Quantitative proteomics reveal the protective effects of Qiang Jing decoction against oligoasthenospermia via modulating spermatogenesis related-proteins

Introduction

Human infertility may bring hidden danger to the harmony and stability of the family, leading to a social problem that has to be settled urgently. In the meantime, it has also become an intractable medical bottleneck because it is difficult to cure. An epidemiological study conducted by Vander Borght et al. reported that 8–12% of married couples experience reproductive disorders, among which male factors are either directly or indirectly involved in approximately 50% of cases (1). Oligoasthenospermia is one of the most common phenotypes of male reproductive disorders that accounts for 60–75% of cases in the clinic (2-4). Until now, the modern treatment of oligoasthenospermia is mainly dependent on drug therapy, such as antioxidant drugs, hormone drugs, and energy supplementation. At present, as the efficiency of drugs is often limited, leading to an urgent need of alternative treatment strategy for male infertility patients.

Traditional Chinese medicine (TCM) has certain advantages and characteristics in the treatment of men with oligoasthenospermia with the modernization, identification, and increased distribution of TCM active compounds (5,6). For example, Runjing decoction alleviates sperm quality and testis damage in oligoasthenospermia rats by inhibiting the RXFP1/AKT/FOXO1 pathway (7). Qiang Jing decoction (QJD) is a TCM prescription of which the major constituents are Cuscuta chinensis seeds, Lycium chinense seeds, Schisandra chinensis seeds, Astragalus membranaceus, Lujiaoshuang (Cervus nippon angles), Codonopsis pilosula, Dipsacus asper roots, Leonurus japonicus, Angelica sinensis, Ostreidae, Shenqu, Ziheche (Placenta Hominis). The components in QJD have functions of anti-oxidation, anti-fatigue, anti-inflammation, improving immunity, and microcirculation, which plays a crucial role in protecting testicular normal function (8,9). For example, Cuscuta chinensis seeds is a commonly used kidney tonic Chinese medicine combination that is widely used in the clinical treatment of male infertility (10). However, protein profiling of QJD in the treatment of oligoasthenospermia is still poorly studied and merits further investigation.

Mass spectrometry (MS)-based proteomics provides feasible tools to find insights into protein expression patterns (11). In this study, we evaluated the effects of QJD on protecting testis tissue from damage and investigated protein expression via tandem mass tag (TMT) proteomics technology in a rat model. The results originating from the testis of oligoasthenospermia rats could serve as resources for future studies related to the molecular mechanism of QJD treatment. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-24-327/rc).

Methods

Rat model and treatment

A total of 45 male Sprague-Dawley (SD) rats (8–10 weeks, 200–220 g) were purchased from Changsha Tianqin Biotechnology Company (Changsha, China). Laboratory animal experiment was approved with certificate No. SCXK2014-0011. Experimental rats were randomly divided into three treatment groups containing control, oligoasthenospermia model, and QJD treatment, with 15 experimental rats in each treatment group (n=15). Rats belonging to oligoasthenospermia model group and QJD group were intraperitoneally injected with cyclophosphamide at 20 mg/kg dissolved in normal saline for 14 consecutive days to make oligoasthenospermia model rats. The control group was treated with the same method but with saline. Since the fifteenth day, rats from the control and model treatment were given normal saline by gavage. Meanwhile, the rats in the QJD treatment will get 12 g/kg/d QJD by gavage for 36 consecutive days until the completion of the process of rat sperm production (12). Rats were injected with 3% pentobarbital sodium to anesthesia after 24 h the final treatment. Their testes were carefully and completely removed by laparotomy, washed with saline three times and flash-frozen in liquid nitrogen, and stored at −80 ℃ until further analysis. A protocol was prepared before the study without registration. All procedures employed were approved by the Animal Ethical and Welfare Committee of The First Affiliated Hospital of Guangxi University of Chinese Medicine (No. DW20240919-200), in compliance with institutional guidelines for the care and use of animals.

Sperm quality analysis

Both epididymides of each rat were removed and cut into small pieces immediately after sample harvest. The samples were subsequently transferred to a tube containing 37 ℃ saline to make sperm diffusion. Then, 10 µL of sperm suspension was smeared on a counting board to determine sperm concentration. The sperm quality analysis was performed by the sperm quality examination system (CFT-9201X, Jiangsu Ruiqi Co., Ltd., Jiangsu, China).

Protein extraction, and digestion

The rat testis sample was rapidly ground into cell powder with liquid nitrogen and then transferred into a 5-mL centrifuge tube. Following this, four volumes of lysis buffer (8 M urea, 1% protease inhibitor mixture) were added to the cell powder, and ultrasonic treatment was performed three times. To completely remove the remaining debris, the sample was centrifuged at 12,000 g at 4 ℃ for 10 min. Subsequently, the supernatant was carefully collected. The bicinchoninic acid (BCA) kit was used for evaluating protein concentration according to the manufacturer’s instructions.

For digestion, the protein solution was reduced with 5 mM dithiothreitol at 56 ℃ for 30 minutes and alkylated with 11 mM iodoacetamide at room temperature for 15 minutes in darkness. Then 100 mM tetraethylammonium bromide (TEAB) was then added into the protein sample until to urea concentration was less than 2M. Finally, the sample was digested with trypsin added at 1:50 trypsin/protein ratio for the first digestion overnight and 1:100 trypsin/protein ratio for a subsequent 4 h-digestion.

TMT labeling and high-performance liquid chromatography (HPLC) fractionation

Peptide was labeled with the TMT kit/iTRAQ kit according to the manufacturer’s protocol. Briefly, one unit of TMT/iTRAQ reagent was un-freeze in acetonitrile and reconstituted. The peptide mixtures were then incubated at room temperature for 2 hours and pooled, desalted, and dried by vacuum centrifugation.

Thermo Betasil C18 (Thermo Fisher, Shanghai, China) column [diameter 5 µm, inner diameter (ID) 10 mm, length 250 mm] was used for the fractionated of the tryptic peptides based on high pH reversed-phase high performance liquid chromatography. Briefly, peptides were first separated into 60 fractions over 60 min with a gradient of 8% to 32% acetonitrile (pH 9.0). Then, the peptides were combined into 6 fractions and dried by vacuum centrifuging for further analysis.

Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS)

The peptides were analyzed using an EASY-nLC 1000 UPLC system equipped with tandem mass spectrometry (MS/MS) in Q Exactive™ Plus (Thermo) using a home-made reversed-phase analytical column (15-cm length, 75 µm i.d.). The tryptic peptides were separated using 0.1% formic acid and 0.1% formic acid in 98% acetonitrile as mobile phases A and B, respectively with a flow rate of 400 nL/min. The gradient was comprised of an increase from 6% to 23% solvent B over 26 min, 23% to 35% in 8 min, and climbing to 80% in 3 min then holding at 80% for the last 3 min. Intact peptides were detected in the Orbitrap at a resolution of 70,000. The m/z scan range was 350 to 1,800 for the full scan. The electrospray voltage applied was 2.0 kV. The fragments were detected in the Orbitrap at a resolution of 17,500 and peptides were then selected for MS/MS using normalized collision energy (NCE) setting as 28. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. The fixed first mass was set as 100 m/z. Automatic gain control (AGC) was set at 5E4.

Bioinformatics analysis

Maxquant search engine (v.1.5.2.8) was used for processing MS/MS data obtained from the LC-MS/MS. Then human UniProt database con-catenated with the reverse decoy database was served for processing tandem mass spectra. The mass tolerance for precursor ions was set as 20 ppm and the mass tolerance for fragment ions was set as 0.02 Da. False discovery rate (FDR) was adjusted to <1% and the minimum score for modified peptides was set to >40. The threshold value of differentially expressed proteins were as follows: unique peptide fold change ≥2; P value ≤0.05. Gene Ontology (GO) (http://www.ebi.ac.uk/interpro/) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kaas-bin/kaas_main; http://www.kegg.jp/kegg/mapper.html) was employed predict each differentially expressed proteins functional characterization and metabolic pathway classification, respectively. Wolfpsort (http://www.genscript.com/psort/wolf_psort.html) was applied to predict the subcellular localization of each identified protein.

Immunohistochemistry

The testis sample was soaked in Bouin fixing solution at room temperature for 48 h. After dehydration, the samples were embedded in paraffin and cut into 5-µm thick sections that were collected on glass slides. After this, the samples were deparaffinized in xylene. A graded series of water and ethanol was used for rinsed and rehydrated, respectively. Endogenous peroxidase activity was blocked by incubation in phosphate-buffered saline (PBS) solution contained 0.3% hydrogen peroxide at room temperature for 30 min. The PBS solution supplemented with 10% normal goat serum was used for the block slides for 1 h. PI3K, Akt, and mTOR expression were detected after overnight incubation at 4 ℃ with antibodies. After washing, the sections were incubated with HRP-conjugated goat anti-rabbit immunoglobulin G (IgG, 1:2,000; Santa Cruz Biotechnology, Texas, USA) in 10% goat serum for 1 hours, counterstained for 10 s with hematoxylin (Gill No. 3; Sigma, Michigan, USA). Confocal microscope (Nikon Eclipse TS100, Tokyo, Japan) was used for the experiment imaging analyses.

Statistical analysis

Significant differences among control, oligoasthenospermia, and QJD treatment groups were evaluated by one-way analysis of variance (ANOVA) with Tukey’s test. All data were shown as mean ± standard deviation (SD), and P≤0.05 was considered significant.

Results

QJD treatment improves sperm quality

The oligoasthenospermia rat model was established based on the modified methods (13). There were very significantly lower sperm density and activity in the model group than in the control group, indicating that the oligoasthenospermia model rats were successfully established. After the QJD intervention, sperm density was elevated compared to the model group and was close to the level of the control group. The improvement in sperm activity rate was similar to that in sperm density (Figure 1). Combined these results with the improvement of sperm quality mentioned above, the QJD can be considered to positively affect the recovery of testicular tissue of rats, ensuring the production of more high-quality sperm.

Figure 1 QJD treatment improves sperm quality. (A) Representative photograph sperm quality of oligoasthenospermia rats induced in different group. (B,C) Demonstrate sperm density, sperm activity rate, respectively. ****, P<0.0001; ns, no significant, P>0.05. QJD, Qiang Jing decoction.

Global proteomic changes in rats testis

To understand the changes in protein abundance of oligoasthenospermia model rats’ response to QJD drug treatment, comparative proteomics-based TMT labeling technology was conducted. After processing MS/MS spectra in Maxquant software, a total of 425,265 spectra were generated from TMT analysis, yielding 50,497 matched peptides, 47,134 unique peptides, 6,861 matched proteins, and 5,533 quantified proteins. All the protein information is listed in Table S1.

Identification and bioinformatics analysis of differently abundant proteins (DAPs) in the oligoasthenospermia model group compared with the control group

According to a criterion of 95% significance and a 1.2-fold cutoff, a total of 56 and 215 proteins were identified as up and down-regulated DAPs between the model and control group, respectively. All the DAPs information are available online: https://cdn.amegroups.cn/static/public/tau-24-327-1.xlsx. GO biological process (BP) analysis indicated that these DAPs primarily engaged in the cellular process, metabolic process, biological regulation, and single organism process. GO cellular component (CC) analysis implied that these DAPs mainly originated from the cell, organelle, macromolecular complex, and membrane-enclosed lumen complex. GO molecular function (MF) analysis revealed that these DAPs engaged in binding, catalytic activity. In the classification of subcellular localization, the nucleus with 125 DAPs, and cytoplasm with 73 DAPs were the first and second largest categories. KEGG pathway analysis indicated that the main DAPs were enriched in the ‘rno04672 intestinal immune network for IgA production’, ‘rno05202 transcriptional mis-regulation in cancer’, and ‘rno05322 systemic lupus erythematosus’ pathway (Figure 2).

Figure 2 Identification and bioinformatics analysis of DAPs in model group compared with the control group. (A) Volcano, (B) GO analysis of DAPs in the testis. (C) KEGG analysis of DAPs in the testis. (D) Subcellular localization analysis of DAPs in the testis. CK, control check; GO, Gene Ontology; DAPs, differently abundant proteins; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Identification and bioinformatics analysis of DAPs in the QJD group compared with the control group

As shown in Figure 3, a total of 28 and 16 proteins were identified as up- and down-regulated DAPs between drug treatment and control groups, respectively. All the DAPs information are available online: https://cdn.amegroups.cn/static/public/tau-24-327-2.xlsx. GO BP analysis showed that these DAPs were most enriched in cellular process, biological regulation, single-organism process, and metabolic process. GO CC analysis implied that these DAPs mainly originated from cells, organelle, macromolecular complex, and membranes. In the meantime, GO MF analysis revealed that these DAPs primarily engaged in binding and catalytic activity terms. As the classification of subcellular localization, cytoplasm with 16 DAPs, extracellular with 9 DAPs, nucleus with 9 DAPs were the first and second largest categories. KEGG pathway analysis indicated that the main DAPs were enriched in the ‘rno04672 intestinal immune network for IgA production’, ‘rno05330 allograft rejection’, ‘rno05320 autoimmune thyroid disease’, and ‘rno05332 graft versus host disease’ pathway.

Figure 3 Identification and bioinformatics analysis of DAPs in drug group compared with the control group. (A) Volcano, (B) GO analysis of DAPs in the testis. (C) KEGG analysis of DAPs in the testis. (D) Subcellular localization analysis of DAPs in the testis. CK, control check; GO, Gene Ontology; DAPs, differently abundant proteins; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Identification and bioinformatics analysis of DAPs in QJD group compared with the oligoasthenospermia model group

As showed in Figure 4, a total of 70 and 28 proteins was identified as up and down regulated DAPs between drug treatment and model group, respectively. All the DAPs information are available online: https://cdn.amegroups.cn/static/public/tau-24-327-3.xlsx. GO BP analysis demonstrated that these DAPs were most enriched in cellular process, biological regulation, single-organism process, and metabolic process. GO CC analysis implied that these DAPs main originated from cells, organelle, macromolecular complex, and membrane. In the meantime, GO MF analysis revealed that these DAPs primarily engaged in binding and catalytic activity terms. In the classification of subcellular localization, the nucleus with 41 DAPs, and cytoplasm with 23 DAPs, were the first and second largest categories. KEGG pathway analysis demonstrated that the main DAPs were enriched in the ‘rno04672 intestinal immune network for IgA production’, ‘rno05330 allograft rejection’, ‘rno05320 autoimmune thyroid disease’, ‘rno05202 transcriptional misregulation in cancer’, and ‘rno05203 viral carcinogenesis’ pathway.

Figure 4 Identification and bioinformatics analysis of DAPs in drug group compared with the model group. (A) Volcano, (B) GO analysis of DAPs in the testis. (C) KEGG analysis of DAPs in the testis. (D) Subcellular localization analysis of DAPs in the testis. GO, Gene Ontology; DAPs, differently abundant proteins; KEGG, Kyoto Encyclopedia of Genes and Genomes.

QJD treatment alters the expression of spermatogenesis-related proteins

Spermatogenesis is a very important process related to male fertility. Here, we identified 20 DAPs related to the spermatogenesis process based on the protein annotation in rat testis of different treatments. Interestingly, we found that almost all of the proteins related to the spermatogenesis were significantly down regulated in oligoasthenospermia model rats compared to the control group, except for protein G3V7X0 (Table 1). These proteins were downregulated with a ratio ranging from 0.677 to 0.832 and a highest ratio of 0.611 being observed with Piwi-like homolog 2 protein (D3ZRE1). However, all these protein expression levels were increased and showed no difference with the control group after the QJD treatment.

Effect of QJD on the PI3K/AKT signaling pathway in oligoasthenospermia rats

The PI3K/AKT signaling pathway was considered as can directly and indirectly maintain and promote spermatogenesis in male testis (14). To understand the possible mechanism of QJD alleviating oligoasthenospermia model rats, we performed Akt, PI3K, and mTOR expression in testis tissue by immunohistochemistry. Immunofluorescence analysis revealed that Akt, PI3K, and mTOR expression levels have significantly decreased in the model group. The expression level of Akt, PI3K, and mTOR was observed significantly increased after QID treatment compared to the control group. In the meantime, we observed that the spermatogenic epithelium of the control and drug group was closely arranged, and active and the lumen of seminiferous tubules was filled with a large number of sperm. However, the spermatogenic epithelium was loose, and the number of sperm was significantly reduced in our model group (Figure 5). These results suggest that the spermatogenic capacity was obviously recovered after the QJD intervention.

Figure 5 Effect of QJD on the PI3K/AKT signaling pathway. (A) The location of Akt, mTOR, and PI3K proteins was determined by immunohistochemistry in testis. (B) Effect of QJD on protein expression levels of Akt, mTOR, and PI3K in oligoasthenospermia rats. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant, P>0.05. Akt, serine and threonine kinase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3 kinase; QJD, Qiang Jing decoction.

Discussion

Male infertility causes significant social and psychological distress and raises a considerable economic burden on patients and healthcare systems (15), which remains poorly understood, and oligoasthenospermia accounts for most of the cases. QJD is one of the most effective TCM prescriptions for male infertility with indications of oligoasthenospermia. However, the molecular mechanism by which QJDs alleviate oligoasthenospermia has not been entirely understand. In the present study, combined with TMT proteomic technology, we examined the effect of QJD on the sperm quality and protein levels in the testis of oligoasthenospermia model rats.

We found that the majority of proteins related to spermatogenesis were significantly downregulated in oligoasthenospermia model rats compared to the control group. It is well known that the testis-specific serine threonine protein kinase family members play important roles in spermiogenesis (16,17). Such as serine threonine protein kinase 2, a specific phosphorylated protein of testicular tissue, showed a significantly weakened trend in a rat model of oligoasthenospermia (18). However, serine-threonine protein kinase 2 expression gradually recovered after treatments with another TCM Qilin pills (QLPs), which is consistent with our result. The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely related to the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme (19,20). In addition, SMCP-deficient mutant mice become infertile due to decreased capability of the spermatozoa to penetrate oocytes and reduced motility of the spermatozoa (19). Our result showed that oligoasthenospermia model rats had lower sperm density and activity with reduced expression level of SMCP. And the QJD can obviously recover the sperm quality and increase the SMCP protein abundance. Other proteins that have a very important role in spermatogenesis also exhibited the same expression trend that was significantly increased after QJD the intervention, such as nuclear autoantigenic sperm protein (NASP), Tudor domain-containing protein (Tdrd), and so on (21-23). Combined with these results, we speculated that QJD can alleviate oligoasthenospermia symptoms by regulating the protein abundance related to the spermatogenesis.

To deeply understand the mechanism of QJD regulating the spermatogenesis, the PI3K/AKT signaling pathway proteins that are involved in spermatogonia stem cells and spermatogonia self-renewal, proliferation, and anti-apoptosis, were analyzed by the immunohistochemistry. Our results demonstrated that the abundance of PI3K, AKT, and mTOR proteins was suppressed in oligoasthenospermia model rats and was induced during the drug treatment. PI3K can phosphorylate 3'hydroxyl of the phosphatidylinositol-3,4-bisphophate (PIP2), converting it to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), which binds to AKT and activate it both in vivo and in vitro (24,25). AKT, which is usually known as a crucial factor in mediating the metabolism of the testis, can inhibit radiation-induced apoptosis in vivo (26). So, we indicated that QJD has a positive effect on PI3K/AKT signaling pathway. The activation of the PI3K/AKT signaling pathway can promote the proliferation and anti-apoptosis of immature Sertoli cells and spermatogonia stem cells that are the main part of seminiferous epithelium (27,28).

Conclusions

In brief, we think that QJD improves sperm density and quality by activating spermatogenesis-related proteins expression and PI3K/AKT signaling pathway in an oligo-asthenospermia model rats. However, we realize that most studies only focus on the whole proteome changes after the drug intervention, owing to the complexity of this process. Future studies should more closely focus on the specific protein involved in improving sperm quality in the testis to discover the details and accurate regulatory mechanism.

Acknowledgments

We thank Key Laboratory of Chinese Medicine Clinical Research for their support to our experiments.

Funding: None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-24-327/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-327/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-327/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-327/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures employed were approved by the Animal Ethical and Welfare Committee of The First Affiliated Hospital of Guangxi University of Chinese Medicine (No. DW20240919-200), in compliance with institutional guidelines for the care and use of animals.

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