1. Introduction
It is generally believed that the negative effects related to reproduction caused by increased parents’ age are mainly attributable to females. However, there is growing evidence that the increase in male age has a significantly negative impact on spermatogenesis, sperm function, fertilization, pregnancy, and offspring health [
1]. Senescence also impacts the entire endocrine system, including the hypothalamic-pituitary-gonadal (HPG) axis, which is critical for the production of hormones such as testosterone and leads to changes in testis physiology as well as fertility status [
2]. In addition, it has been demonstrated that aging can attenuate the function of blood-testis barrier (BTB), which is constituted by coexisting tight junction (TJ) between adjacent Sertoli cells (SCs). The prominently lowered expression of TJ-related proteins (Claudin-11, ZO-1, and Occludin) has been detected in natural [
3] and iatrogenic ageing (such chronic D-galactose exposure [
4]) rodent models, suggesting that aging indeed damages the BTB function in the testes to induce spermatogenesis dysfunction.
Autophagy refers to the dynamic of self-protection and cell defense mechanism that serves as a valid routine to remove hazardous and toxic matters by cells [
5]. It has been increasingly evidenced that autophagy in SCs plays an essential role in normal generation of sperms and fertility of males [
6]. It has also been recently researched that autophagy is weakened in rat testes during aging [
4]. The damaged cells or organelles together with cumulated metabolic wastes may destroy efficient autophagy modulation as the age increases [
7]. Oxidative stress is defined as a state of imbalance between excessive oxidant (free) radicals and insufficient degradation of those radicals by antioxidant systems as an in-house defense mechanism [
8]. Notably, when an organism becomes senescent, the oxidative stress-induced lipoprotein degeneration on cell membrane and organelle impairment emerge as crucial factors for organ and cell functional decline, while autophagy relieves the impairment attributable to reactive oxygen species (ROS) accumulation during senescence [
9,
10].
As testified by enormous studies, there is a relationship between excess ROS production in cells and the infertility of males besides testis injury [
11,
12]. To be more specific, it is also a pivotal player in TJ impairment in SCs [
13]. It is argued that NLRP3 inflammasomes can be activated by ROS as a leading triggering factor [
14]. As a multiprotein complex with Caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NLRP3 as the constituents, NLRP3 inflammasome not only mediates Caspase-1 activation but also subsequently promotes IL-1
β plus IL-18 to mature and release [
13]. The latest studies have manifested that such typical cytokines may induce or worsen the inflammation in cells or organs due to their functions to mediate inflammatory responses in diversified cells, thus promoting disease progression [
15].
Classified as mesoderm-derived stem cells, bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate into various types of cells involving muscle cells, osteoblasts, adipocytes, and chondrocytes. A growing number of studies have demonstrated that transplanted MSCs could increase the reproductive ability during natural aging or in modeling of senescent animals by virtue of agents [
16,
17]. A few investigations conducted in recent years have demonstrated that BMSCs can secrete exosomes (Exos), which are special membranous vesicles in nano-size possessing similar functions to MSCs [
16]. Being small membrane-bound vesicles with a diameter of 30–100 nm, Exos can bind to the recipient cell membrane by virtue of internal membrane contents and deliver a variety of biomolecules (nucleic acids, proteins, lipids, etc.). Although BMSC-derived Exos (BMSC-Exos) have been considered as a promising therapeutic tool for resisting aging [
18], the mechanism by which Exos improve the prognosis has not been entirely understood. Thus, the beneficial effect of BMSC-Exos on aging-induced TJ impairment was explored in the present study through both in vitro experiment and mouse model. The data obtained thereof will provide useful information for developing a new therapeutic approach to improve fertility in elder boars.
2. Materials and Methods
2.1. Collection of Porcine Testes
A total of 3 young (2–3 years old) and 3 old (5–6 years old) Landrace pigs were selected from the local station for testis collection. Samples (size: approximately 3 × 3 cm2) acquired from the middle testes were directly cryopreserved (−80 °C) by liquid N2 for subsequent extraction of proteins as well as RNAs. The remaining testes were subjected to fixation using glutaraldehyde (2.5%) or paraformaldehyde (4%) for histological, immunohistochemical, and echocardiographic analyses.
2.2. Porcine BMSC Separation, Character Determination, and Differentiation
After flushing with DMEM, the bone marrow acquired from the tibia and femur of 1-month-old Landrace boars was separated through 5 min of 800× g centrifugation. Next, the sediments produced underwent inoculation into DMEM (1 × 105 cells/cm2) composed of fetal bovine serum (FBS, 10%, Hyclone Laboratory, Logan, UT, USA) mixed with penicillin (100 U/mL) and streptomycin (100 μg/mL) under a humidified atmosphere (37 °C) containing 5% CO2. After initial plating, the medium replacement was conducted every 3–4 d. The cells with about 80–90% confluence were passaged for further expansion. Finally, CD29, CD44, and CD45 were selected for characterization using immunofluorescence analysis, so as to detect the classical biomarkers of BMSCs.
The multipotent differentiation potential from porcine into osteoblasts and adipocytes was evaluated. Adipogenic or osteogenic differentiation complete media provided by Cyagen Biosciences (Suzhou, China) were utilized to replace the culture medium of BMSCs passaged to the 3rd generation. Subsequent to 14-day differentiation culture induction, intracellular lipids together with calcium were evaluated for accumulation using oil red O staining plus alizarin red staining (Sigma-Aldrich, St. Louis, MO, USA), respectively.
2.3. Porcine SC Segregation and Culture
Normal Landrace boars (1 month old in age) were chosen to obtain the testicular tissues, followed by washing in streptomycin (100 mg/mL) + penicillin (100 IU/mL) added three times into phosphate buffer saline (PBS). Later, isolation and cultivation of SCs were performed in accordance with a slightly modified previous method. Under sterile conditions, the culture medium was used for rinsing of every testis, with the tunica albuginea discarded. The separated testicular parenchyma was divided into sections followed by 15 min of digestion using collagenase type IV (1 mg/mL) at 37 °C. After washing three times in PBS, the convoluted seminiferous tubules were collected under a stereo microscope, followed by an additional 30 min of tissue digestion using collagenase type IV. Next, the cell pellets subjected to 5 min 1000× g centrifugation plus three times of culture medium rinsing were resuspended via 10% FBS-containing DMEM in a humidified incubator under 5% CO2 + 95% air at 37 °C. Being cultured for 72 h, hypotonic Tris-HCl solution (20 mM, pH 7.4) was applied to treat the cells, from which residual germ cells were eliminated through 2 min of gentle shaking, followed by discarding of Tris-HCl solution. Lastly, immunofluorescence analysis was adopted for characterization with SOX9, so as to measure the classical biomarkers of SCs.
With the confluence reaching 80%, a 6-well plate cell seeding was implemented (density: nearly 1.5 × 105 cells/well) for 24 h of 37 °C cultivation using the humidified incubator under 5% CO2 plus 95% O2.
2.4. Purification and Identification of Exos
Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) was the supplier of murine BMSCs. Following overnight inoculation in 25 cm2 culture bottles to realize 80% confluence before use, porcine and murine BMSCs were washed 3 times in PBS in addition to 24 h of serum-free DMEM culture. Exo extraction from the culture media was implemented following the recommendations offered by the manufacturer (BB-3901, Shanghai Bestbio Biotechnology Co., Ltd., Shanghai, China).
The porcine BMSC-Exos were resuspended in 30 μL of PBS, from which specimens (10 μL) were loaded onto a copper mesh for 1 min, and then filter paper was employed to absorb the liquid. Later, the copper mesh was reacted for 1 min in uranyl acetate (phosphotungstic acid, 10 μL), from which the filter paper was used for liquid elimination. Subsequent to room-temperature drying for several minutes, electron microscopy was adopted for imaging (80 kV) to examine the specimens.
2.5. PKH26 Staining for Exos
BMSC-Exos received 15 min of PKH26 (Sigma) labeling in the dark (37 °C) and were washed three times in PBS prior to 10,000× g and 4 °C centrifugation for 2 h. Later, the prepared SCs were co-cultured for 6 h with the labeled Exos (10 μg/mL). Next, DAPI (C1002, Beyotime, Nanjing, China) counterstaining of the cells was accomplished followed by washing one time in PBS to identify their nuclei. The uptake of MSC-Exos by SCs was observed under a fluorescence microscope.
2.6. Cell Treatment
Prior to experiments, serum-free DMEM was applied to culture porcine SCs for 12 h which underwent 48 h of D-galactose (D-gal, 12.5, 25 or 50 g/L in final concentrations for aging induction, HY-N0210, Med Chem Express, Monmouth Junction, NJ, USA), and/or BMSC-Exos (20 μg/mL) processing. For inhibition experiments, 2 h of cell incubation was conducted prior to treatment with or without the supplement of autophagy inducer rapamycin (Rapa, HY-N0210, 200 nM), ROS scavenger acetylcysteine (NAC, HY-B0215, 5 mM), autophagy inhibitor chloroquine (CQ, HY-17589A, 50 μM), AMPK inhibitor Compound C (CC, HY-13418A, 10 μM), NLRP3 inhibitor MCC950 (HY-12815, 10 μM), and IL-1 receptor antagonist (IL-1Ra, HY-P72566, 20 ng/mL) offered by Med Chem Express.
2.7. Measurement of Autophagic Flux
Based on the manufacturer’s instructions, the mCherry-GFP-LC3 reporter plasmid (C3011, 1 μL/mL) provided by Beyotime (Nanjing, China) was selected for SC transfection to determine the autophagic flux. Thereafter, the cells underwent grouping and processing through the aforementioned methods. Fluorescence microscopy was performed to observe the cell images.
2.8. Senescence-Associated β-Galactosidase (SA-β-Gal) Staining
The SA-β-gal staining kit purchased from Beyotime (C0602, Nanjing, China) was utilized to implement SA-β-gal staining in accordance with the protocol formulated by the manufacturer. The SA-β-gal-positive cells were stained blue. Finally, an optical microscope (Olympus-DP73, Tokyo, Japan) was employed to count positive cells.
2.9. ROS and Antioxidant Assessment
The GSH assay kit (S0073, Beyotime) was adopted to examine the SCs of the GSH level according to the manufacturer’s protocol. By reference to the manufacturer’s instructions, DCFH-DA (S0033, Beyotime) was used for total ROS level measurement. SCs (5000 cells/well at concentration) were plated in a 96-well microplate and processed as indicated. Next, the DCFH-DA (10 μmol/L)-loaded cells were placed for 30 min away from light (37 °C) and gently cleaned 3 times in PBS. The fluorescence microscope (Nikon, Tokyo, Japan) together with a microplate reader (Gemini XPS, Molecular Devices, Gothenburg, Sweden) were employed to detect total ROS for the fluorescence intensities.
2.10. IL-1β Determination
Enzyme linked immunosorbent assay (ELISA) was executed to measure the culture medium for IL-1β concentration as per the detailed procedures described in Porcine IL-1β ELISA Kit (JL21874, Jianglaibio, Shanghai, China). Afterward, all samples experienced duplicate measurement to obtain the 450 nm absorbance, and the values of negative controls (sample-free blanks) were subtracted. A total of 1 pg/mL IL-1β was set as the minimum detectable concentration.
2.11. Animal Grouping and Age Modeling
The processing of all experimental animals was accomplished in compliance with relevant regulations published by the China Council on Animal Care, with the procedures all accomplished by reference to the Guidelines of the Animal Ethics Committee of Beijing University of Agriculture [Permit No.: SYXK(JING)2021-0001].
A total of 40 ICR male mice aged 7–8 weeks old provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were grown in a controlled humid (40–70%) animal house with a 12 h dark/light cycle at (20–25 °C). The mice were subjected to 1-week acclimatization with free access to water in addition to food throughout the study, followed by allocation into four groups: D-gal group (n = 10), BMSC group (n = 10), control group (n = 10), and BMSC-Exos group (n = 10). D-gal (200 mg/kg/day) was subcutaneously injected into the mice from the D-gal group daily for 60 d, while saline was administered in an equal volume to the control group for 60 d. On days 30 and 45, the therapeutic groups were infused with 100 μg BMSC-Exos in addition to 1 × 106 BMSCs from the tail vein.
On the last day, with general anesthesia (induced by 150 mg/kg pentobarbital sodium injected intraperitoneally) achieved, all mice were killed. The left testis was preserved at −80 °C immediately following excision to receive biochemical analysis, whereas the right one was fixed in 4% paraformaldehyde or 2.5% glutaraldehyde for histological, immunohistochemical, and echocardiographic analyses.
2.12. Western Blotting Analysis
After collection and ice-cold PBS washing, the tissues and cells were treated with ice-cold PMSF (1 mM)-containing RIPA lysis buffer. Western blotting assay was conducted as previously described [
4]. The primary antibodies against p-AMPK (Ser485) (1:1000 dilution; #2537; Cell Signaling Technology, Danvers, MA, USA), AMPK (Ser485) (1:1000 dilution; #2532; Cell Signaling Technology), p-ERK1/2 (Thr202/Tyr204) (1:1000 dilution; #9101; Cell Signaling Technology), ERK1/2 (1:1000 dilution; #9102; Cell Signaling Technology), p-AKT (Ser473) (1:1000 dilution; #4060; Cell Signaling Technology), AKT (1:1000 dilution; #4691; Cell Signaling Technology), p-mTOR (Ser2448) (1:1000 dilution; #5536; Cell Signaling Technology), mTOR (1:1000 dilution; #2983; Cell Signaling Technology), ZO-1 (1:1000 dilution; bs-1329R; Bioss, Woburn, MA, USA), Occludin (1:1000 dilution; bs-10011R; Bioss), Claudin-11 (1:1000 dilution; bs-21509R; Bioss), Beclin-1 (1:1000 dilution; 11306-1-AP; Proteintech, Rosemont, IL, USA), LC3 (1:1000 dilution; 14600-1-AP; Proteintech), NLRP3 (1:1000 dilution; 19771-1-AP; Proteintech), ASC (1:1000 dilution; 10500-1-AP; Proteintech), Caspase-1 (1:1000 dilution; 22915-1-AP; Proteintech), IL-1
β (1:1000 dilution; #12703; Cell Signaling Technology), and
β-actin (1:3,000 dilution; bs-0061R; Bioss) were utilized. The HRP-conjugated goat anti-rabbit secondary antibody (diluted at 1:3000, bs-0295-HRP; Bioss) was utilized. ECL solution was used for band examination, and the ImageJ 1.44p was adopted for signal quantification.
2.13. Histological Analysis
The porcine and murine testicular tissues embedded in paraffin were sliced into 4 μm sections and subjected to hematoxylin-eosin staining. The optical microscope (Olympus-DP73, Tokyo, Japan) was utilized to evaluate the dynamic changes for histological analysis of the testes.
2.14. Immunohistochemical Staining
Immunohistochemical staining was conducted based on the procedures in our previous report [
4]. Specifically, the overnight incubation (4 °C) of testicular tissue sections was executed by virtue of the rabbit polyclonal antibody against ZO-1 (diluted at 1:100; bs-1329R; Bioss) or LC3 (1:100 dilution; 14600-1-AP, Proteintech) in combination with the mouse monoclonal SOX9 antibody (diluted at 1:100; ab76997; abcam, an SC-specific marker). Later, the tissue sections, washed 3 times in PBS, were cultured for 45 min using the FITC-coupled goat anti-rabbit IgG (H+L) antibody (1:200 dilution; HS111, TransGen, Beijing, China) and the PE-labeled goat anti-mouse IgG (H+L) antibody (1:200 dilution; HS221, TransGen, Beijing, China). Lastly, the cell nuclei received DAPI staining based on the previously described methods, followed by observation and photography of the sections under the Olympus-DP73 optical microscope (Tokyo, Japan).
2.15. Evaluation of Oxidative Stress in Testes
The level of lipid peroxidation marker, MDA, in addition to the activity of enzymatic antioxidants, SOD and CAT, were examined to appraise the oxidative stress in murine testes in accordance with the instructions of commercially available kits for MDA (A003-1), SOD (A001-1), and CAT (A007-1) (Jiancheng Bioengineering Institute, Nanjing, China).
2.16. Transmission Electron Microscopy (TEM)
The 2.5% glutaraldehyde-fixed testicular tissues received 1 h of a third fixation (4 °C) in osmium tetroxide (1%), prior to rehydration, embedding, slicing, and uranyl acetate + citrate double staining. Finally, the transmission electron microscope (Hitachi H-7500, Hitachi Ltd., Tokyo, Japan) was applied for the ultrastructure observation of the TJs formed by adjacent SCs plus autophagy monitoring in SCs.
2.17. Data Analysis
Data are shown as the mean ± SD, and results were analyzed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). Normal distribution data were analyzed by unpaired Student’s t-test for comparisons between two groups or by one-way ANOVA with a Student–Newman–Keuls test for pairwise comparisons between three or more groups. A non-parametric test was used when data were not normally distributed. Data were considered statistically significant at p value < 0.05.
4. Discussion
During spermatogenesis, the developing germ cell-enclosed SCs perform the function of modulating germ cell development and spermatogenesis based on the functions of providing nutrients while participating in BTB formation. The impaired or decayed testicular function and structure emerges along with increasing age, thus disrupting the BTB, aggravating seminiferous epithelium damage, and finally resulting in spermatogenesis dysfunction and male infertility [
3,
4]. It was discovered through the present study that BMSC-Exos might restore the TJ function of senescent porcine SCs and the testes of aging mouse model. The underlying mechanism is that BMSC-Exos exert their protective effect by enhancing autophagy to repress ROS production and subsequent NLRP3 inflammasome activation in SCs.
Under normal physiological and pathological circumstances, autophagy in SCs has an essential effect on their survival and function [
22]. It has been recognized that autophagy-related protein 5 (ATG5) and ATG7 are important players in autophagosome biogenesis. To be specific, ATG5 or ATG7 knockout in testicular SCs mitigates autophagy, thus affecting the fertility of male mice [
22]. Previous studies also argued that moderate autophagy reduces the apoptosis of aging cells and improves their survival, but senescent cell damage may be aggravated by excessive autophagy [
7]. As denoted by a number of other studies, the upregulated autophagy alleviates TJ dysfunction in aging-induced blood-brain barrier [
23]. According to previous studies, several hallmarks of aging, including reduced longevity, worsened oxidative stress, exacerbated mitochondrial dysfunction, and decreased completion of the autophagic flux, are observed from the premature aging characteristics under induction by chronic D-gal exposure, which are similar to those of natural aging [
19]. In this study, D-gal dose dependently decreased TJ function and autophagy in porcine SCs. More importantly, the autophagy inducer rapamycin blocked the D-gal-induced autophagy degradation and promoted TJ function, while the lysosomal inhibitor CQ aggravated the D-gal-induced autophagy and TJ dysfunction. These data supported the notion that insufficient autophagy exerts an upstream role in D-gal-induced TJ function in porcine SCs.
Numerous investigations have demonstrated the protective effects of MSCs on such reproductive organs as ovary and testis [
15,
16]. As for the testis, MSC transplant can delay testis aging and increase androgen secretion [
20]. The central mechanism of such a process involves paracrine instead of MSC propagation and differentiation [
17,
20]. Exos have become the most explored paracrine substance over the past years, which has extensive application as a treatment strategy concerning degenerative diseases like osteoarthritis (OA) [
24] and premature ovarian failure (POF) [
25]. Exos functioning as lipid nanovesicles can directly penetrate the blood-brain barrier and reach the lesion sites easily [
26]. It has been verified that in the case of diseased neurons undergoing toxic protein aggregate exposure, BMSCs facilitate autophagy, thereby improving neuronal survival [
27]. Moreover, a very recent study elucidated that MSC-Exos can reduce the death of ischemic cardiomyocytes, which has relation to the mitigation of ischemia-induced autophagy [
28]. The data from this study testified that Exos excreted by BMSCs were conducive to autophagy and TJ function in D-gal-induced senescent porcine SCs. In vivo experiment also demonstrated that the stimulatory effect of BMSC-Exos on autophagy and TJ function in the testes of aging mouse model is similar to that of BMSC transplants. These results forecast that BMSC-Exos serve as a positive regulator of TJ function and its mechanism is partly dependent on the stimulation of autophagy.
ROS, another type of pathology commonly detected in numerous infertile men, has been recognized as the key player in triggering NLRP3 inflammasome formation [
13]. The NLRP3 inflammasome-related signaling pathway stimulates IL-1
β and other pro-inflammatory mediators to start generation and subsequently accelerates the release of TNF-
α and other pro-inflammatory cytokines, thereby leading to systemic chronic inflammation due to aging [
29]. NLRP3 inflammasomes have been proven by a previous study to be activated by ROS generation, which cause D-gal-triggered learning and memory impairment in mice [
30]. Moreover, it has been elaborated that the ROS generation stimulates the NLRP3 inflammasome activation to facilitate cardiocyte aging [
31]. On the other hand, large quantities of investigations have confirmed the roles of MSCs or MSC-Exos in affecting ROS generation and NLRP3 inflammasome activation, including injury or inflammation [
32,
33]. The data of this study demonstrated that old porcine testes presented obvious increases in ROS level and NLRP3 inflammasome activation by comparison with the young ones, implying that ROS and inflammation have impacts on the old testes. Moreover, BMSC-Exos ameliorated ROS generation and NLRP3 inflammasome activation in D-gal-induced aging models were established both in vitro and
in vivo. NAC, MCC950 (NLRP3 inhibitor), and IL-1Ra were examined to validate whether the interference of BMSC-Exos in ROS generation and NLRP3 inflammasome activation has correlation with the reversal of D-gal-induced TJ dysfunction. According to the results of this study, NAC, MCC950, and IL-1Ra could all rescue the expressions of ZO-1, Occludin, and Claudin-11 in D-gal-induced aging SCs. The above findings explicitly corroborate that the suppressed ROS production and NLRP3 inflammasome activation are involved in the rescue of TJ in senescent SCs by BMSC-Exos.
Autophagy is a crucial player in clearing ROS, misfolded proteins, proinflammatory cytokines, and ATP that trigger the activation of NLRP3 inflammasomes [
34,
35]. In addition, it has been manifested that the packaging and degradation of NLRP3 inflammasome components ASC and NLRP3 by autophagy-related proteins have been testified through increasing evidence [
35]. It is crucial that autophagy directly eliminates mature IL-1
β as well [
36]. It was unveiled through this study that rapamycin alleviated ROS generation and NLRP3 inflammasome activation in D-gal-induced aging porcine SCs, while CQ alone aggravated and blocked the suppression of BMSC-Exos on ROS generation and NLRP3 inflammasome activation. Based on these data, BMSC-Exos have become a feasible strategy for curing D-gal-associated TJ dysfunction in SCs via autophagy promotion while repressing ROS production induced NLRP3 by inflammasome activation.
Furthermore, the AMPK/mTOR signaling pathway is known to be intimately related to the autophagy-modulated TJ function in intestinal epithelium barrier [
37] or blood-brain barrier injury [
38]. BMSC-Exos in this study were proven to increase the p-AMPK/AMPK ratio but decrease the p-mTOR/mTOR ratio in the in vivo and in vitro aging models. Moreover, the CC-induced AMPK inhibition exerts a reversing effect on the BMSC-Exos-mediated increase in autophagic flux and TJ function as well as the decrease in ROS production and NLRP3 inflammasome activation, suggesting that autophagy enhancement in D-gal-induced aging porcine SCs by BMSC-Exos treatment alleviates ROS production plus subsequent NLRP3 inflammasome activation to recover TJ function, which is minimally and partially realized by regulating the AMPK/mTOR signaling pathway.
There are several limitations of our current study. Firstly, it is possible that our in vivo experiments were not sufficient since we were not able to test the BMSC-Exos function in aging porcine models. A future study will examine the aging porcine model in depth, in order to reveal the administration and dosage of Exos. In addition, numerous studies have found that miRNAs or proteins in the Exos play a major role in many disease models [
39,
40]. Hence, the effects of individual RNA, miRNA, and related proteins in Exos need to be determined in future work, so as to validate their respective roles in the abovementioned effects.