Adult ICR male mice, purchased from SPF (Beijing) Biotechnology CO., Ltd, were maintained with The Animal Research Committee of the Institute of Zoology, Beijing, China. Mice were housed in 12 h alternating light/dark cycles, with free access to water and food. All experiments were conducted with the approval of the Animal Research Committee of the Institute of Zoology, Chinese Academy of Sciences. Mice were killed under standard protocols, and all efforts were made to minimize suffering.

Mice were given a single round, intraperitoneal injection of either busulfan (35 mg/kg, Sigma, St. Louis, MO, United States) or DMSO (Sigma), according to previously described methods (Bucci and Meistrich, 1987; Qu et al., 2020). After 4 weeks, the testis weight, epididymal sperm count, and testis histology were evaluated and the phenotypes were checked. The remaining mice were then separated into three groups. The first group was given daily oral administration of 0.5 g/kg Oyster peptides, the second group was given daily oral administration of 0.5 g/kg Perilla purple peptides, and the control group was given daily oral administration of equal amounts of water. Opp and Ppp were provided by the Beijing Engineering Research Center of Protein and Functional Peptides. After 6 weeks, the testis weights, epididymal sperm count, and testis histology were evaluated.

After 10 weeks of busulfan treatment, the mice were sacrificed and epididymides were sampled for sperm analysis as previously described (Wang et al., 2014). In brief, the caudal epididymis was dissected from each group of mice. Spermatozoa were squeezed out from the caudal epididymis and incubated for 30 min at 37°C under 5% CO₂. The incubated sperm medium was then diluted 1:500 and transferred to a hemocytometer for counting using a Primo Star microscope (Zeiss). The sperm motility assays were performed as previously described (Jimenez et al., 2010). Briefly, 10 μL aliquots of the sperm sample were taken before dilution and placed into 70 μm-deep glass cell chambers (Leja Products BV, NieuwVennep, Netherlands). The chambers were imaged using an Olympus BX51 microscope (Olympus, Tokyo, Japan) through a 20× phase objective and maintained at 37°C on a water bath. The viewing areas of each chamber were captured using a CCD camera. The samples were analyzed via computer-assisted semen analysis using the Mini-tube Sperm Vision Digital Semen Evaluation system. Different sperm motility parameters were analyzed.

Non-fixed sperm were spread on precoated slides with 10 μL for morphological observation and immunostaining. After air drying at room temperature, PBS (pH 7.4) was used to wash the slides three times, with 5 min of rest time between washes. The spermatozoa were then fixed in 4% (w/v) paraformaldehyde (PFA; P1110, Solarbio, Beijing, China) at room temperature for 10 min, and the slides were again washed with PBS three times, with 5 min of rest time between washes. After this, the slides were treated with 0.1% Triton X-100 for 10 min, rinsed with PBS three times, and blocked using 5% bovine serum albumin (BSA, AP0027, Amresco, Framingham, United States) in PBS for 30 min. The primary antibody was added to the slides and incubated at 4°C overnight. This was followed by incubation with FITC- or TRITC-conjugated secondary antibody at 1:200 for 1 h at 37°C. Next, the slides were washed in PBS, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; D3571, Life Technologies). Images were taken immediately using a LSM 780 microscope (Zeiss, Germany), LSM 880 Fast Ariyscan, or SP8 microscope (Leica, Germany). The antibodies used in this study are summarized in Table 1.

Testes harvested from Opp, Ppp, and control mice were homogenized in cold RIPA buffer (Solarbio, R0010) and supplemented with 1 mM phenylmethylsulphonyl fluoride (PMSF, Amresco, 0754) and complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland, 04693116001). The homogenates were centrifuged at 12,000 rpm for 15 min. Protein lysates (25 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane by electrolysis. Proteins obtained from lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). The membranes were then incubated in TBS-T (10 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1% Tween-20) containing 5% non-fat milk at room temperature for 1 hour and stained with the appropriate primary and secondary antibodies, which are summarized in Table 1. After the final washes with TBS-T, the membranes were developed using the ODYSSEY Sa Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Testes from each group were dissected immediately after euthanasia, fixed in 4% paraformaldehyde (PFA; Solarbio, Beijing, China, P1110) for at least 24 h, and stored in 70% ethanol. They were then embedded in paraffin, cut into 5 μm sections, and mounted on glass slides. After deparaffinization, the slides were stained with hematoxylin and either eosin or periodic acid-Schiff (PAS) for histological analysis. The slides were scanned by Leica Aperio VESA8. The stages of seminiferous the epithelium cycle and spermatid development were detected as previously described (Hess and Renato de Franca, 2008).

All data are reported as the mean ± SD. GraphPadPrism9 software (GraphPad Software, La Jolla, CA, United States) was used for the graphing and statistical analysis of data to determine whether the data conform to the normal distribution. The statistical significance of the differences between the various groups was measured by Student’s t-test with an unpaired, two-tailed distribution. The statistical significances were defined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

To explore the effects of food-derived high arginine peptides on the spermatogenesis process, an NOA mouse model was developed with treatment by the genotoxic agent, busulfan (Bucci and Meistrich, 1987). Briefly, the mouse model was established by single intraperitoneal administration of busulfan in wild-type 8-week-old male mice, or DMSO as control group. After 4 weeks of administration, the success rate of modeling was then detected. Subsequently, busulfan-induced NOA mouse were treated with high arginine peptides for six consecutive weeks, and the testis histology was then detected (Figure 1A).

After 4 weeks of busulfan treatment, the body weights of the busulfan-treated mice were considerably lower than that of the control group (Figure 1C). To learn more about the phenotypes of busulfan-treated mice, we then examined the testis structure from both macroscopic and histological standpoints. Here, we found significant differences in the testis size, weight, and testis/body weight ratio between the control and busulfan-treated mice (Figures 1B,D,F). Hematoxylin and eosin (H&E) staining revealed that the seminiferous tubules of the busulfan-treated mice were almost empty. The spermatozoa in the cauda epididymis were analyzed, and we found that busulfan-treated mice had no or few spermatozoa in the epididymal lumen (Figure 1E). We also examined the spermatozoa released from the cauda epididymis of the busulfan-treated mice and found that the sperm count and motility were much lower than that of the control mice (Figures 1G,H). Thus, we successfully established a busulfan-induced azoospermic mice model.

To characterize the potential functions of Opp and Ppp on spermatogenesis, the busulfan-treated mice were further treated with Opp and Ppp for another 6 weeks, while the control group was only administrated with water (vehicle of peptide administration) (Figure 1A). We found that the testes size (Figure 2A) and weight (Figure 2B) of Opp- and Ppp-treated mice were significantly increased compared with the control mice. Interestingly, the body weight of both the Opp and Ppp groups were also significantly increased (Figure 2C). Thus, there were no significant differences in the testis/body weight ratio between the control and the Ppp-treated groups (Figure 2D).

The H&E histological analysis further revealed that thinner seminiferous epithelia were found in the testes of control mice while most of the tubules in Opp- and Ppp-treated testes contained all stages of spermatid development in the seminiferous epithelium (Figure 2E). The results of the histological analysis showed that the diameters of round seminiferous tubules in the Opp- and Ppp-treated groups were observably thickened compared to the control group (Figure 2F). In the control group, 80% of the atrophic seminiferous tubules remained, while the number of atrophic seminiferous tubules decreased by less than 20% in the Opp-treated groups and 40% in the Ppp-treated groups (Figure 2G). Further staining with periodic acid-schiff (PAS)-hematoxylin demonstrated that all stages of germinal epithelium from spermatogonia to spermatozoa were recovered in Opp- and Ppp-administered groups compared to the almost empty control groups (Figure 3A). Close examination by peanut agglutinin (PNA) staining (to show the development of acrosome) found that acrosomes were well-developed in the Opp- and Ppp-treated groups, but not in the control mice (Figure 3B). Consistent with the PNA staining results, the expression of SYCP3, a meiotic cell-specific protein, was also dramatically increased in the seminiferous tubules of the Opp- and Ppp-treated groups compared to those of the control group (Figures 3C,D). These results were further supported by western-blot detection of DDX4 (Figures 3C,E). Taken together, these results demonstrate that both Opp and Ppp can promote the recovery of impaired meiosis and spermatogenesis after busulfan treatment.

It has previously been reported that busulfan treatment leads to abnormalities in mice sperm heads (Bucci and Meistrich, 1987). To determine the effect of the Opp and Ppp treatment on these abnormalities, we used H&E staining to examine transverse sections of the cauda epididymis. Here, we discovered that the Opp- and Ppp-treated animals had more epididymal sperm than the control mice, which had only a few aberrant spermatozoa and numerous detached premature germ cells in the epididymal lumen (Figure 4A). We further examined the spermatozoa released from the caudal epididymis and found the sperm count in Opp- and Ppp-treated mice was dramatically increased compared with that of the control mice (Figure 4B). In addition, sperm motility was measured by computer-assisted semen analysis (CASA). We found that the Opp-treatment could promote sperm motility significantly when compared with that of the control mice (Figure 4C). Single-sperm immunofluorescence was utilized to examine the morphological properties of the spermatozoa using the acrosome-specific marker, Sp56 (Zp3r). Morphological evaluation revealed that many spermatozoa from control mice had more irregularly-shaped sperm heads and tails compared to the Opp-treatment and Ppp-treatment groups (Figures 4D–F). These results suggest that the administration of both Opp and Ppp could at least partially restore epididymal sperm morphology, spermatozoa count, and motility.

Spermatogenesis starts from spermatogonia, which are the only stem cells in the germline that undergo either self-renewal or differentiation (Kanatsu-Shinohara and Shinohara, 2013). It has been reported that treatment with busulfan kills most of the germ cells, but not spermatogonia (Bucci and Meistrich, 1987). Thus, we further investigated whether the therapeutic effects of Opp and Ppp were related to spermatogenesis from the endogenous population of spermatogonia cells. Immunofluorescence staining was performed to examine the expression of the undifferentiated spermatogonia marker, promyelocytic leukaemia zinc finger (PLZF), and proliferating cell nuclear antigen (Ki67), a marker of cell proliferation (Wang et al., 2019). We found that the number of PLZF⁺Ki67⁺ spermatogonia was dramatically increased in Opp and Ppp-treated seminiferous tubules (Figures 5A–D), while almost no PLZF⁺Ki67⁺ spermatogonia were detected in the control mice (Figures 5A–D).

Furthermore, we examined differentiating spermatogonia markers, such as c-KIT, in every mouse group (Figure 5E). In each testicular cross-section of the Opp- and Ppp-treated groups, the number of c-KIT positive cells was higher than that of the control mice (Figures 5F,G). To further confirm the effects of Opp and Ppp on germ cell proliferation, we examined another general cell proliferation marker, proliferating cell nuclear antigen (PCNA). Here, we found that PCNA positive cells were rarely detected in the control seminiferous tubules, while the PCNA positive cells were increased in Opp- or Ppp-treated tubules (Figures 5H–J). Thus, all these results suggest that Opp and Ppp might promote spermatogonia proliferation, and restore spermatogenesis.

It has been reported that busulfan treatment might also affect the function of Sertoli cells (Cai et al., 2016; Panahi et al., 2020). Therefore, we then examined the impact of Opp and Ppp on Sertoli cells by detecting WT1, a Sertoli cell-specific marker (Rotgers et al., 2018). After busulfan treatment, DDX4, a germ cells marker, was barely detected in the seminiferous tubules, while only WT1 positive cells were found at the basement layer of the tubules in the control group (Figure 6A). In contrast, after treatment with either Opp or Ppp, a vast number of DDX4 positive cells were found, suggesting the germ cells have been at least partially restored (Figure 6A). The essential function of Sertoli cells is to form the BTB, which not only separates seminiferous tubules into the basal and luminal compartments, but also provides a specialized microenvironment for spermatogenesis (Cheng and Mruk, 2012).

To explore the effects of Opp and Ppp on the BTB after busulfan treatment, the expression and localization of some BTB component proteins such as occludin, basal ectoplasmic specialization β-catenin, connexin 43 (Cx43) and junction proteins Zona occludin 1 (ZO-1) were examined by immunofluorescent analysis. In the control group testes, occludin, β-catenin, Cx43 and ZO-1 were all still diffusively-distributed (Figures 6B–E), while in both Opp- and Ppp-treated testes, ZO-1, β-catenin, occludin, and Cx43 were all localized tightly to the BTB sites, which were adjacent to the base of the membrane (Figures 6B–E). Together, these results suggest that Opp and Ppp might either directly or indirectly promote BTB recovery after busulfan treatment.