*2.5. Effects of OPs on TP-Induced Reproductive Hormone Level*

Male reproductive hormones regulate the process of spermatogenesis. As shown in Figure 3, the concentration of serum testosterone and estradiol was significantly increased in the TP group, and the levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were significantly decreased compared with the control group (*p* < 0.01). Obviously, it was shown that TP disturbed the serum hormone level of mice compared with the controls. As compared with the TP group, the treatment of OPs restored the serum testosterone and estradiol level of mice, which were close to the control group. Additionally, a significant increase in FSH and LH levels was seen in the TP+OPs-H group compared with the TP group (*p* < 0.01). VE as a positive control significantly restored the TP-induced serum reproductive hormone (testosterone, estradiol, and FSH) disorder in mice.

**Figure 3.** Effects of OPs on serum reproductive hormone level of ICR mice induced by TP. (**A**) Serum testosterone level, (**B**) serum estradiol (E2) level, (**C**) serum follicle stimulating hormone (FSH) level, (**D**) serum luteinizing hormone (LH) level. The data were expressed as mean ± SEM, n = 10. Compared with the control group, # *p* < 0.05 and ## *p* < 0.01; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

#### *2.6. OPs Increased Testicular Marker Enzyme Activity and Reduced Oxidative Stress Induced by TP*

Oxidative stress is a cause of testis injury which was also found in the TP-induced mice model (Figure 4A–C). TP raised the lipid peroxidation product malondialdehyde (MDA) and disrupted the antioxidative system of the testis, including the enzyme superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). OPs treatment reduced the level of MDA and increased the activity of SOD and GSH-Px, and especially the middle and high two-dose groups of OPs showed extremely significant changes (*p* < 0.001). Meanwhile, VE treatment as a positive control also significantly reduced oxidative stress in testicular tissue induced by TP.

**Figure 4.** Effects of OPs on testicular marker enzymes and bio markers of oxidative stress in testes tissues of ICR mice induced by TP. (**A**) superoxide dismutase (SOD) level, (**B**) glutathione peroxidase (GSH-Px) level, (**C**) malondialdehyde (MDA) level, (**D**) lactate dehydrogenase (LDH) level, (**E**) alkaline phosphatase (ALP) level, (**F**) acid phosphatase (ACP) level. The data were expressed as mean ± SEM, n = 10. Compared with the control group, ## *p* < 0.01 and ### *p* < 0.001; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

Activities of testicular marker enzymes such as lactate dehydrogenase (LDH), acid phosphatase (ACP) level, and alkaline phosphatase (ALP) are considered as functional indicators of spermatogenesis and testicular development. The alterations of testicular marker enzyme activity could affect the energy metabolism pathway, thus interfering with the energy supply of spermatogenesis. As were shown in Figure 4D–F, TP significantly suppressed the level of testicular marker enzymes in testis tissue, including LDH, ALP, and ACP (*p* < 0.01). As compared with the TP group, middle and high doses of OPs significantly increased the activity of LDH (*p* < 0.001), and different dose of OPs ameliorated the activity of ALP and ACP. Meanwhile, VE treatment significantly increased the activity of testicular marker enzymes (LDH and ALP).

#### *2.7. Effects of OPs on the Testicular Apoptotic Induced by TP*

The results of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining of cell apoptosis were shown in Figure 5. There were few apoptotic cells in the testicular tissue of the control group, which is common during spermatogenesis. However, in the TP group, the proportion of the apoptotic cells was significantly increased despite the loss of total cells in seminiferous tubules (Figure 5B,C). VE and OPs treatment significantly ameliorated the decrease in the total number of cells in seminiferous tubules induced by TP (Figure 5B). Meanwhile, VE and high dose of OPs treatment reduced the number and proportion of the apoptotic cells (Figure 5C).

To further elucidate the testicular apoptotic events, the protein expression of Bcl2, Bax, caspase-3, and PARP reliable apoptotic markers were analyzed. As shown in Figure 6, TP stimulated the upregulation of Bax, caspase-3, cleaved caspase-3, and cleaved PARP, and inhibited the expression of Bcl-2. The upregulated Bax, caspase-3, cleaved caspase-3, and cleaved PARP were significantly inhibited by treatment of OPs at three doses. VE treatment downregulated the activation of cleaved caspase-3. The decrease in Bcl-2 expression was significantly upregulated by VE and high dose of OPs treatment. These findings suggested that OPs ameliorated TP-induced apoptosis in testicular tissue.

**Figure 5.** Effects of OPs on apoptotic index in testes tissues of ICR mice induced by TP. (**A**) Apoptotic cells in testicular tissue sections were detected by TUNEL assay (apoptotic cells: green fluorescence, cell nucleus: blue fluorescence). (**B**) The total number of cells in each group compared by Control group were shown. (**C**) The ratios of apoptotic cell were shown. The data were expressed as mean ± SEM, n = 3. Compared with the control group, ### *p* < 0.001; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

**Figure 6.** Effects of OPs on protein expression levels of markers of apoptosis in TP-induced testis tissues of male ICR mice. (**A**) Electrophoresis images of Bcl-2, Bax, Caspase-3, Cleaved Caspase-3, PARP, and GAPDH protein; (**B**) the quantitative densitometric analysis of Bcl-2, Bax, Caspase-3, Cleaved Caspase-3, and PARP proteins. The data were expressed as mean ± SEM, n = 3. Compared with the control group, ## *p* < 0.01 and ### *p* < 0.001; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

### *2.8. Effects of OPs on Related Proteins Expression in the Nrf2 and JNK Pathways*

Detection of the levels of related protein factors in the testicular tissue via Western blot was illustrated in Figures 7 and 8. As displayed in Figure 7, administration of TP led to the downregulation of Nrf2, Keap1, HO-1, and NQO1 expression in mice testis tissues, concomitant with upregulated expressions of p-JNK and p-JNK/JNK, and total JNK protein expression was not affected by TP treatment, as shown in Figure 8. ImageJ software was used to obtain optical density values of the protein bands. The decrease in Nrf2, Keap1, HO1, and NQO1 protein expression were upregulated by treatment of OPs at middle and high doses. VE as a positive control upregulated the Nrf2, HO1, and NQO1 protein expression. The treatment with OPs downregulated the activation of p-JNK to a nearly normal level.

**Figure 7.** Effects of OPs on the Nrf2/Keap1 signaling pathway in TP-induced testis tissues of male ICR mice. (**A**) Electrophoresis images of Nrf2, Keap1, HO-1, NQO1, and GAPDH protein; (**B**) The quantitative densitometric analysis of Nrf2, Keap1, HO-1, and NQO1 proteins. The data were expressed as mean ± SEM, n = 3. Compared with the control group, ## *p* < 0.01 and ### *p* < 0.001; compared with the TP group, \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

**Figure 8.** Effects of OPs on the JNK signaling pathway in TP-induced testis tissues of male ICR mice. (**A**) Electrophoresis images of JNK, p-JNK and GAPDH protein; (**B**) the quantitative densitometric analysis of p-JNK/JNK bands. The data were expressed as mean ± SEM, n = 3. Compared with the control group, ### *p* < 0.001; compared with the TP group, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

#### **3. Discussion**

In this study, male infertility disease model mice were successfully established by TP induction. In the model, the deteriorated sperm quality, altered testicle histomorphology, disordered hormone levels, the decreased activity of testicular marker enzyme, and the triggered testis oxidative stress and germ cells apoptosis were observed. The observed altered sperm quality and testicle histomorphology in this study is consistent with the previous reports [8,13,30]. Gastric administration of appropriate dose of OPs could reverse these abnormalities. The results showed that OPs treatment in TP-induced mice has beneficial effects on the testis index, sperm parameters, histological structure of testis, hormone level, testicular marker enzyme activity, and oxidative stress, as well as testicular

apoptosis. The mechanism of the protective effects of OPs may be through inhibiting the oxidative stress by Nrf2 and JNK pathways, increasing the expression of Bcl2, and reducing the level of cell apoptosis by suppressing the expression of the apoptotic markers Bax, caspase-3, and PARP.

Oxidative stress due to toxic substances is considered to be closely related to male infertility [31]. Production of reactive oxygen species (ROS) and consequent oxidative damage have been established as mechanisms for TP toxicity [13]. It is well known that oxidative stress leads to sperm dysfunction by inducing peroxidation damage to the plasma membrane. In this study, TP-induced increased MDA levels and decreased GSH-Px and SOD levels in mice, suggesting oxidative stress, which was consistent with the reports of some researchers [8,13,32]. As spermatozoa are high in polyunsaturated fatty acids (PUFAs), it is more sensitive to oxidative damage than other cells [33]. Furthermore, the generation of ROS decreased the number of spermatogonia cells in the testis, and it is thought to be detrimental for spermatogenesis [34]. In this study, the sperm count and motility of mice were significantly decreased, and the sperm deformity rate was increased, induced by TP. The significant reduction in spermatogonia was also observed on histopathological assay. The severe impairment of sperm characteristics induced by TP is closely related to the obvious oxidant/antioxidant imbalance in testis. The treatment of OPs effectively reduced the deterioration of sperm quality, which may be related to the amelioration of oxidative stress in testicular microenvironment as manifested by decreasing in the level of MDA and increasing in the activity of SOD and GSH-Px. It suggested that OPs exert significant antioxidant effects in testicular tissue, including the increase in activities of antioxidant enzymes and decrease in lipid peroxidation. It has been reported that peptides (2-20 amino acids) can completely cross the intestinal barrier and perform biological functions in tissues, while peptides the size of 5–16 amino acids show potent antioxidant activity [28,35], which is manifested in its ability to carry out DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging [16,20]. Combined with the results of animal experiments, the main peptides of OPs with 6–14 amino acid residues in length may also show antioxidant activity, suggesting that its alleviation of ROS-induced testicular injury may be related to its antioxidant function. In addition, compared with other hydrophilic amino acids, hydrophobic amino acids have higher antioxidant activity in peptides, and its high amounts in OPs may play an important role in reducing the oxidative damage of the testis caused by ROS [36].

Decreased activity of antioxidant enzymes could induce the accumulation of ROS and lead to oxidative stress. The expression of antioxidant enzymes including HO-1, NQO1, and SOD were mainly mediated by the Nrf2 pathway. Under normal conditions, Nrf2 is mainly regulated by the repressor protein Keap1 in the cell cytoplasm. The disturbance of interaction between Nrf2 and Keap1 or degradation of Keap1 affect the further activation of downstream antioxidant enzyme expression by Nrf2 [37]. Recent studies have shown that the regulation of endogenous antioxidant system through the Nrf2 pathway significantly reduces oxidative stress-induced apoptosis of Sertoli cells and testicular injury [32]. In this study, TP-induced decrease the expression of NQO1 and HO-1 protein by decreasing Nrf2 and Keap1 expression was consistent with the results of the previous study [8]. Pretreatment with OPs may inhibit the decrease in antioxidant enzyme expression by activating Nrf2 expression. This finding suggests that the protective effect of OPs on testicular injury might be related to the upregulation of Nrf2 expression.

Previous studies showed that TP could promote the production of ROS in Sertoli cells, thus further activating the JNK pathway, which triggered the mitochondrial-mediated apoptosis pathway [13]. In vivo results showed that TP reduced testicular weight, destroyed the microstructure of the testis, disrupted the enzyme activity, increased MDA levels, activated JNK phosphorylation, and promoted testicular tissue apoptosis. In this study, treatment of OPs significantly reduced the activation of JNK phosphorylation, which indicated that OPs may protect testis against oxidative stress by mediating the JNK pathway.

Mitochondria are responsible for cells energy metabolism and are the main targets of ROS production and regulation of apoptosis. In our study, it was found that OPs may reduce the excessive apoptosis of testicular cells mediated by the mitochondrial apoptosis pathway, as shown in the results of TUNEL assay and the Western blotting of apoptosisrelated proteins (Bax, Bcl-2, caspase-3, and PARP). Both the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax regulate apoptosis by controlling the permeability of the mitochondrial membrane. OPs treatment significantly upregulated the expression of Bcl-2, downregulated the expression of Bax, thereby regulating cell permeability, further inhibiting the activation of Caspase-3 and reducing the Caspase cascade reaction. Several studies have shown that increased ROS accumulation may lead to increased permeability of the mitochondrial outer membrane and then activate mitochondria-dependent apoptosis signaling pathway to induce cell apoptosis. At present, few studies have shown that OPs play a direct role in the inhibition of cell apoptosis, while in the activation of the apoptosis process, it is believed that oxidative stress-induced damage can trigger apoptotic signaling procedures, leading to cell death. Therefore, it indicated that OPs may mediate mitochondria-dependent apoptosis signaling pathways to reduce cell apoptosis and tissue damage by reducing oxidative damage of mitochondrial lipids caused by ROS. In our study, it was also showed that OPs reversed severe testicular tissue damage by ameliorating testicular apoptosis by assessing the testis histopathology.

Sertoli cells, as nursing cells, regulate the processes of spermatogenesis by providing nutritional support and a suitable situation for the survival and development of germ cells [38]. The altered testicular enzyme activities induced by TP may have resulted from the impairment of Sertoli cell function and disrupted metabolism in mice [39]. LDH enzyme is involved in the process of glycolysis and gluconeogenesis and controls the synthesis of the main energy source of germ cells. The ACP enzyme is mainly distributed in the cytoplasm of Sertoli cells, which is involved in protein synthesis and related to the phagocytosis of Sertoli cells. ALP is involved in the synthesis of nucleic acids, nucleoproteins, and phospholipids, such as in the cleavage of phosphate esters and in mobilizing carbohydrates and lipid metabolites to be used by spermatozoa [40]. In this study, TP inhibited the activities of LDH, ACP, and ALP, which not only interfered with the energy supply process of aerobic and anaerobic glycolysis but also disturbed the energy utilization of testis. OPs treatment improved the energy metabolism of testicular tissue and the energy supply of spermatogenesis by increasing the enzyme activity of LDH, ACP, and ALP. In addition, metabolic disorders may be due to the disintegration of mitochondrial membrane ultrastructure caused by mitochondrial lipid peroxidation. It has been found that lipid peroxidation induced by oxidative stress produces a strong cytotoxic effect in the testis, leading to the damage of nucleic acid, protein, carbohydrates, and lipids in cells. Spermatogenesis is reduced due to the resulting disturbances in energy metabolism, oxidative phosphorylation, tricarboxylic acid cycle, and glycolysis. Previous studies have shown that OPs could protect TM4 Sertoli cells from toxic damage induced by TP, including improving the cell viability of TM4 cells, reducing the production of intracellular ROS and lipid peroxidation, and enhancing the antioxidant activity of TM4 cells [41]. Combined with the results of this study, it was found that OPs ameliorated TP-induced metabolic disorders by inhibiting mitochondrial lipid peroxidation.

Spermatogenesis is a multistep process that could be disturbed by multifaceted factors. Expect for oxidative stress, hormone imbalance can affect the process of spermatogenesis [42]. Destructive endogenous hormone signaling might mediate the process of spermatogenesis and lead to low sperm count [43]. LH and FSH are considered to be key factors in the regulation of testis function. LH is related to produce testosterone by stimulating the Leydig cells and both FSH and testosterone in turn regulate Leydig cells activity and stimulate germ cell proliferation and differentiation by stimulating the Sertoli cells. Estradiol is converted from circulating testosterone by enzyme aromatase; therefore, its concentration is affected by the level of testosterone. However, dysregulation of circulating estradiol may lead to inhibition of LH production not beneficial for spermatogenesis [44]. In this study, TP increased the levels of serum testosterone and estradiol, and decreased serum LH and FSH levels in mice. The treatment of OPs restored the serum hormone level of mice to the normal, and reduced the disturbance of hormones on spermatogenesis in mice. Additionally, previous studies had found that oyster peptides improved the level of serum androgen induced by cyclophosphamide [45].

The identification of the active components of oyster peptides is of great interest in exploring new strategies for the treatment or prevention of testicular and sperm damage. Previous studies showed that some antioxidant peptides were identified from oysters, such as Pro-Val-Met-Gly-Asp, Glu-His-Gly-Val, and Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu [16,46]. These antioxidant peptides may play an important role in preventing oxidative damage in the testis, resulting in reduced sperm quality. However, other active ingredients still need to be identified by Sephadex gel chromatography, HPLC, mass spectrometry, and other purification methods.

In conclusion, the underlying mechanism of OPs in the potential protective effect on testis and sperm is attributed to the synergistic modulations, and these results deepen the understanding of the potential improvement of male reproductive function by OPs, thus demonstrating the potential application of OPs in functional foods (Figure 9).

**Figure 9.** Possible mechanism underlying the protective effects of OPs intervention on TP-induced testicular damage in mice. By detecting MDA, antioxidant enzymes and related proteins in the testis, as well as sexual hormone levels in serum, the anti-effect of OPs was shown to be related to Nrf2/Keap1, JNK, and Bcl-2/Bax pathways.
