Lycium barbarum Polysaccharide Ameliorates Heat-Stress-Induced Impairment of Primary Sertoli Cells and the Blood-Testis Barrier in Rat via Androgen Receptor and Akt Phosphorylation

Male infertility induced by heat stress has been attracting more and more attention. Heat stress not only causes apoptosis of spermatocytes but also has adverse effects on Sertoli cells, further damaging spermatogenesis. Lycium barbarum polysaccharide (LBP) is the main bioactive component of Lycium barbarum, which has a protective effect on male reproduction, but its mechanism is still unclear. In this study, our results proved that LBP blocked the inhibitory effect on the proliferation activity of Sertoli cells after heat stress, reversed the dedifferentiation of Sertoli cells induced by heat stress, and ameliorated the structural integrity of the blood-testis barrier. In addition, it increased the expression of the androgen receptor and activated Akt signaling pathway to resist heat-stress-induced injury of Sertoli cells.


Introduction
More than half of the childbearing couples could not have children due to male infertility [1,2], which is induced by varieties of causes including the spermatogenic quantitative or qualitative defect, catheter obstruction or dysfunction, and hypothalamic-pituitary axis disorders [3,4]. Among these situations, abnormal spermatogenesis is the primary culprit of impaired male fertility. Spermatogenesis is a temperature-dependent process [5,6]. For most mammals, normal spermatogenesis entails the temperature in the scrotum to be lower than body temperature. After heat treatment to local testis, male animals display testicular damages, including local testis tissue hypoxia [7,8], germ cells apoptosis [9], blood-testis barrier (BTB) dysfunction, and reduced sperm count and quality [10][11][12]. us, scrotal temperature increases and spermatogenesis is impaired, leading to male infertility.
Spermatogenesis depends on mature Sertoli cells (SCs). In the seminiferous tubule, SCs provide structural support and supply nutrients, functional proteins, and cytokines for spermatogenesis [13,14]. e number of SCs is proportional to the number of germ cells [15]. Occludin and zonula occludens-1 exist between adjacent SCs [16] and compose BTB which provides a suitable microenvironment for spermatogenesis [17,18]. e decrease of occludin and (or) zonula occludens-1 causes the damage of BTB integrity, negatively effecting spermatogenesis [15,19].
Androgen receptor (AR) is a type I steroid receptor. Androgens have to bind to AR before they can regulate the development of germ cells and SCs. Expressed in SCs, an androgen receptor (AR) plays a crucial role in spermatogenesis [20,21]. Studies demonstrated that the loss of AR directly influences the maturation and the final quantity of SCs [22,23], as well as spermatogenesis [24]. Besides, the absence of AR causes increased permeability of BTB in vitro or in vivo [25,26].

Primary SC Isolation, Culture, and Heat Treatment.
Sertoli cell isolation was according to the previous method [33]. Briefly, the testicular tissue was obtained from the testis and washed twice in PBS precooled at 4°C. After fully centrifuged at 1000 rmp about 5 min, the sediment was treated with collagenase IV (0.5 mg/mL) for 5 min. After washing twice and certification for 5 min, the sedimentation was secondly digested with trypsin (0.05%) for 5 min. FBS in the same velum was added to stop digestion. en, the sedimentation was filtrated through a 100-mesh filter and centrifugated at 1000 rmp for 5 min. e cells were washed twice with DMEM/F12, collected in the culture medium (DMEM plus F12 with 10% FBS and 1% penicillin-streptomycin), and cultured at 35°C in a CO 2 incubator (5% CO 2 / 95% air). After 40 h culturing, the medium was replaced to remove unattached germ cells, and 12-24 h later, when cells were confluent, they were ready for the following experiments. LBP was dissolved and diluted with PBS into different concentrations and then was added in SCs at the dose of 25 mg/L, 50 mg/L, and 100 mg/L. Cells in the control group and heat-stress group were added with the same volume of PBS. After 24 h, the control group was cultured at 35°C for another 20 minutes, and the other groups were treated in a 43°C water bath for 20 minutes.

Evaluation of Cell
Viability. MTT assay has been widely used for measuring cell viability. e obtained absorbance value (OD value) under the specific wavelength is directly proportional to the number of living cells. SCs were seeded at 5 × 104 per well in 96-well plates in DMEM/F12 supplemented with 10% FBS. Cells were treated with LBP in different concentrations or PBS for 24 h. After removing some supernatant, an MTT regent (5 mg/ml) was added to each well for four hours. en SCs were treated with DMSO by shaking for 15 min at room temperature, and the OD value was measured at 570 nm by using a microplate reader FLUO Star Omega (BMG Labtech, Offenburg, Germany). e blank group had no cells in wells and was used as the zero point of absorbance. Also, the absorbance value of each group divided by the control group was cell viability (%).

Immunohistochemistry.
e immunohistochemistry protocol in our experiment followed that described previously [34]. SCs were first fixed in a 4% polyoxymethylene solution and then treated with 0.5% triton X-100 and 0.3% hydrogen peroxide, respectively. After three washes in PBS, cells were blocked with 10% goat serum to suppress the nonspecific antigen and then incubated in the primary antibody of Ki67 (1 : 200) or AR (1 : 200) overnight at 4°C. e next day, after three washes in PBS, the biotinylated secondary antibodies were added for 15 min at 37°C. After three washes in PBS, the horseradish enzyme labeling streptavidin working solution was added for 15 min at 37°C. Immunostaining was developed with the diaminobenzidine kit and counterstained with hematoxylin. Six nonoverlapping fields were selected for each group to take pictures. Image-Pro Plus (Version 6.0, Media Cybernetics, Bethesda, MD, USA) software was used to process the images and count the positive cells for statistical analysis.

Statistical Analysis.
Each experiment was repeated at least three times. Statistical analysis was performed with the software SPSS version 20.0 (IBM, Albuquerque, NY, USA). Data that conformed to normal distribution or approximate normal distribution are expressed as means ± standard error of the mean (SEM). One-way analysis of variance was used for analyzing the data in different groups, and the pairwise comparisons were tested by the Tukey multiple comparison test. P < 0.05 was considered as significant, and P < 0.01 was considered as highly significant.

e Changes in Cell Viability of SCs after Different Concentrations of LBP Treatment.
To observe the effect of LPB on cell viability and select appropriate drug concentrations, we detected the OD value by MTT. As shown in Figure 2, compared with the control group, the cell viability of SCs at 25 mg/L, 50 mg/L, and 100 mg/L LBP treatment groups significantly increased (P < 0.05 or P < 0.01), while the differences in other LBP groups were not statistically significant (P > 0.05). erefore, these three concentrations were selected as the concentration of LBP drug groups.  Evidence-Based Complementary and Alternative Medicine 3 LBP group (P < 0.05 or P < 0.01), and the 50 mg/L LBP and 100 mg/L LBP group have no significant change. Compared with the heat-stress group, the positive signal of Ki67 in LBP treatment groups showed a noticeable increase (P < 0.01). ese data indicated that LBP treatment could resist the reduction of Ki67 expression induced by heat stress; however, the 25 mg/L LBP group still had a noticeable difference compared to the control group, and 50 mg/L LBP and 100 mg/L LBP could improve the proliferation activity of SCs after heat stress to the level of normal statement.

LBP Inhibits the Dedifferentiation of SCs after Heat
Stress. To observe the effect of LBP on the differentiation of SCs, we tested the expression of CK-18 ( Figure 4). e expression of CK-18 in the heat-stress group significantly increased (P < 0.05 or P < 0.01) when compared with the control group. Moreover, compared with the heat-stress group, the expression of CK-18 in LBP treatment groups (25,50, and 100 mg/L) decreased significantly (P < 0.01).

LBP Maintains the Integrity of BTB after Heat Stress.
To clarify the effect of LBP on BTB, we detected the TJ-associated protein, occludin ( Figure 5(a)) and zonula occludens-1 ( Figure 5(b)). As shown in Figure 5, compared with the control group, the expression of occludin and zonula occludens-1 in the heat-stress group significantly decreased (P < 0.05 or P < 0.01). Compared with the heat-stress group, the expression of occludin and zonula occludens-1 in LBP treatment groups increased significantly (P < 0.01) in a dosedependent manner (P < 0.05 or P < 0.01).

LBP Maintains the Expression of AR in SCs after Heat
Stress. Testosterone only works when combining to the androgen receptor. To determine the AR in SCs and to better understand the mechanism of AR, we analyzed AR with immunohistochemistry staining (Figure 6(a)) and western blot (Figure 6(b)). Figure 6(a) shows that AR mainly expresses in the nucleus and few are expressed in the cytoplasm of mature SCs. As shown in Figure 6, the expression of AR did significantly decrease (P < 0.01) after heat treatment.   Evidence-Based Complementary and Alternative Medicine Compared with the heat-stress group, the expression of AR in LBP treatment groups increased significantly (P < 0.01).

LBP Maintains the Akt Phosphorylation in SCs after Heat
Stress. We all know the critical role of the Akt signaling pathway in cell activity. It has been found that there is an interaction between AR and Akt phosphorylation at Ser473 [37]. Consequently, we tested the expression of p-Akt (Ser473) to explore the effective way of LBP on SCs (Figure 7). Compared with the control group, the expression of p-Akt (Ser473) in the heat-stress group significantly decreased (P < 0.01). Compared with the heat-stress group, the expression of p-Akt (Ser473) in LBP treatment groups increased significantly (P < 0.01). ese data indicated that Akt phosphorylation at Ser473 was involved in the protective effect of LBP on SCs and BTB.

Discussion
Sertoli cells are the most crucial somatic cells for spermatogenesis. e number and maturations of SCs determine the spermatogenesis. In this study, our data indicate that LBP could resist the decrease of proliferation activity, inhibit the dedifferentiation of SCs after heat stress, and more importantly, preserve BTB integrity and permeability by maintaining AR and phosphorylated-Akt (Ser473).

Evidence-Based Complementary and Alternative Medicine
Lycium barbarum fruits, as a traditional Chinese medicine and health food for people, have been used to nourish the kidney and improve fertility for thousands of years [38,39]. e polysaccharide is the primary active component responsible for those biological activities in L. barbarum fruits [28]. Also, LBP has been reported to possess a wide range of pharmacological activities, including antioxidant, anticancer, and neuroprotective effects, immune regulation, and others [40]. Recently, there are some reports about the effects of LBP on male fertility, for example, increasing the serum testosterone level and decreasing apoptosis of germ cells. However, the effects of LBP on SCs and BTB are rarely reported. us, this study intends to investigate the effects and underlying mechanisms of LBP on SCs and BTB.
Temperature is an essential controller for reproductive activity and testicular homeostasis [41]. Only can physiological scrotal hypothermia guarantee the normal spermatogenesis in most mammals [42]. Despite that SCs are more tolerant to heat than germ cells, heat stress still can cause damaged structure and dysfunction of SCs [43], resulting in spermatogenic arrest and weak fertilizing capacity in vivo and in vitro [10,44]. Some evidence and our previous study proved that local testis heat treatment (43°C for 20 min) successfully leads to dyszoospermia of monkey and rodents [33,45,46]. Furthermore, compared with other modeling methods of spermatogenesis disorder, heat-stressinduced impairment on testis is reversible, which is very suitable to study the sequence and interaction between SCs and germ cells.
We first selected three suitable concentrations of LBP by MTT ( Figure 2) to treat Sertoli cell. As we previously said, the number of SCs directly determines the number of sperm. Ki67, a nuclear antigen closely related to cell mitosis, is often considered as a marker of cell proliferative activity [47,48]. For rat SCs, the number of SCs keeps increasing after birth, as well as the number of Ki67 positive cells, while it began to decline at the age of 90 days [49], which suggest SCs of 60-day-old rats are not fully mature and some of them still can proliferate. Intervention on SCs of 60-day-old rats can affect the final number of SCs in seminiferous tubules. erefore, we detected the expression of Ki67 to observe the proliferation activity of SCs. e result confirmed that LBP could preserve the cell proliferative activity of SCs after heat stress (Figure 3). Furthermore, only mature SCs can support spermatogenesis. CK-18 is a cytoskeleton molecule, which expresses in the prepubertal SCs and gradually disappears after puberty on mammals [50,51]. High expression of CK-18 indicates that SCs are immature and dysfunctional [52]. Our results imply that LBP treatment could inhibit the re-expression of CK-18 after heat stress ( Figure 4) to prevent the dedifferentiation of SCs. e formation of BTB occurs at the beginning of puberty, and BTB is partly composed of tight junction (TJ), which directly affects the permeability [53]. Occludin is a highly phosphorylated transmembrane TJ-associated protein [54], believed as the initiator of BTB formation [55], and it is related to the initiation of spermatogenesis as well [56,57]. Zonula occludens-1, a peripheral transmembrane protein, forms a link between the transmembrane proteins and the cytoskeletal compartment [58,59] to maintain the integrity of BTB and support the migration and release of germ cells [60]. Reports have shown that increased permeability and dysfunction of BTB are blamed for the loss of occludin and (or) zonula occludens-1, resulting in harmful influence to spermatogenesis [61,62]. erefore, we selected occludin and zonula occludens-1 as the molecule marker of BTB integrity. Our data indicated that LBP could maintain the expression of them in SCs to ameliorate the heat-stress-induced increasing permeability and dysfunction of BTB ( Figure 5).
It is well known that testosterone and AR are the decisive factors for maintaining male fertility and secondary sexual characteristics. AR is believed as a crucial upstream factor in controlling the development of SCs and the forming of BTB [63]. AR deficiency caused failure of SCs maturation [64] and decrease of TJ-associated protein expression [10,11,65]. In the present study, our data demonstrated that LBP could against the decrease of AR induced by heat stress, which was consistent with the change of tight junction protein, but opposite to CK-18 ( Figure 6). Furthermore, through in vivo experiments, it was found that LBP increased the serum testosterone level in rats. We concluded the increase of testosterone and AR can improve their binding efficiency, which may be an important reason for LBP to ameliorate heat-stress-induced damage of SCs dedifferentiation and increase of BTB integrity and permeability.
Akt/protein kinase B (PKB), a serine/threonine kinase, is a mediator in the growth and proliferation of Sertoli cells [66,67]. Akt is activated by phosphorylation on threonine 308 ( r308) and serine 473 (Ser473), with phosphorylation at Ser473 resulting in maximal Akt activity [68]. Upregulated  Figure 8: Effects and mechanism of LBP on Sertoli cells and BTB after heat stress. AR could directly interact with PI3K regulatory subunit p85α to activate kinase Akt [37,73]. LBP maintained the expression of Ki67 and TJ protein and suppressed the re-expression of CK-18 by resisting the decrease of AR and maintaining phosphorylation of Akt in Sertoli cells after heat stress.

Evidence-Based Complementary and Alternative Medicine 7
Akt phosphorylation could promote the expression of Ki67 [69]. Besides, studies showed that the Akt signaling pathway is related to the expressions of occludin and zonula occludens-1 [70,71], and activation of Akt by enhancing phosphorylation of p-Akt ( r308) and p-Akt (Ser473) can effectively prevent the destruction of the TJ barrier [72]. Also, after blocking the Akt signaling pathway, AR transduction was blocked into the testosterone signaling pathway in SCs, and Akt phosphorylation at Ser473 is the key molecule in the pathway of AR trafficking [37,73]. In our results, the expression p-Akt (Ser473) of heat-stress SCs decreased while it increased after LBP treatment (Figure 7), as well as AR. erefore, we concluded that the Akt signaling pathway involves in the effect of LBP on ameliorating heatstress-induced damages in SCs and BTB. In summary, our study indicates that LBP can preserve the expression of Ki67 and occludin and zonula occludens-1 and inhibit the expression of CK-18 to prevent heatstress-induced impairment of Sertoli cells and BTB through maintaining AR and Akt phosphorylation at Ser473 (Figure 8). Also, it provides the experimental evidence for clinical prevention of male reproductive heatstress injury.
Data Availability e data supporting the conclusions of this article are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Jian Guo conceived of the study and directed the work; Suqin Hu performed the experiments; Dianlong Liu, Sijia Liu, and Chunrui Li also contributed to the experiments; Suqin Hu drafted the manuscript; Jian Guo made the critical revisions and improvements for the manuscript; and Suqin Hu and Jian Guo finalized the paper.