Studying the effect of hyperoside on recovery from cyclophosphamide induced oligoasthenozoospermia

Abstract Oligoasthenozoospermia is becoming a serious problem, but effective prevention or treatment is lacking. Hyperoside, one of the main active ingredients in traditional Chinese medicine, may be effective in the treatment of oligoasthenozoospermia. In this study, we used cyclophosphamide (CTX: 50 mg/kg) to establish a mouse model of Oligoasthenozoospermia to investigate the therapeutic effect of hyperoside (30 mg/kg) on CTX-induced oligoasthenozoospermia. All mice were divided into four groups: blank control group (Control), treatment control group (Hyp), disease group (CTX) and treatment group (CTX + H). Mice body weight, testicular weight, sperm parameters and testicular histology were used to assess the reproductive capacity of mice and to explore the underlying mechanism of hyperoside in the treatment of oligoasthenozoospermia by assessing hormone levels, protein levels of molecules related to hormone synthesis and transcript levels of important genes related to spermatogenesis. Treatment with hyperoside significantly improved sperm density, sperm viability and testicular function compared to untreated oligoasthenozoospermia mice. In mechanism, treatment with hyperoside resulted in significant improvement in pathological changes in spermatogenic tubules, with an increase in testosterone production, and upregulations of Protein Kinase CAMP-Activated Catalytic Subunit Beta (PRKACB), Steroidogenic Acute Regulatory Protein (STAR), and Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17A1) for testosterone production. Hyperoside also promoted the cell cycle of germ cells and up-regulated meiosis and spermatogenesis-related genes, including DNA Meiotic Recombinase 1 (Dmc1), Ataxia telangiectasia mutated (Atm) and RAD21 Cohesin Complex Component (Rad21). In conclusion, hyperoside exerted protective effects on oligoasthenozoospermia mice by regulating testosterone production, meiosis and sperm maturation of germ cells.


Introduction
Infertility has become a serious health problem worldwide, part of which is attributed to oligoasthenozoospermia, mainly manifested as spermatogenesis disorder, sperm quantity reduction, sperm motility deficiency (Skakkebaek et al. 2016;Amory et al. 2017;Liang et al. 2021).The incidence of oligoasthenozoospermia has increased year by year due to psychological hazards, environmental pollution, smoking, chemotherapy drugs and other factors (Levine et al. 2022;Marchiani et al. 2019).However, its pathogenesis has not been discovered and there is little effective treatment in clinical practice.
Spermatogenesis, which spermatozoa develops from spermatogonial stem cells (SSC) in the testis (Wang et al. 2018;Sohni et al. 2019), is a highly ordered physiological process.It could be divided into three main stages: (1) the mitotic phase that a single SSC ultimately give rise to spermatogonias (SPGs) that are capable of entering meiosis and forming precursor spermatocytes after several rounds of mitosis; (2) the meiotic phase that spermatocytes (SPCs) undergo complex chromosomal behavior during the first stage of meiosis, followed by two rounds of cell division to produce round spermatid (RS); (3) the postmeiotic phase that round spermatids elongate and mature to motile spermatozoa (Xiong et al. 2022).At the same time, the highly ordered physiological process is inseparable from the precise regulation of hormones, especially testosterone produced by Leydig cells (LCs) (Albuquerque et al. 2013).When testosterone is insufficient, spermatogenesis will halt during meiosis, so that a few germ cells develop to round spermatid and elongated live spermatids are not formed (Bonanno et al. 2016).
Spermatogenesis is vulnerable to chemotherapeutic drugs, environmental contamination and other factors.Cyclophosphamide (CTX), an alkylating agent, is one of the chemotherapeutic drugs which is widely used in treating multifold cancers, it gives excellent therapeutic effects but it also produces side effects on spermatogenesis and further causes male infertility (Ganjalikhan Hakemi et al. 2019;Delessard et al. 2020).CTX-treated mice showed a significantly reduction of serum testosterone levels, sperm concentration and motility (Ceribas ¸i et al. 2010).
Chinese medicine Cuscuta chinensis has been used for thousands of years for the treatment of human fertility and reproduction, and hyperoside, a natural flavonoid and an active constituent of Cuscuta chinensis has a variety of pharmacological and biological activities, such as antibacterial, antitumor, anti-inflammatory, antioxidant, neuroprotective and liver protective effects (Yen et al. 2008;Donnapee et al. 2014).Additionally, it has been reported that hyperoside supplementation in preservation media surpasses vitamin C protection against oxidative stress-induced damages in human spermatozoa (Moreira et al. 2022).Current studies have shown that hyperoside is involved in the regulation of steroids hormones secretion and lipidic metabolism (Nie et al. 2019).Moreover, methanolic extract of Carissa opaca leaves containing hyperoside exerted functional recovery of male sexual dysfunction and reproductive impairment induced by CCl4 in male mice by regulating the hormonal endocrine activity (Sahreen et al. 2013).Therefore, hyperoside may have a promising application in the treatment of oligoasthenozoospermia.
The purpose of this study was to determine the effect of hyperoside on the recovery of spermatogenesis after CTX treatment in mice and to develop an effective method to protect the reproductive function of patients with oligoasthenozoospermia.

Induction of an animal circadian disruption model and treatment
Male mice were treated to hyperoside and CTX for seven days, as described in the Figure 1A.We measured body weight every day and daily food consumption.The weight gain and final body weight were significantly lower in the mice exposed to CTX (groups: CTX and CTX þ H) than control and Hyp groups (Figure 1B).Similarly, the daily food intake of mice with exposure to CTX gradually decreased compared with the control and Hyp groups (Figure 1C).However, there were no difference in the end body weight and daily food intake of mice between the CTX group and CTX þ H group.

Hyperoside restored CTX-induced oligoasthenozoospermia
To assess the fertility of mice treated by CTX and hyperoside, we compared the testicular and epididymal weight, testicular and epididymal index, and sperm parameters (sperm concentration, sperm viability and sperm motility).After CTX exposure, testicular weights and testicular index reduced significantly compared with the control and Hyp group (Table 1).The testicular weight and index increased after treatment with hyperoside compared with the CTX group (Table 1).Upon histological examination of the testis (Figure 2B), testicular histopathology was consistent with the testicular index.The intact seminiferous tubules showed the normal stages of spermatogenesis in the control and Hyp groups.Compared to the control group, the CTX exposure induced hyperemia around the seminiferous tubules, Leydig, Sertoli cells and spermatogenetic, shed, reduced and layers lessened, and seminiferous epithelium cells arrayed loosely in mice that did not receive hyperoside.Moreover, CTX disrupts the development of germ cells at various stages of seminiferous tubules: in the CTX group, the percentage of SPC and LS were reduced, and the percentage of RS was increased compared to control and Hyp groups (Figure 2C).In addition, sperm parameters were consistent with testicular histopathology.Compared with the control group and Hyp group, the sperm concentration, sperm viability and sperm motility decreased after CTX exposure (Figure 2A).Therefore, we successfully established a mouse model of oligoasthenozoospermia, and testicular tissue and sperm parameters were significantly damaged.However, hyperoside significantly reversed the damage of CTX.In the CTX þ H group, the sperm parameters (Figure 2A) and the testicular tissues (Figure 2B) showed significant and almost complete recoveries compared to the CTX group.These results suggested that CTX disrupts spermatogenesis: SPC stagnated at meiosis, and RS could not mature into LS, which led to an increase in the percentage of SPC and RS, while the percentage of LS decreased.However, hyperoside improve the meiotic I biological processes of germ cells destroyed by CTX.SPC and RS successfully completed the meiotic phase and sperm maturation, which resulted in a decrease in their percentages; similarly, RS successfully matured into LS, which increased the percentage of LS (Figure 2C).Altogether, the data in this section showed that CTX damaged the reproductive organs and sperm parameters of mice, but hyperoside reversed the damage of CTX-induced oligoasthenozoospermia mice.

Hyperoside increased testosterone in oligoasthenozoospermia mice
Hormone imbalance correlates with male infertility.However, it remained unclear whether hyperoside also affects hormone synthesis.After exposed to CTX, the Before treating the mice every day, the body weight and food weight of the mice were recorded on the day.CTX (50 mg/kg) intraperitoneal injection and hyperoside (30 mg/kg) gavage lasted 7 d, and hyperoside gavage started one day later than CTX intraperitoneal injection.On the 8th day, it was humanely sacrificed 2 h after oral administration of hyperoside, and the testis and epididymis were collected for analysis.(B) the weight gain (g), (C) food intake of mouse model (g).level of testosterone in the serum and testis were significantly lower than control and Hyp groups, while the level of testosterone in the CTX þ H was significantly restored by hyperoside (Figure 3A,B).Next, we assessed the effects of hyperoside treatment on the upstream hormones and key proteins of testosterone production.The results in Figure S1 showed that there is not significant effect on LH and FSH with CTX and hyperoside.Protein levels of PRKACB Protein Kinase CAMP-Activated Catalytic Subunit Beta (PRKACB), Steroidogenic Acute Regulatory Protein (STAR) and Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17A1) tested by Western Blot were reduced in the CTX group compared to the control and Hyp groups.
In contrast, the proteins level of CYP17A1, PRKACB, and STAR were increased in the CTX þ H group compared to the CTX group (Figure 3C,D).These results demonstrated that CTX disrupts the synthesis of testosterone, but hyperoside positively restored levels in the CTX þ H group by upregulating key proteins involved PRKACB, STAR and CYP17A1 in the testis of oligoasthenozoospermia mice.

Depression germ cell development potential and dysfunction in oligoasthenozoospermia
To explore the target of hyperoside in treating Oligoasthenozoospermia, we analyzed the mRNA expression on testis samples in patients with severely impaired and normal spermatogenesis according to the Gene Expression Omnibus website.The functions of the differently expressed genes were analyzed using Go and KEGG pathway analysis.KEGG analysis showed that Ubiquitin mediated proteolysis and Tyrosine metabolism were the most relevant down-regulated pathways (Figure 4A).GO enrichment top 20 terms down-regulated indicated spermatid differentiation, spermatid development and biological processes related to meiosis (Figure 4B).In addition, the Oligoasthenozoospermia model and normal mice testis were collected for RNA-seq analysis.The functions of the differently expressed genes were analyzed using Go and KEGG pathway analysis.KEGG analysis showed that down-regulated genes were mainly enriched in the cell cycle, p53 signaling pathway, Progesteronemediated oocyte maturation and DNA replication (Figure 4C).GO analysis showed that down-regulated genes were mainly enriched in the meiotic cell cycle and other meiosis-related biological processes (Figure 4D).Combining the Go analysis of patients and mice, we found that the oligoasthenozoospermia is mainly related to meiosis I (meiotic cell cycle; meiosis I; meiosis I cell cycle process; meiotic nuclear division; meiotic cell cycle process; nuclear division; organelle fission) and the later stages of sperm maturation (germ cell development; spermatid development; spermatid differentiation) (Figure S2A).We also constructed the protein-protein interaction (PPI) networks of biological processes include meiosis-related (Figure S2B,C) and later stages of sperm maturation (Figure S2D).Bioinformatics results show that the mouse model of oligoasthenozoospermia has the same biological processes as patients with oligoasthenozoospermia: Germ cell stagnation during meiosis and impaired sperm maturation.

Hyperoside improve the meiotic processes of germ cells in oligoasthenozoospermia mice
Bioinformatics and testicular histology results suggested that the oligoasthenozoospermia mouse model has germ cell arrest during meiosis and impaired sperm maturation.However, the testicular histology results indicated that hyperoside improved the meiotic I biological processes of germ cells destroyed by CTX (Figure 2C).To explore the involved molecules by which hyperoside improve meiosis processes, 14 genes involved in all 7 meiosis I biological processes were investigated By PCR and immunohistochemistry analysis, including spermatogenesis associated 2 (Spata2), HORMA domain containing (Hormad), Ataxia Telangiectasia Mutated Proteins (Atm); and RAD21 Cohesin Complex Component (Rad21) (Figure 5A).
The PCR analysis indicated that the mRNA levels of Spata2, Hormad1, Rad21 and Atm were significantly lower in the CTX group compared with the control and Hyp groups, while their mRNA levels upregulated in the CTX þ H group compared with the CTX group (Figure 5B).Consistently, Immunohistochemistry analysis of the testicular tissue indicated that the protein levels of RAD21 and ATM in the CTX group decreased compared with the control and Hyp groups,

Hyperoside improve the sperm maturation processes in oligoasthenozoospermia mice
To explore the involved molecules by which hyperoside promotes sperm maturation processes, 8 genes involved in all 3 sperm maturation biological processes were investigated, including DNA Meiotic Recombinase 1 (Dmc1) and predicted gene 773 (Gm773) (Figure 6A).Our PCR analysis indicated that the mRNA levels of Gm773 and Dmc1 were significantly lower in the CTX group compared with the control and Hyp groups, while their expression upregulated in the CTX þ H group compared with the CTX group (Figure 6B).Consistently, Immunohistochemistry analysis of the testicular tissue indicated that the Dmc1 expression levels in the CTX group decreased compared with the control and Hyp groups, while hyperoside treatment upregulated the protein level of DMC1 (Figure 6C,D).These results indicated that hyperoside probably promote sperm maturation in oligoasthenozoospermia by upregulating Dmc1 expression.

Discussion
Male infertility is a common male disease.In recent years, the number of oligoasthenozoospermia has been increasing as sperm quality has plummeted (Inhorn and Patrizio 2015).Currently, the hormone replacement therapy used in clinical practice is ineffective and associated with a range of adverse reactions (Rastrelli et al. 2014), thus seeking natural, safe and efficient remedies is of great significance for people with oligoasthenozoospermia.CTX is a chemotherapeutic agent with certain male reproductive toxicity.
In this study, we found that one dose of CTX (50 mg/Kg BW) produced oligoasthenozoospermia in mice during adulthood (including reductions in body weight, food intake, testis weight, testicular index, sperm parameters, testosterone, and testicle damage observed by histology), which is consistent with many previous studies (Azhar et al. 2021;Liu et al. 2021;Solomon et al. 2021).Hyperoside, a flavonoid from Cuscuta sinensis, has been explored to treat some diseases (Wang et al. 2022).In this study, hyperoside treatment remarkably reversed CTX-induced reproductive damages in testis weight, testicular index, sperm parameters, testosterone, and testicle damage observed by histology, which suggested that CTX was detrimental to male fertility, and hyperoside treatment can improve this effect.Therefore, we set out to explore the underlying mechanism by which hyperoside improves spermatogenesis.
In this study, cell count results of testicular tissue revealed increases of SPC and RS and a reduction of LS.It suggested that CTX disrupts the meiotic phase of germ cell and the sperm maturation phase, resulting in an increase in SPC and immature RS, and a decrease in mature sperm LS.These results are in harmony with some previously studies which showed that the transition from the meiotic G2 phase to the MI phase was significantly disturbed in the case of acute exposure to CTX, and the number of meiotic and meiotic/post-meiotic cells was also significantly reduced (Lu et al. 2012).The study of Velez de la Calle et al. found that CTX-treated (at different ages) rats had significantly reduced testicular weight and spermatogonia (Velez de la Calle et al. 1989), which is consistent with our study.However, CTX þ hyperoside significantly decreased the proportion of SPC and RS compared to the CTX group.The percentage of LS was also higher in the CTX þ H group compared to the CTX group.Based on our data, we proposed that hyperoside treatment may protect germ cell development in testes, and ameliorate reproductive function injury in oligoasthenozoospermia mice induced by CTX.Moreover, the beneficial effects of hyperoside on male germ cell development were mainly due to the recovery of testosterone (Sahreen et al. 2013).
Hormones are the main factors that regulate testicular function.Stable and normal hormone levels are the guarantee of normal sperm production.The literatures have proved that the production of testosterone regulated by numerous enzymes in the testis is a complex and sophisticated process (Yu et al. 2020).Testosterone is produced by Leydig cells under the regulation of LH and FSH (Scott et al. 2007).In the first step, LH receptors activate the cAMP/PRKACB pathway after binding LH.In the second step, after PRKACB acts on STAR, the cholesterol is transferred to the inner mitochondrial membrane by STAR, which is the rate-limiting step of steroid hormone biosynthesis.Cholesterol entering the mitochondria is subsequently converted into pregnenolone by the cholesterol side cleaves cytochrome P450 (CYP11A1).In the third step, pregnenolone is transported by CYP17A1 and 17b-hydroxysteroid dehydrogenase (HSD17b) to the smooth endoplasmic reticulum, where it is converted to testosterone (Walker 2021).In this study, we found that CTX decreased the levels of testosterone in mice blood and testis samples, while hyperoside reversed this change.Furthermore, the results of Western blot showed that hyperoside increased the protein levels of PRKACB, STAR and CYP17A1 in the CTX þ H group, which was decreased in the CTX group.These findings suggested that hyperoside can regulate testosterone production by up-regulating the level of testosteronerelated proteins including PRKACB, STAR and CYP17A1, to improve sperm development (Figure 7).
Testosterone, as a paracrine factor, also diffuses into the seminiferous tubules and then maintain spermatogenensis at normal levels (Cooke and Walker 2021;Walker 2021).In the absence of testosterone, the meiotic phase of germ cells is arrested, immature germ cells were prematurely displaced from Sertoli cells, and round sperm are unable to mature into long sperm (O'Shaughnessy 2014).The disruption of these testosterone-dependent steps results in the failure of spermatogenesis and infertility (Smith and Walker 2014;O'Donnell et al. 2022).As mentioned above, in oligoasthenozoospermia model mice, the meiotic phase of germ cells was arrested and sperm maturation was impaired.We suspect that this was due to testosterone production being disrupted by CTX.The purpose of our study was to provide treatment options for clinically oligoasthenozoospermia patients, so we analyzed transcriptomics in patients with spermatogenic disorders.GO analysis showed that spermatogenesis disorder in patients with oligoasthenozoospermia was related to the meiotic including meiotic cell cycle and other meiosis-related biological processes, and sperm maturation stages including spermatid differentiation, spermatid development, which was very consistent with GO analysis of oligoasthenozoospermia model mice induced by CTX.Previous studies have shown that both stagnation during meiosis and abnormalities in the development of the haploid reproductive system affect the final production of sperm, which is consistent with our findings (Azhar et al. 2021).Moreover, it is worth noting that the meiosis process and sperm maturation process are most easily destroyed by external factors (Lu et al. 2012;Abofoul-Azab et al. 2019).Spata2, Dmc1, Hormad, Rad21, Atm and Gm773 play important roles in spermatogenesis, homologous recombination, division recombination, DNA repair DNA recombination repair and sperm differentiation biological processes during meiosis and sperm maturation, respectively (Prieto et al. 2002;Akter et al. 2021;Lee and Paull 2021;Guan et al. 2020;Masola et al. 2022;Yang et al. 2021).Q-PCR experiments and immunohistochemical assays were used to find the molecular targets of hyperoside to promote germ cell meiosis and sperm maturation process.The results showed that hyperoside treatment up-regulated the expression levels of meiosis-related genes Spata2, Dmc1, Hormad, Rad21 and Atm, and proteins level of RAD21 and ATM.In addition, hyperoside also up-regulated the expression levels of sperm maturation-related genes Dmc1 and Gm773, and DMC1 protein levels.
In conclusion, hyperoside can reduce the decrease of sperm quantity and quality as well as testicular tissue damage in CTX-induced oligoasthenozoospermia mice, and protect reproductive function by upregulating testicular levels, regulating germ cell meiosis and sperm maturation process.Hyperoside promotes the levels of testosterone in Leydig cells by regulating the production of important proteins (PRKACB, STAR, CYP17A1).Testosterone further promotes the meiosis of spermatogenic cells and sperm maturation, but the exact mechanism still needs to be further investigated by in vitro experiments.Ultimately, this study will provide natural, efficient and safe spermatogenic drugs for the treatment of oligoasthenozoospermia.

Animals
Six to eight week-old male C57BL/6 mice were obtained from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences and housed at the Medical Experiment Center of Lanzhou University.The relative humidity of the animal room was controlled at about 70%, the temperature was controlled at about 22 C, and the regular light and darkness were 12 h each.During the whole study period, water and food were provided free of charge.All mice were fed for 5d to start the formal experiment.

Treatments
Cyclophosphamide provided by SHIFEN BIOLO GYCOLC TECHNOLOGY co., LTD (Shanghai, China) was intraperitoneally injected into mice for the oligoasthenozoospermia model.Hyperoside was provided by SHANGHAI SHIFEN BIOLOGYCOLC TECHNOLOGY co., LTD (Shang Hai, China).In this study, we chose 30 mg/kg of hyperoside by oral gavage to treat oligoasthenozoospermia (Liu et al. 2019;Qiu et al. 2019;Fan et al. 2022).According to previous studies, mice was intraperitoneally injected by CTX at a dose of 50 mg/kg (Fan et al. 2022).Fluid volume for intraperitoneal injection and oral is 150-200ll/mouse/day (10 ll/g, total volume < 400 ll/mouse).The solution of CTX and hyperoside was freshly prepared in NaCl solution every day.We randomly assigned 48 mice into four groups, with 12 mice assigned to each of the following, control group, Hyp group (30 mg/kg hyperoside), CTX group (50 mg/kg CTX), and CTX þ H group (50 mg/kg CTX, 30 mg/kg hyperoside).Before treating the mice every day, we recorded the body weight and food weight of the mice on the day.CTX intraperitoneal injection and hyperoside gavage lasted 7 d.On the 8th day, it was humanely sacrificed 2 h after oral administration of hyperoside, and we collected the testis and epididymis for analysis.In order to anesthetize mice, we chose a dose of 50 mg/kg to inject 1% pentobarbital solution (Tsukamoto et al. 2015).The testicular weight was measured, and the relative testicular organ index was calculated by dividing the total weight of the two testicles (mg) by body weight (g).

Sperm analysis
Fresh epididymis was cut into pieces along the lumen and incubated in PBS with 10% (w/v) bovine serum albumin (Every Green; Zhejiang, China) at 37 C for 5 min to prepare sperm suspension.Add the sperm suspension to a preheated blood counting pool (Furuide, Qingdao, China) to count.The calculation of sperm parameters was based on previous studies (Fan et al. 2022) 100%; sperm concentration (10 6 /ml) ¼ the count total sperm/4 Â 10 6 /ml; sperm viability: the count live sperm/the count total sperm Â 100%, and using a graded semi-quantitative scale(a ¼ rapid motility; b ¼ slow motility; c ¼ nonprogressive motility; d ¼ immotile sperm).

Histological evaluation of testes and immunofluorescence
The testis and epididymis of mice were fixed with a 4% formaldehyde solution.For histological examination, 4 lm thickness was sliced followed by hematoxylin and eosin (HE; Servicebio, Wuhan, China) according to standard histopathological methods.Images were observed under a microscope to assess the testicular damage.Two complete seminiferous tubules were selected for each image and the numbers of SPC, RS and LS were calculated.We dewaxed the paraffin sections by xylenol, rehydrated them in ethanol series, and hydrogen peroxide treatment (Liu et al. 2021).Then, we incubated the sections by 0.25% trypsin for 10 min.In order to block the binding of nonspecific proteins, the sections were incubated for 10 min.The sections were then incubated at 4 C overnight by primary antibodies (1:100 dilution) including Rad21, Atm and Dmc1 (Baijia, Taizhou, China).After washing three times with PBS, sections were incubated with HRP-labeled goat anti-rabbit protein (1:100 dilution) (Baijia, Taizhou, China).HRP was then observed with streptomyces antibiotic peroxidase and DAB.Finally, the sections were stained with hematoxylin.Collect images with a microscope.Six regions were randomly selected from each picture.
The average optical density of each group was calculated using Image J software.

RNA-seq and bioinformatics analysis
Trizol reagent (Servicebio, Wuhan, China) was used to extract total RNA from testicular samples.Sequencing was performed using the Illumina HiSeq2000 system (Novogene, Beijing, China).The data after quality control were analyzed subsequently.The data set in June 2021 was obtained from the GEO data portal (https://www.ncbi.nlm.nih.gov),including transcriptome analysis data of 10 normal and 10 damaged testes.Limma package in R software was used to identify the differentially expressed genes (DEGs) of the control group and CTX group samples, normal spermatogensis and 10 impaired spermatogensis testis samples.We analyzed the DEG functional spectrum by R Bioconductor/cluster Profiler package and Gene Ontology (GO) and predicted protein-protein interaction networks by the Search Tool for Interacting Genes/Proteins (STRING), and illustrated gene function by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.The significance threshold was set to adjusted p .05 and jlog2FCj>1 as the cutoff point to screen differential genes.

Quantitative real-time PCR
Total RNA was reverse transcribed using PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, RR047A, Beijing, China).Quantitative realtime PCR (QRT-PCR) was used to assess changes in mRNA levels.QRT-PCR was performed using Light Cycler SYBR Green I Master Mix according to the manufacturer's instructions using the Light Cycler real-time PCR instrument (Bio-Rad, The United States).In this study, a 10 ll reaction system volume was used, following the PCR conditions previously studied.We use actin as an internal reference for analyzing gene expression.The 2 ÀDDCt method was used to calculate relative mRNA expression levels.Gene primer sequences are listed in Table 2.

Western blot analysis
After the testicular tissue was washed with normal saline solution, 100 mg of tissue was weighed, and an appropriate amount of RIPA lysis solution (Servicebio, Wuhan, China) was added, fully ground, and centrifuged at 4 C at 12,000 r/min for 30 min.The supernatant of the tissue was sucked out and the loading buffer (Servicebio, Wuhan, China) was added to determine the expression levels of STAR, CYP17A1 and PRKACB proteins (Bioss, Beijing, China).The image is analyzed by image software, and we get the gray ratio of the image.

Hormone detection
Blood samples were collected directly from the heart, and the serum were separated for hormone detection.The levels of testosterone, LH and FSH were quantitatively determined by ELISA kits (JINGMEI, Jiangsu, China).Proteins were extracted from the testis and testosterone levels were quantified by ELISA kit.

Statistical analysis
All results were expressed as the mean ± SEM.SPSS version 22.0 software and GraphPad Prism version 8.4.2 for Windows was used for data analysis and the differences between groups were analyzed by unpaired t tests.p < .05 was defined as a significant difference; p < .01 was a highly significant difference.

Ethics approval
This study makes the use of male C57BL/6 mice, and all the experimental protocol for the use of animal was approved by the Ethics Committee of the First Hospital of Lanzhou University (LDYYLL2019-44).

Figure 1 .
Figure 1.Experimental scheme for manipulations, treatments and monitoring of mice.(A) Scheme of experiment design.Before treating the mice every day, the body weight and food weight of the mice were recorded on the day.CTX (50 mg/kg) intraperitoneal injection and hyperoside (30 mg/kg) gavage lasted 7 d, and hyperoside gavage started one day later than CTX intraperitoneal injection.On the 8th day, it was humanely sacrificed 2 h after oral administration of hyperoside, and the testis and epididymis were collected for analysis.(B) the weight gain (g), (C) food intake of mouse model (g).

Figure 2 .
Figure 2. Effects of Hyperoside on sperm parameter and testicular histopathology (A) Sperm concentration, viability and motility.Unpaired t test was used, data are represented as mean ± SEM (n ¼ 6).Ã p < .05,ÃÃ p < .01. (B) Testicular histopathology.H&E staining is shown in the Figure.The intact seminiferous tubules showed the normal stages of spermatogenesis in the control and Hyp groups.Compared to control group, the CTX exposure induced the hyperemia around the seminiferous tubules, Leydig, Sertoli cells and spermatogenetic, shed, reduced and layers lessened, and seminiferous epithelium cells arrayed loosely in mice that did not receive hyperoside.(Bar ¼ 100 lm, H&E stain, 200Â.Red arrows refer to congested blood between seminiferous tubules; Red Ã refer to spermatogenetic, leydig and sertoli cells reduced, shed and layers lessened; Red triangle refer to loss of spermatogenic cells at all levels in seminiferous tubules; yellow arrows refer to spermatocytes (SPCs); green arrows refer to round spermatid (RS); orange arrows refer to loog spermatozoa (LS)).(C) Percentage of SPC, RS and LS/seminiferous tubules view.Unpaired t test was used, data are represented as mean ± SEM (n ¼ 6 per group, about 6 seminiferous tubules were examined for each mouse).Ã p < .05,ÃÃ p < .01;ÃÃÃ p < .001,ÃÃÃÃ p < .0001.

Figure 3 .
Figure 3. Effects of Hyperoside on the level of proteins involved in testosterone synthesis and the concentration of testosterone.(A, B) The content of testosterone in testes and serum.Unpaired t test was used, data are represented as mean ± SEM (n ¼ 5), Ã p < .05,ÃÃ p < .01. (C) The protein levels of PRKACB, STAR and CYP17A1 using Western blot analysis.PRKACB, Protein Kinase CAMP-Activated Catalytic Subunit Beta; STAR, Steroidogenic Acute Regulatory Protein; CYP17A1, Cytochrome P450 Family 17 Subfamily A Member 1. (D) The relative abundance of PRKACB, STAR and CYP17A1 in different groups.Unpaired t test was used, Data are represented as mean ± SEM (n ¼ 3), Ã p < .05,ÃÃ p < .01.

Figure 4 .
Figure 4. Overview of the RNA-seq data for mouse testes and human testes.(A) The KEGG pathway enrichment analysis of down-regulated genes in the Non obstructive azoospermia-vs-Obstructive azoospermia (n ¼ 10).(B) The GO analysis of down-regulated genes in the Non obstructive azoospermia-vs-Obstructive azoospermia (n ¼ 10).(C) The KEGG pathway enrichment analysis of down-regulated genes in CTX-vs-Control group (n ¼ 3).(D) The GO analysis of down-regulated genes in CTX-vs-Control group (n ¼ 3).

Figure 7 .
Figure 7.The potential mechanism diagram of Hyperoside improving oligoasthenozoospermia.Hyperoside promotes the levels of testosterone in Leydig cells by regulating the production of important proteins (PRKACB, STAR, CYP17A1).

Table 1 .
Efects of hyperoside on testicular and epididymal in the mice.

Table 2 .
Primers used for quantitative RT-PCR.