
The UNAM Bioethical Committee approved all experimental procedures.