Elevation in water salinity can threaten the spermatogenesis and fertility of freshwater animals. The role of the renin–angiotensin system (RAS) in regulating spermatogenesis has attracted considerable attention. Our previous study found that red-eared sliders (Trachemys scripta elegans), could survive in 10 PSU water for over 1 year. To understand the chronic impact of salinity on testicular spermatogenesis and underlying mechanisms, male T. s. elegans were subjected to treatment with water of 5 PSU and 10 PSU for a year, and spermatogenesis and regulation of the RAS signal pathway was assessed. Results showed induced inflammation in the testes of T. s. elegans in the 10 PSU group, as evidenced by a decrease in the number of testicular germ cells from 1586 to 943. Compared with the control group, the levels of proinflammatory genes, including TNF-α, IL-12A and IL-6 were elevated 3.1, 0.3, and 1.4 times, respectively, in animals exposed to 10 PSU water. Testicular antiapoptotic processes of T. s. elegans might involve the vasoactive peptide angiotensin-(1–7) in the RAS, as its level was significantly increased from 220.2 ng ml−1 in controls to 419.2 ng ml−1 in the 10 PSU group. As expected, specific inhibitor (A-779) for the Ang-(1–7) acceptor effectively prevented the salinity-induced upregulation of genes encoding anti-inflammatory and antiapoptotic factors (TGF-β1, Bcl-6) in the testis of the 10 PSU animals, whereas it promoted the upregulation of proinflammatory and proapoptotic factors (TNF-α, IL-12A, IL-6, Bax and caspase-3). Our data indicated that Ang-(1–7) attenuates the effect of salinity on inflammation and apoptosis of the testis in T. s. elegans. A new perspective to prevent salinity-induced testis dysfunction is provided.

In recent years, coastal freshwater salinization has become an emerging global problem due to sea level rise caused by global warming and seawater intrusions caused by extreme weather (Horton et al., 2014; Kaushal et al., 2021; Mengel et al., 2016). This implies that many extant coastal freshwater animals may be affected to some degree by increased salinization of freshwater ecosystems (Hasan et al., 2017; Leite et al., 2022). Importantly, variations in salinity can pose a serious threat to biological functions and reduce the fertility of freshwater animals. In fish, the elevated salinity can hinder its fertility via its impact on hormone secretion, and testis and ovary development (Bosker et al., 2017; Cruz Vieira et al., 2019). Although some attention has been given to the effect of salinity changes on animal fertility, most of which has focused on fish (Green et al., 2020) and shellfish (Long et al., 2017; Nichols et al., 2021), there is a shortage of research on reptiles and amphibians.

The red-eared slider (Trachemys scripta elegans), listed among the 100 worst worldwide invasive species, is a freshwater turtle native to the USA and northeastern Mexico (Gibbons et al., 1979; Lowe et al., 2000). In the face of increasing salinity, T. s. elegans attempts to regulate the necessary water and ion balance in cells and tissues through a series of physiological mechanisms, such as enhancing Na+/K+-ATPase activity (Zhang et al., 2014), regulating energy metabolism systems (Hong et al., 2019), activating antioxidant defenses (Ding et al., 2019) and the unfolded protein response (Li et al., 2021), thus prolonging its survival time. Experiments on the tolerance of T. s. elegans to the salinity of water environments have shown that most die in 35 PSU (seawater salinity) within 30 days, while they survive in 15 PSU for at least 3 months (Zhang, 2014). With time elapsed, the sliders display symptoms such as edema, tissue and organ damage, and inevitably death when exposed to 15 PSU for more than 3 months.

However, if the environmental salinity is maintained at 10 PSU, these turtles can survive for over a year (our unpublished data). Additionally, female T. s. elegans inhabiting low salinity environments (<15 PSU) maintain reproductive function (Gibbons et al., 1979; Yang, 2015). Consequently, water conditions with 10 PSU are more appropriate to study the chronic effects of water salinity on the fertility of T. s. elegans in their natural state.

Spermatogenesis is an indicator for evaluating the effects of ecological factors on animal fertility (Gabrielsen and Tanrikut, 2016). To date, research on the regulation of spermatogenesis has focused chiefly on the hypothalamic–pituitary–gonadal (HPG) axis (Yang et al., 2021). Although the renin–angiotensin system (RAS), as an endocrine system, is best known for its role in regulating water–salt balance and blood pressure in vertebrates (Nishimura, 2017; Stroth and Unger, 1999), accumulating evidence shows that RAS is also indispensable in the regulation of fertility in males (Gianzo and Subiran, 2020; Herr et al., 2013; Leung and Sernia, 2003). Preliminary transcriptomic analysis revealed that salinity exposure significantly enriches the RAS pathway in the liver of T. s. elegans (accession no. PRJNA612601, Fig. S1). The RAS pathway includes angiotensinogen (AGT) and angiotensin I (Ang I), which are acted on by renin and angiotensin-converting enzyme (ACE), respectively, to form the bioactive metabolite angiotensin II (Ang II). Ang II eventually exerts its actions through Ang II type 1 and 2 receptors (AT1R and AT2R) (Paul et al., 2006; Reis and Reis, 2020). It is imperative to note that while the RAS component has been demonstrated in the freshwater turtle Pseudemys scripta (=T. s. elegans) (Cipolle and Zehr, 1984; Stephens, 1981; Zehr et al., 1981), there is a marked lack of information regarding its relationship with spermatogenesis in this species.

The canonical RAS theory has been expanded with the identification of new components of the RAS: ACE2, an ACE homolog, is responsible for the conversion of Ang II to angiotensin-1–7 [Ang-(1–7)], which activates the G protein-coupled receptor of angiotensin-(1–7) (Mas) receptor (MasR) (Santos et al., 2019). An experimental study demonstrated that the Ang-(1–7)/MasR axis is downregulated in the testes of men with impaired spermatogenesis, suggesting a possible role for Ang-(1–7) in the regulation of male fertility (Reis et al., 2010). Several studies have revealed that Ang-(1–7) is associated with processes such as testosterone synthesis, inflammation, and apoptosis in the testes (Al-Maghrebi and Renno, 2016; Leal et al., 2009; Xu et al., 2007). In particular, the impact of air pollution and COVID-19 on spermatogenesis through the ACE2/Ang-(1–7)/MasR pathway has been highlighted in recent studies (Montano et al., 2021; Seymen, 2021). However, it remains unclear whether changes in salinity in the aquatic environment affect testicular spermatogenesis in freshwater animals via this pathway. This information must be urgently obtained to enhance our understanding of the subject.

The main purpose of this study was to investigate the role of RAS, which participates in regulating spermatogenesis after salinity stress. We first evaluated the effects of salinity on the testes by the histomorphological changes and the inflammation and apoptosis gene expression in the testes after salinity stress of T. s. elegans. Next, we assessed the response of testicular RAS to salinity. To test the hypothesis that the testicular Ang-(1–7) in the RAS regulates spermatogenesis under salinity stress, we administered the Ang-(1–7) Mas receptor antagonist (A-779) to T. s. elegans and measured the inflammation and apoptosis-related gene expression changes in testes after salinity stress.

Turtle husbandry and experimental design

Sixty-three healthy adult male T. s. elegans (age 6 years; mass=550±50 g) were obtained from a turtle farm (Hongwang Ltd, Haikou, China). The individuals were reared under the standard facility conditions with constant light (12 h:12 h light:dark cycle), temperature (27±2°C), pH (7.5±0.2), and dissolved oxygen (8.5±0.3 mg l−1) and were fed commercial diets twice a week. After acclimation, 27 turtles were used in experiment I. Thirty-six turtles were used in experiment II.

In experiment I (Fig. 1), the turtles were randomly distributed in the experimental tanks into three groups and chronically exposed to the following: freshwater group (S0), 5 PSU group (S5), and 10 PSU group (S10) for 1 year (n=9/group). The water salinity was produced using sea salt crystals (Hailong Brand, Hailan Marine Biological Auxiliary Factory, Jiangxi, China), monitored daily, and adjusted as needed.

Fig. 1.

Design of study to investigate the role of Angiotensin-(17) in regulating spermatogenesis in Trachemys scripta elegans under salinity stress.

Fig. 1.

Design of study to investigate the role of Angiotensin-(17) in regulating spermatogenesis in Trachemys scripta elegans under salinity stress.

In experiment II (Fig. 1), the turtles were randomly distributed in the experimental tanks into two groups and chronically exposed to the following: freshwater group (S0) and 10 PSU group (S10) (n=18/group). After 1 year, these two groups (S0 and S10) were treated with A-779 and normal saline (NS), respectively. These turtles were divided into four groups (n=9/group): S0+NS, S0+A-779, S10+NS, and S10+A-779. NS (1 ml kg−1 day−1) or A-779 (300 ng kg−1 day−1) was administered subcutaneously for 4 consecutive weeks. A-779 was diluted in NS to prepare the working solution (0.3 mg ml−1), and the NS and A-779 were purchased from Kelun (Guangdong, China) and AbMole (Shanghai, China), respectively.

All animal protocols described herein were performed according to the guiding principles of the Hainan Provincial Ecological Environment Education Center Experimental Animal Ethics Committee (No: HNECEE-2019-005).

Sample collection and processing

Following Li et al. (2023), T. s. elegans were sampled in August and September. As described in Fig. 1, animals were weighed and generally anesthetized using 20% ethyl carbamate (1.6 g kg−1) after various treatments. Then, the turtles were euthanized, and samples were collected on ice. Blood was collected from individuals in 5 ml tubes for ELISA analyses. Testis tissues were taken, and the weights were determined directly. The gonadosomatic index (GSI) was calculated as follows: GSI=testes mass (g)/body mass (g). The testis samples from three individuals were fixed with paraformaldehyde for histological and apoptotic analysis, while the testes from nine individuals were frozen in liquid nitrogen and stored at −80°C.

Histological observation

To examine the histopathological changes in T. s. elegans testicular tissues, Hematoxylin and Eosin (HE) staining was performed based on the method described by Ding et al. (2021). Briefly, the fixed testes were dehydrated with an ethanol gradient. Subsequently, the dehydrated samples were cleared in xylene and embedded in paraffin wax. The testicular sections (4 μm) were continuously sliced with a Leica slicer (Leica, RM2016, Germany) and stained with HE. Finally, images were taken using a light microscope (Nikon, Eclipse E100, and DS-U3, Japan). Three testicular sections per animal were analyzed; 6 standardized fields were selected from each testicular section. The number of seminiferous tubules and the number of germ cells per field were manually counted.

Gene expression analysis

Total RNA was extracted from isolated testes using the TRIzol® Reagent Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the standard protocol. The concentration and integrity of the RNA were measured using a NanoDrop™ One/OneC spectrophotometer (Thermo Scientific, MA, USA) and 1.2% agarose gel electrophoresis, respectively. cDNA was synthesized from 1 μg mRNA using a cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the Genious 2X SYBR Green Fast qPCR Kit (ABclonal Technology, Wuhan, China) in a LightCycler 480 SYBR® (Roche) with specific primers for target genes as given in Table S1. All primers were designed based on the gene sequences in our T. s. elegans transcriptome data (accession no. PRJNA612601) using the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and then synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). β-Actin was used as the internal control, and the relative quantification of mRNA expression was calculated by the 2−ΔΔCt method.

Apoptosis detection

To detect apoptosis changes in T. s. elegans testicular tissues, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. Different groups of paraffin-embedded testis tissue were serially sectioned (5 μm), and the standard method was applied according to the protocols of the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). The testis slices were dewaxed, and the proteinase K working solution was added and incubated at 37°C for 25 min. The slices were added to the permeabilized working solution, incubated at room temperature for 20 min, and then washed three times with PBS. After equilibrating at room temperature, the TDT reaction mixture was poured into tissue slices and incubated at 37°C for 2 h in the dark. Then, the cells were incubated with DAPI solution at room temperature for 10 min. Finally, coverslips were mounted with an antifade mounting medium after washing with PBS. The sections were observed under a fluorescence microscope (Nikon Eclipse C1), and the cells in the testes exhibiting green nuclear staining were considered positively apoptotic cells. We used ImageJ imaging software (National Institutes of Health, Bethesda, MD, USA, Java 8) to quantify the green fluorescence intensity of TUNEL-positive cells in the testis. The percentage of apoptotic cells was calculated for each section as the ratio of total TUNEL-positive cells to whole DAPI-positive cells. Three testicular sections per animal were analyzed; 10 standardized fields were selected from each testicular section.

Biochemical analysis

To determine the levels of Ang II and Ang-(1–7) in plasma and testicular tissues (n=6), ELISA was performed. First, testis tissue was homogenized with 100 mg in 1 ml of NS. Then, the plasma and tissue supernatants were obtained by centrifugation at 4°C for 15 min (3000 rpm). Thereafter, the Ang II and Ang-(1–7) contents were measured using ELISA kits (Zike Biological Technology Co. Ltd, Shenzhen, China) according to the manufacturer's protocols. Optical density (OD) was recorded at 450 nm using a microtiter plate reader within 15 min. Samples were tested in triplicate.

Statistical analysis

All data were statistically analyzed using Excel 2019 and SPSS 22.0 software. Before analyzing the data, the Homogeneity Variances Test (HeV) and Kolmogorov–Smironov Test (KeS) were used to test the uniformity and normality, respectively. If the data were normally distributed, one-way ANOVA followed by Duncan's multiple range test was used to calculate the significance of differences among groups. If the data were not normally distributed, the Kruskal–Wallis H test was used to calculate the significance of differences among groups. Values were considered significant at P<0.05. Values are expressed as the mean±s.e.m. Statistical graphs were generated using GraphPad Prism software version 9.0.

Salinity stress induces histopathological damage in the testis

There was no significant difference in the GSI among the S0, S5 and S10 groups (P>0.05) (Fig. S2). However, there was a difference in the histological structure of testes among these three groups (Fig. 2). In freshwater controls (S0), the testicular architecture and morphology were normal, with spermatogenic cells at all developmental stages tightly connected in seminiferous tubules (Fig. 2A). Typical sperm columns radiating from the bottom of the basement membrane to the lumen were formed, and a considerable number of spermatids was contained in the lumen (Fig. 2A). There was no significant change in the histological structure of the testes of T. s. elegans in the S5 group compared with the control group (S0) (P>0.05) (Fig. 2B,C). However, in the S10 group, the arrangement of seminiferous tubules was looser and more disturbed, the structure was deformed and some seminiferous tubules were atrophied. The layers of testicular spermatogenic epithelia were severely exfoliated and disordered in the S10 group compared with the S0 group (P<0.05) (Fig. 2A,B). Moreover, in the S10 group, there was a significant reduction in germ cells, and exfoliated germ cells were observed in the lumen (P<0.05) (Fig. 2A,C). These results suggested that T. s. elegans exhibited high adaptability in the S5 group, while morphological and functional damage of the testes occurred in the S10 animals. These adverse effects were more severe as the water salinity increased.

Fig. 2.

Histological structure of the testis in T. s. elegans following exposure to salinity for 1 year. (A) Testicular histopathology was evaluated by H&E staining in T. s. elegans exposed to control (S0), 5 PSU (S5) and 10 PSU (S10) conditions. Ge, seminiferous epithelium; It, interstitium; Lu, lumen; Spg, spermatogonia; Spc, spermatocytes; Rspm, round spermatid; Espm, elongated spermatid; Spz, spermatozoa. Black star: layers of seminiferous tubules that are severely exfoliated, with loose cell alignment. Red star: exfoliated germ cells in the lumen. Scale bars: 100 μm (left), 20 μm (right). (B) The number of seminiferous tubules from spermatogenic epithelia exfoliated per histological section. (C) The number of germ cells per histological section. Data on quantification are based on the average of 6 fields observed in each testicular section. Data are shown as means±s.e.m. (n=3). Different letters (a, b) indicate that there is a significant difference between different groups (P<0.05).

Fig. 2.

Histological structure of the testis in T. s. elegans following exposure to salinity for 1 year. (A) Testicular histopathology was evaluated by H&E staining in T. s. elegans exposed to control (S0), 5 PSU (S5) and 10 PSU (S10) conditions. Ge, seminiferous epithelium; It, interstitium; Lu, lumen; Spg, spermatogonia; Spc, spermatocytes; Rspm, round spermatid; Espm, elongated spermatid; Spz, spermatozoa. Black star: layers of seminiferous tubules that are severely exfoliated, with loose cell alignment. Red star: exfoliated germ cells in the lumen. Scale bars: 100 μm (left), 20 μm (right). (B) The number of seminiferous tubules from spermatogenic epithelia exfoliated per histological section. (C) The number of germ cells per histological section. Data on quantification are based on the average of 6 fields observed in each testicular section. Data are shown as means±s.e.m. (n=3). Different letters (a, b) indicate that there is a significant difference between different groups (P<0.05).

Inflammation and apoptosis in the testis under salinity stress

The mRNA expression levels of the proinflammatory cytokine IL-12A were elevated in the T. s. elegans testes after stress to salinity (S5 and S10 groups) (P<0.05) (Fig. 3). In particular, expression levels of the proinflammatory cytokines TNF-α and IL-6 in the S10 group were significantly higher than those in the S0 group (P<0.05). S5 and S10 animals decreased the expression levels of the anti-inflammatory cytokine IL-10 below the levels in the control group (P<0.05). In contrast, the mRNA expression level of TGF-β1 was significantly increased after stress to salinity (S5 and S10 groups) (P<0.05) and was highest in the S5 group. In addition, the expression level of the antiapoptotic gene Bcl-2 was markedly increased in the S5 animals (P<0.05), and the Bcl-6 level was significantly increased in both the S5 and S10 animals compared with the controls (P<0.05) with the highest expression in the S5 group.

Fig. 3.

Relative mRNA expression of genes involving inflammation and apoptosis in testes of T. s. elegans following exposure to salinity for 1 year. Different letters (a–c) indicate that there is a significant difference between different groups (P<0.05, n=6).

Fig. 3.

Relative mRNA expression of genes involving inflammation and apoptosis in testes of T. s. elegans following exposure to salinity for 1 year. Different letters (a–c) indicate that there is a significant difference between different groups (P<0.05, n=6).

The number of TUNEL-positive cells increased with increasing salinity. Apoptotic cells in spermatocytes were observed in the S5 and S10 groups (Fig. S3A). Notably, the apoptosis rates of testicular tissue in the S5 and S10 animals were higher than in the S0 group. However, there was no significant difference (P>0.05) (Fig. S3B).

Salinity stress activates RAS in testicular tissue

There was no significant change in Ang II and Ang-(1–7) levels in plasma among the three groups (S0, S5 and S10) (P>0.05), whereas the levels of Ang II and Ang-(1–7) in the testis of the salinity-treated groups (S5 and S10) were significantly higher than those in the controls, especially Ang-(1–7), which in the S5 and S10 animals was 2.1 and 1.9 times as abundant as that in S0 group, respectively (P<0.05) (Fig. 4A). The results show that the local RAS in the testis of T. s. elegans is activated in the animals exposed to 5 and 10 PSU.

Fig. 4.

Effect of salinity exposure on the RAS in the testes of T. s. elegans. (A) Ang II and Ang-(1–7) concentrations in plasma and testes of T. s. elegans following exposure to salinity for 1 year (n=6). (B) mRNA expression level of RAS-related genes (AGT, Renin, ACE, ACE2, AT1R, AT2R and MasR) in testes of T. s. elegans following exposure to salinity for 1 year (n=6). Different letters (a,b) indicate that there is a significant difference between different groups (P<0.05).

Fig. 4.

Effect of salinity exposure on the RAS in the testes of T. s. elegans. (A) Ang II and Ang-(1–7) concentrations in plasma and testes of T. s. elegans following exposure to salinity for 1 year (n=6). (B) mRNA expression level of RAS-related genes (AGT, Renin, ACE, ACE2, AT1R, AT2R and MasR) in testes of T. s. elegans following exposure to salinity for 1 year (n=6). Different letters (a,b) indicate that there is a significant difference between different groups (P<0.05).

The expression of AGT in the testis was significantly increased in the S10 group compared with the controls (P<0.05). In addition, no significant changes were observed in the expression of ACE, AT1R, or MasR among the three groups (P>0.05). Notably, the mRNA expression levels of genes encoding renin, ACE2 and AT2R in the S5 and S10 animals were significantly higher than those in the control group (P<0.05) (Fig. 4B). In particular, the expression level of ACE2 in the testes of the S5 and S10 groups was approximately 6.0- and 5.6-fold higher than that of the S0 group, respectively.

Ang-(1–7) attenuates salinity-induced inflammation and apoptosis in the testis

The mRNA expression levels of proinflammatory cytokines (TNF-α, IL-12A and IL-6) in testes were significantly elevated after salinity stress (S10+NS) (P<0.05) (Fig. 5). However, treatment with A-779 further significantly increased the mRNA expression levels of the proinflammatory cytokines TNF-α, IL-12A and IL-6 and markedly reduced the expression levels of the anti-inflammatory cytokine TGF-β1 in the testis after salinity stress (S10+A-779) compared with the S10+NS group (P<0.05). In addition, instead of decreasing, the expression level of the antiapoptotic gene Bcl-6 was significantly elevated in the S10+NS group compared with the S0+NS animals (P<0.05). However, the addition of A-779 significantly promoted the expression level of genes encoding the proapoptosis markers Bax and Caspase-3. It markedly inhibited the expression of Bcl-6 after salinity stress (S10+A-779 group) compared with the S10+NS group (P<0.05). These changes supported our assumption that Ang-(1–7) regulated inflammation and apoptosis in the testes of T. s. elegans under salinity stress.

Fig. 5.

Relative mRNA levels of inflammatory factors (TNF-α, IL-12A, IL-6, and TGF-β1) and apoptotic genes (Bax, Caspase-3, Bcl-2, and Bcl-6) in testes of T. s. elegans treated with A-779 for 1 month (n=6). Different letters (a–d) indicate that there is a significant difference between different groups (P<0.05).

Fig. 5.

Relative mRNA levels of inflammatory factors (TNF-α, IL-12A, IL-6, and TGF-β1) and apoptotic genes (Bax, Caspase-3, Bcl-2, and Bcl-6) in testes of T. s. elegans treated with A-779 for 1 month (n=6). Different letters (a–d) indicate that there is a significant difference between different groups (P<0.05).

There is an increase in the extent and intensity of freshwater salinization worldwide (Horton et al., 2014; Mengel et al., 2016). This environmental problem commands more attention because increased water salinities can threaten the fertility of freshwater species, including turtles, and have the capacity to change entire population structures (Cruz Vieira et al., 2019; Jeppesen et al., 2015). The testes are the most vital reproductive organ in the male reproductive system and play an essential role in hormone secretion and spermatogenesis. Histopathological analysis showed that the testes in the T. s. elegans exposed to 5 PSU were not significantly different from those in the control group (S0), which revealed that a salinity level of the water environment below 5 PSU has no significant effect on the number of spermatogenic cells or the structure of the testes. Some mechanism might exist in T. s. elegans testes to regulate spermatogenesis to reduce the negative impact of elevated salinity in the water, thus improving their tolerance to a brackish water environment. Nevertheless, the testes of the S10 group showed tissue damage, including vacuolation of the seminiferous epithelium, disordered germ cell arrangement, and exfoliation in this study. Similar results were obtained by Cruz Vieira et al. (2019), who found that high salinity levels increased the presence of empty spermatogenic cysts in the seminiferous tubules, enlarged the interstitial tissue, caused hyperplasia of Leydig cells, and led to inflammatory infiltrate of euryhaline in Nile tilapia Oreochromis niloticus. It has been shown that spermatogenesis and fertility in mice are particularly impaired by testicular inflammatory events (Jin et al., 2018). Our observations indicate that T. s. elegans exhibits high tolerance in the lower salinity environment (<5 PSU), whereas the high-salinity environment (10 PSU) will disrupt the normal structure of the testes and induce testicular inflammation.

The overexpression of inflammatory cytokines is a marker of tissue damage. Our study has shown that expression levels of the proinflammatory cytokines TNF-α and IL-6 were significantly upregulated in the 10 PSU animals, indicating that the testes were in an inflammatory state. Additionally, salinity stress (S5 and S10 groups) promoted the expression of the anti-inflammatory cytokine TGF-β1 in the testis, especially in the 5 PSU group, which may be the main reason why the testicular structure of the turtles in this group was not altered. This is because T. s. elegans may resist salinity-induced damage by promoting the expression of anti-inflammatory cytokines to maintain normal testicular development and spermatogenesis in the low-salinity water environment. In the S10 group, this balance between the expression levels of anti-inflammatory and proinflammatory cytokines in the testes was disturbed, inducing inflammatory events in the testes, which resulted in structural damage to the testes of T. s. elegans.

Both Bcl-2 and Bcl-6 belong to the B-cell lymphoma (Bcl) family of proteins that prevent apoptosis, while Bax, as a proapoptotic protein, is a member of the Bcl-2 family of proteins (Morrill and He, 2017). Caspase-3 is one of the best-known markers of apoptosis and is proteolytically cleaved from pro-caspase-3 (Bernard et al., 2019). In our study, there was no significant change in the mRNA expression of proapoptotic genes (Bax and Caspase-3) or the apoptosis rates of testicular tissue (by TUNEL staining) in the testes after salinity stress (S5 and S10 groups). Interestingly, there was a significant increase in the mRNA expression of antiapoptotic genes (Bcl-2 and Bcl-6) in the 5 PSU animals, which may be attributed to some physiological mechanism existing in the turtle to prevent apoptosis. Previous studies have shown that the RAS system can modulate the inflammatory and apoptosis process of the testis by affecting cytokine production and release (endothelin 1), cellular migration (neutrophil influx) and signaling pathways (NF-κB and JNK pathways) (Capettini et al., 2012; Li et al., 2017; Meneses et al., 2014; Silveira et al., 2010).

To verify the interaction among RAS, inflammation and apoptosis of T. s. elegans under salinity stress, we analyzed the levels of Ang II and Ang-(1–7) in both the plasma and testis. The results showed that the levels of Ang II and Ang-(1–7) increased significantly only in the testes of T. s. elegans after salinity stress (S5 and S10 groups), indicating that the local RAS in testis tissue is probably associated with the process of testicular inflammation and apoptosis after salinity stress. Increasing evidence suggests that the local tissue RAS is found in both male and female genital organs (Aykan et al., 2020; Herr et al., 2013). The RAS plays a relevant role in the pathogenesis of the inflammatory disease, and proinflammatory actions appear to be the effects of Ang II. For instance, human testicular peritubular cells (HTPCs) stimulated with Ang II exhibit significantly increased IL-6 mRNA levels and IL-6 secretion within hours (Welter et al., 2014). In addition, Ang II causes apoptosis of adult hippocampal neural stem cells (HCNs) through AMPK-PGC1α signaling (Kim et al., 2017). Thus, considering that locally generated Ang II amplifies the immune-mediated inflammatory process (Neri Serneri et al., 2004), it can be suggested that salinity stress promoted the level of Ang II in testis, which in turn increased the expression of proinflammatory cytokines and ultimately led to inflammation in the testis of T. s. elegans (Fig. 6). Notably, no significant apoptosis was observed in the testicular tissues of T. s. elegans after salinity stress. Simultaneously, the data presented unequivocally demonstrated that salinity stress markedly elevated Ang-(1–7) levels in T. s. elegans testes, surpassing Ang II levels. Previous studies have shown that administering exogenous Ang-(1–7) before reperfusion of the ischemic testis can prevent damage to spermatogenesis by antiapoptosis and antioxidative stress mechanisms (Al-Maghrebi and Renno, 2016). Combined with these results, it is reasonable to believe that elevated levels of Ang-(1–7) may predominate in testes and have the potential to regulate spermatogenesis through antiapoptosis actions after the stress of T. s. elegans to salinity.

Fig. 6.

Proposed model for how Ang-(1–7) attenuates the effect of brackish water environments on inflammation and apoptosis in the testes of T. s. elegans. ACE, angiotensin-converting enzyme; AGT, angiotensinogen; Ang, angiotensin; AT1R, Ang II type 1 receptor; Bcl-6, B-cell lymphoma 6; IL, interleukin; MasR, Mas receptor; TGF-β1, transforming growth factor-β; TNF-α, tumor necrosis factor-α.

Fig. 6.

Proposed model for how Ang-(1–7) attenuates the effect of brackish water environments on inflammation and apoptosis in the testes of T. s. elegans. ACE, angiotensin-converting enzyme; AGT, angiotensinogen; Ang, angiotensin; AT1R, Ang II type 1 receptor; Bcl-6, B-cell lymphoma 6; IL, interleukin; MasR, Mas receptor; TGF-β1, transforming growth factor-β; TNF-α, tumor necrosis factor-α.

It is well known that AGT, which acts as a substrate for renin, is the starting point for the RAS. Renin is a protease produced in the kidney that cleaves AGT to make the inactive decapeptide Ang I, so it is considered the rate-limiting enzyme of Ang II synthesis (Liu et al., 2019). In our study, the levels of AGT and renin exhibited a significant increase in the testes of T. s. elegans after salinity stress (S5 and S10 groups). Our results agree with previous findings that the changes in testis RAS may have an essential role in spermatogenesis impairments (Fang et al., 2018; Manção Santos et al., 2022). Remarkably, a significant upregulation of ACE2 expression was observed in the testis after salinity stress. ACE2 is considered the main Ang-(1–7)-forming enzyme (Simões Silva et al., 2013). This result implies that ACE2, which is elevated under salinity stress, leads to the upregulation of Ang-(1–7). Ang-(1–7), after binding to its receptor MasR, exerts its anti-inflammatory effects through attenuated NF-κB activity, phospho-p38 MAPK, proinflammatory cytokines, phospho-JNK, phospho-ERK1/2 and PI3K (Jiang et al., 2012; Santos et al., 2013; Souza and Costa-Neto, 2012; Su et al., 2006; Xue et al., 2012; Zhang et al., 2016). Thus, we hypothesize that the counter-regulatory axis composed of ACE2, Ang-(1–7) and MasR opposes action of the ACE/Ang II/AT1R branch and is involved in the anti-inflammatory and antiapoptotic processes of testicular spermatogenesis.

We next treated two groups (S0 and S10) of T. s. elegans with Ang-(1–7) Mas receptor antagonist (A-779) to obtain solid evidence that Ang-(1–7) participated in regulating inflammation and apoptosis of testis after salinity stress. As shown in our study, the treatment of T. s. elegans with A-779 after salinity stress resulted in a significant increase in the expression of proinflammatory factors (TNF-α, IL-12A and IL-6) and proapoptotic genes (Bax and Caspase-3) but a substantial reduction in anti-inflammatory (TGF-β1) and antiapoptotic genes (Bcl-6) (Fig. 6). This finding indicated that administration of A-779 after salinity stress resulted in Ang-(1–7) molecular blockade, that is, the anti-inflammatory and antiapoptotic effects were alleviated. Notably, Ang-(1–7) inhibited the inflammation and apoptosis of testis tissue after salinity stress, which is due to its interaction with its receptor Mas. Likewise, a recent study (Zhang et al., 2018) found that added Ang-(1–7) can ameliorate seawater stimulation-induced apoptosis, and this effect was related to the JNK pathway. In addition, Li et al. (2008) reported that the ACE2/Ang-(1–7)/MasR axis exhibited significant antiapoptotic action in pulmonary fibrosis, which was consistent with the results in our study. Thus, we demonstrated that Ang-(1–7) attenuates testicular inflammation and apoptosis in response to salinity stress.

Conclusion

In summary, Ang-(1–7) exerts a significant protective effect on the testicular tissues of T. s. elegans under salinity stress. The testicular tissues of T. s. elegans appear normal after prolonged exposure to low salinity (5 PSU). As the salinity level in the water increased (10 PSU), the testes presented histological abnormalities and displayed elevated expression of proinflammatory cytokines (TNF-α, IL-12A, and IL-6). This indicates that chronic exposure to salt concentrations 10 PSU induces testicular inflammatory events. However, the testicular tissue did not undergo significant apoptosis in the S10 group. The testicular anti-inflammatory and antiapoptotic processes of T. s. elegans primarily involved the local RAS that was activated in testes following salinity stress, which was associated with upregulated Ang-(1–7) levels in the RAS after salinity stress. We demonstrated that Ang-(1–7) protected testicular tissue against increased inflammation and apoptosis induced by chronic salinity stress (S10), as the addition of A779 significantly reduced the expression of anti-inflammatory and antiapoptotic genes. The data we have collected is crucial for evaluating the impact of salinity on the fertility of freshwater turtles and suggesting a focus for future studies on the RAS regulation of testis development and spermatogenesis.

Author contributions

Conceptualization: N.L., M.H.; Methodology: N.L., Q.Z.; Validation: Q.Z., S.D.; Formal analysis: N.L., W.R.; Investigation: S.D., W.R.; Resources: H.S., L.D.; Writing - original draft: N.L.; Writing - review & editing: L.D., M.H.; Visualization: N.L.; Supervision: L.D., M.H.; Funding acquisition: N.L., L.D., M.H.

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers 32160251, 31960226, 32271557), Hainan Natural Science Foundation (grant number 320RC598), and the Hainan Innovation Graduate Research Project (grant number Qhyb2021-54).

Data availability

All relevant data can be found within the article and its supplementary information.

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Competing interests

The authors declare no competing or financial interests.

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