Immunodistribution of RFamide-related peptide-3 (RFRP-3) during the seminiferous epithelium cycle in a desert rodent Psammomys obesus
Introduction
Spermatogenesis is a cyclic and highly coordinated process that begins with spermatogonial proliferation, proceeds through two meiotic divisions, and is followed by spermiogenesis in which haploid spermatids develop into spermatozoa that will thereafter be released into the lumen of seminiferous tubule by spermiation (Johnson et al., 1991; Russell, 1990). The germ cells are organized in distinct and regular cellular associations known as stages of the seminiferous epithelium cycle (SEC) (Leblond and Clermont, 1952a, 1952b). Often, the classification of these stages depends on two methods; the first one, that according to the tubular morphology system (Berndston, 1977; Johnson et al., 1991; Leblond and Clermont, 1952a, 1952b; Russell, 1990), and the second one, that according to the development of the acrosomic system (Leblond and Clermont, 1952a, 1952b; Russell, 1990).
The reproductive function in vertebrates depends on Gonadotropin-Releasing Hormone (GnRH) which orchestrates the cascade of reproductive events by stimulating the Hypothalamic-Pituitary-Gonadal axis. Gonadotropins, Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH), synthesized in the anterior pituitary gland under the GnRH effect (Schally et al., 1971) control directly the testicular activity. LH and FSH are released into the circulation and activate the expression of their receptors on Leydig and Sertoli cells, respectively, to stimulate testosterone secretion and spermatogenesis (Shalet, 2009).
It was believed that GnRH is the only neuropeptide that regulates gonadotropins synthesis and release. In 2000, Tsutsui's group successfully identified a new hypothalamic dodecapeptide which, unlike GnRH, actively inhibits the release of gonadotropins in the brain of Japanese quail, and called it Gonadotropin-Inhibitory Hormone (GnIH) (Tsutsui et al., 2000). The mammalian ortholog of avian GnIH, RFamide-Related Peptide (RFRP), possesses a common LPXRF amide (XL or Q) motif at its C terminal (Tsutsui, 2009; Tsutsui et al., 2010a). RFRP precursor peptide produces both RFRP-1 and RFRP-3 (Clarke et al., 2008; Fukusumi et al., 2001).
At the cellular level, two specific G protein-coupled receptors (GPRs), GPR147 and GPR74, have been identified (Elshourbagy et al., 2000; Hinuma et al., 2000). GnIH induces via GPR147 a decrease of gonadotropin common α-and β-subunit production and release, this effect is due to an action on both the GnRH system and the anterior pituitary gland (Tsutsui et al., 2010a; Tsutsui, 2009; Ubuka et al., 2006). Alternatively, its action via GPR74 inhibits the transcriptional activity of cAMP response element (Elshourbagy et al., 2000).
In addition, different effects of RFRP-3 on LH secretion have been reported (Hu et al., 2019). As in birds, RFRP-3 also acts by inhibiting gonadotropins release in various mammalian species (Clarke et al., 2008; Ducret et al., 2009; Kriegsfeld et al., 2006; Tsutsui et al., 2010a, b; Sari et al., 2009; Tsutsui and Ubuka, 2020). On the other hand, a stimulatory effect on the gonadotropins secretion has been demonstrated in the male Syrian hamster (Ancel et al., 2012) and in mice (Ancel et al., 2017, 2012).
GnIH (RFRP-3) and its receptors are also expressed in the gonads and accessory reproductive organs of birds (Bentley et al., 2008; Maddineni et al., 2008; McGuire and Bentley, 2010) and mammals (Singh et al., 2011b; Zhao et al., 2010) including humans (Oishi et al., 2012), potentially acting in an autocrine/paracrine manner (Wang et al., 2018). In mice, the expression of RFRP-3 in the testis has been demonstrated (Anjum et al., 2012) and its effect on steroidogenesis and on GnRH receptor (GnRHR) and LH-receptor (LHR) expression has been evaluated (Anjum et al., 2014). RFRP-3 and its receptors are also detected in Syrian hamster (Zhao et al., 2010) and Gerbillus tarabuli (Hamidatou Khati et al., 2018; Khati Hamidatou et al., 2018) testes during the SEC, suggesting a role on spermatogenesis and androgens synthesis (Zhao et al., 2010). The effects of RFRP-3 depend on gonadotropins presence in primates and rats (Anjum et al., 2014; Oishi et al., 2012; Squicciarini et al., 2018). RFRP-3 expression is affected by photoperiod and reproductive conditions (Zhao et al., 2010). Previous findings indicated that the expression and the function of this peptide differ across sex and species, and depend on the physiological status of the latter (Ancel et al., 2017, 2012; Anjum et al., 2014; Bentley et al., 2017, 2008; Hamidatou Khati et al., 2018; Johnson et al., 2007; Khati Hamidatou et al., 2018; Osugi et al., 2014; Singh et al., 2011a, 2011b; Tsutsui and Ubuka, 2020; Tsutsui et al., 2018; Zhao et al., 2010). Add to that, GnIH (RFRP-3) inhibits not only reproduction but also sexual and aggressive behaviors (Kriegsfeld et al., 2015; Tsutsui et al., 2013; Tsutsui and Ubuka, 2020; Ubuka et al., 2013).
The Sand rat, Psammomys obesus (P. obesus), is a diurnal, herbivore desert rodent that displays a seasonal reproductive cycle with a breeding period from autumn through early spring and a resting phase from late spring through summer. The reproductive physiology of P. obesus well documented in male, however, remains insufficient. In fact, little information on spermatogenesis is available (Sprando et al., 1999). The aim of this study was firstly to describe the stages of SEC in P. obesus by histological and morphometric analysis, and secondly, to determine the cellular localization and the level expression of RFRP-3 during the cycle by immunohistochemistry in order to estimate its functional importance in the different steps of spermatogenesis.
Section snippets
Animals and sampling
The animals studied were five adult males (P. obesus) captured during the breeding season in Beni-Abbes, a desertic region situated in the North-Western Algerian Sahara (30° 4′ 48″ N, 2° 6′ 0″ W). They were weighed and their mean body weight was 176.8 ± 3.76 g. Animals were sacrificed by decapitation following an intraperitoneal injection of 25 % Urethane solution (1.5 g/Kg I.P = 0.4 mL per 100 g of body weight) in the late morning regarding their diurnal state. Testes were immediately removed,
Stages of the SEC
The SEC in P. obesus was distinctly grouped into 14 stages (Table 1). The morphometric data indicated a significant increase of the seminiferous epithelium thickness from stage V through IX (P < 0.05) with a significant decrease at stage VIII (P < 0.05) compared to V-VII stages (Fig. 2; Table 1). For the rest of the cycle, the seminiferous epithelium had a constant thickness (Fig. 2). In parallel, the lumen was virtual at stages V, VI and VII (Fig. 3; Table 1), however, it reappeared at stage
Discussion
The histological and morphometric study of the SEC in the wild desert rodent P. obesus, carried out for the first time, identified 14 stages with 19 steps of spermiogenesis. A single stage was found per seminiferous tubule cross-section. This finding is in perfect agreement with the data of rat (Leblond and Clermont, 1952a) and Gerbilus tarabuli (Hamidatou Khati et al., 2018). Moreover, in mammals, there are other patterns observed, with distinct arrangements between the different generations
Conclusion
The SEC in P. obesus is distinctly grouped into 14 stages according to the tubular morphology method, and only one single stage is present in one seminiferous tubule section. The morphometric parameters evaluated in this species show that the measurement of seminiferous epithelium is more effective for spermatogenic activity evaluation than the others parameters, since it indicates significant variations during the cycle. Testicular expression of RFRP-3 in P. obesus during the breading season,
Authorship contributions
Conception and design of study: N. Djouahra, E. N. Moudilou, J. M. Exbrayat, S. Hammouche;
acquisition of data: : N. Djouahra, S. Hammouche;
analysis and/or interpretation of data: N. Djouahra, E. N. Moudilou, J. M. Exbrayat, S. Hammouche;
Drafting the manuscript: N. Djouahra, S. Hammouche;
revising the manuscript critically for important intellectual content: N. Djouahra, S. Hammouche.
Approval of the version of the manuscript to be published (the names of all authors must be listed): N. Djouahra,
CRediT authorship contribution statement
Nassima Djouahra: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Writing - original draft, Writing - review & editing. Elara N. Moudilou: Conceptualization, Investigation, Methodology, Validation, Visualization. Jean-Marie Exbrayat: Conceptualization, Funding acquisition, Investigation, Resources, Supervision, Validation, Visualization. Sadjia Hammouche: Conceptualization, Data
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This work undertaken in team 3 of the LRZA Laboratory in collaboration with General Biology-Reproduction and Comparative Development Laboratory UCLy/EPHE, Lyon, France deserves to underline the investment of the two team leaders, engineers and hunters from the Beni Abbes station (Algeria). Let them be here all thank.
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Progress in research on the reproductive function in the sand rat (Psammomys obesus): A review
2023, General and Comparative EndocrinologyCitation Excerpt :A recent work studied the immunodistribution of RFamide-related peptide-3 (RFRP-3), the recently discovered mammalian ortholog of avian gonadotropin-inhibitory hormone (GnIH), in the testis of P. obesus. RFRP-3 was expressed in both testicular compartments, the seminiferous tubules and the interstitial tissue, with however a more prominent signal in the Leydig cells (Djouahra et al., 2020). During the SEC, RFRP-3 was highly detectable in late pachytene spermatocytes at stages XI–XII, and less detectable in spermatids at stage IX, suggesting an involvement of RFRP-3 in the control of SEC in P. obesus.
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