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Year : 2019  |  Volume : 3  |  Issue : 4  |  Page : 199-204

Extracellular vesicles in mouse testes elevate the level of serum testosterone

1 Institute of Reproductive Medicine, School of Medicine, Institute of Reproductive Medicine, Nantong University, Nantong 226001, China
2 Department of Gynecology and Obstetrics, Center for Reproductive Medicine, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China

Date of Submission17-Sep-2019
Date of Web Publication2-Jan-2020

Correspondence Address:
A-Juan Liang
Department of Gynecology and Obstetrics, Center for Reproductive Medicine, Tongji Hospital, Tongji University School of Medicine, 389, Xincun Road, Putuo District, Shanghai 200065
Fei Sun
School of Medicine, Institute of Reproductive Medicine, Nantong University, 19 Qixiu Road, Nantong 226001
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.274549

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Objective: Testosterone plays an essential role in maintaining spermatogenesis and male fertility, and the primary known source of testosterone is testicular Leydig cells, which are regulated by luteinizing hormone (LH). However, whether any other ways of testosterone secretion exist still remains unknown.
Methods: Transmission electron microscopy was used to detect testicular extracellular vesicles (EVs), which were isolated by an ultracentrifuge process. Separately, the concentrations of follicle-stimulating hormone (FSH), LH, and testosterone were measured by enzyme-linked immunosorbent assay.
Results: Some EVs were found by tail vein injection to be present in mouse testes that elevate the circulating testosterone and LH levels in the blood, but do not affect FSH. Separately, they also promote testosterone production in the TM3 Leydig cell line in vitro. To determine whether the EVs from spermatogonia were involved in the secretion of testosterone, we used spermatogonial stem/progenitor cell line C18-4 cells and revealed that C18-4 cells promote production of testosterone in the TM3 Leydig cell line using the EVs.
Conclusions: EVs in mouse testes likely originate from spermatogonia and involved in the regulation of the serum testosterone. Our results provide a new mechanism for the regulation of testosterone production.

Keywords: Extracellular Vesicles; Spermatogonial Stem Cell; Testosterone

How to cite this article:
Yun DM, Gao S, Lin Y, Wu XL, Liang AJ, Sun F. Extracellular vesicles in mouse testes elevate the level of serum testosterone. Reprod Dev Med 2019;3:199-204

How to cite this URL:
Yun DM, Gao S, Lin Y, Wu XL, Liang AJ, Sun F. Extracellular vesicles in mouse testes elevate the level of serum testosterone. Reprod Dev Med [serial online] 2019 [cited 2020 Apr 1];3:199-204. Available from: http://www.repdevmed.org/text.asp?2019/3/4/199/274549

  Introduction Top

Spermatogenesis is a continuous process that takes place within the seminiferous tubules of the testes and determines male fertility. Spermatogenesis can be divided into three distinct phases: mitosis, meiosis, and spermiogenesis. In the first phase, the spermatogonial stem cells lie adjacent to the basement membrane of the seminiferous tubules and divide by way of mitosis, resulting in another stem cell and a differential spermatogonium. The stem cells can renew themselves, and the differential spermatogonia undergo a species-specific number of mitotic divisions, with the final division resulting in differentiated Type B spermatogonia. Type B spermatogonia divide to form preleptotene spermatocytes that lose contact with the basement membrane and commence the process of meiosis, eventually completing meiosis to form round spermatids, and after undergoing extensive differentiation (spermiogenesis), the differentiated elongated spermatids are released into the tubule lumen.

Spermatogenesis is tightly controlled by the hypothalamic–pituitary–testicular axis; the hypothalamus secretes gonadotropin-releasing hormone (GnRH) to govern the spermatogenesis. GnRH stimulates the pituitary to release two gonadotropin hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH provokes  Sertoli cells More Details to express testosterone receptors via FSH receptors and supports spermatogenesis through the completion of meiosis but not germ cell maturation independent of testosterone.[1] Testosterone is secreted by adult Leydig cells under LH stimulation, and in the absence of testosterone stimulation, spermatogenesis does not process beyond the meiosis stage.[2] Basically, testosterone is critical to spermatogenesis in men.

It is established that the concentration of testicular testosterone is several degrees higher than that in the peripheral circulation.[3],[4] However, the question of how testosterone is precisely regulated is less defined because insufficient or excess production is detrimental.[5] In addition to the fact that the LH stimulates Leydig cells to secrete testosterone, when LH binds LH receptor on the surface of Leydig cells, cyclic adenosine monophosphate is produced, which activates the protein kinase A pathway to regulate the expression levels of the proteins and enzymes involved in steroidogenesis.[6],[7] It is reported that the serum testosterone concentration was significantly decreased when germ cells are lost,[8] and the serum testosterone of infertile men with Sertoli cell only syndrome was also significantly lower than that in controls. These studies indicated that germ cells may regulate the production of testosterone, but the regulation is unclear.

Extracellular vesicles (EVs) are a heterogeneous group of lipid bilayer-delimited particles that are released from all tissues and organs in both healthy and pathologic individuals. EVs include proteins, lipids, and nucleic acids and play a key role in communication between cells and in the transport of diagnostically significant molecules. Cells can release many vesicles including ectosomes, microparticles, and shedding microvesicles. In order to bring harmonization to the field, all of these vesicles were included under the EV umbrella.[9] In this study, we found that EVs in the testes impact the testosterone level in the blood and proved that the EVs from C18-4 cells stimulate the TM3 Leydig cell line to secrete testosterone in vitro.

  Methods Top


C57BL/6 mice were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China), and the mice were maintained in a 12 h dark/light cycle under temperature at 23°C ± 2°C and relative humidity of 45%–55%. The animals were fed with ad libitum access to food and water. All experiments on animals were performed under the guidelines of the Animal Care and Use Committee of the Nantong University Medicine School. Thirty 8-week-old male mice were randomly divided into three groups (10 animals per group). Three groups underwent a tail vein injection of saline, serum exosome, and testicular EVs (1 × 1,011 particles), respectively. After 8 h, the blood was collected and stored in −80°C. This study was approved by the Ethics Committee of the Nantong University Medicine School.

Isolation and analysis of testicular extracellular vesicles

Ten adult mice were euthanized by cervical dislocation. Next, their testes were dissected and decapsulated, then digested with 2 mg/mL of collagenase IV and 2 mg/mL of hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 20 min. The samples were centrifuged at 3,000 g for 10 min to remove the cells, centrifuged at 10,000 g for 30 min at 4°C to remove cell debris, filtered using a 0.22-μm filter (Millipore, Burlington, MA, USA), and then centrifuged at 100,000 g for 90 min in an SWT32 swinging bucket rotor (Beckman Coulter, Brea, CA, USA) at 4°C to precipitate the EVs. The pellets were washed with phosphate-buffered saline (PBS) and finally collected following centrifugation at 100,000 g for 90 min at 4°C, with resuspension in 20 μL of PBS. The purified EVs were analyzed using Zeta Particle Metrix equipment (Particle Metrix GmbH, Meerbusch, Germany), after which point, they were stored at −80°C. The EVs were resuspended in PBS for subsequent analysis.

Cell culture and exosome isolation

Spermatogonial stem/progenitor cell line C18-4 cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% (v/v) FBS, 2 mmol/L of l-glutamine, 100 μg/mL of streptomycin, and 1% (v/v) of penicillin and streptomycin (Life Technologies, Carlsbad, CA, USA) at 37°C in 5% CO2. The TM3 cells were cultured with DMEM/F12 medium supplemented with 2.5% (v/v) inactivated FBS, 5% (v/v) inactivated horse serum, and 1% (v/v) penicillin and streptomycin and incubated at 37°C with 5% CO2. For EV preparation, first, the C18-4 cells were grown in exosome-free media for 72 h. The media from the cells was then collected, centrifuged at 3,000 g for 30 min, centrifuged at 10,000 g for 30 min at 4°C, filtered using 0.22-μm filter, and then centrifuged at 100,000 g for 90 min in an SWT32 swinging bucket rotor at 4°C to precipitate the EVs. Next, the resulting samples were washed with PBS, then collected by centrifugation at 100,000 g for 90 min at 4°C and resuspended in 20 μL of PBS.


Cells were plated in a 12-well plate and on 12-well Transwell polyester permeable supports (Corning Inc., Corning, NY, USA) with 0.4-μm pore size. Free cells were cocultured for 2 days, whereas cells on the permeable supports were treated with GW4869 (20 μmol/L) or dimethyl sulfoxide and then cocultured with TM3 cells for 2 days. Permeable supports of the control group received media +GW4869. Media were spun at 16,500 g for 20 min, and the supernatant was collected to measure the testosterone.

Transmission electron microscopy

Testicular samples were fixed in 2.5% glutaraldehyde in 0.1 mol/L of HEPES buffer (pH = 5) overnight at 4°C and then postfixed in 1% osmium tetroxide. Rinsed with PBS, subjected to dehydration in an ethanol gradient, and then embedded in Epon 812 (Sigma-Aldrich, St. Louis, MO, USA). Ultrathin sections obtained using an Ultracut R ultramicrotome (Leica) were stained with uranyl acetate and lead citrate.

EVs were fixed in 4% paraformaldehyde and absorbed by Formvar-carbon-coated electron microscopy grids, washed three times with PBS, further fixed with 1% glutaraldehyde for 5 min, and then stained with 4% uranyl acetate for 30 min. Following drying at room temperature, images of the micrographs were captured using a transmission electron microscope (H-7560; Hitachi, Tokyo, Japan) at 80 kV.

Western blot analysis

The testicular EVs were lysed using RIPA lysis buffer (Beyotime, China) and incubated on ice for 30 min. The protein concentrations were measured using a bicinchoninic acid (BCA) protein assay (Beyotime, China). A total of 20 μg of protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was blocked in TBST containing 5% nonfat milk at room temperature for 1 h, and then incubated with the primary antibodies cluster of differentiation (CD) 9 (1:2,000, Proteintech, Rosmont, IL, USA), Thy1 (1:2,000, Proteintech, Rosmont, IL, USA), Gfra1 (1:2,000, Proteintech, Rosmont, IL, USA), and CD81 (1:2,000, Proteintech, Rosmont, IL, USA) overnight at 4°C. After the membrane was washed three times, it was incubated with secondary antibodies (1:1,000, Abcam, Cambridge, UK) for 1 h at room temperature. All the antibodies are diluted in 5% nonfat milk. After washing three times, immunoblots were visualized using ECL substrate (Thermo Scientific, Waltham, MA, USA) and the ImageQuant LAS4000 Mini software (GE Healthcare Life Sciences, Chicago, IL, USA).

Enzyme-linked immunosorbent assay

Supernatants were analyzed using enzyme-linked immunosorbent assay kits (Mlbio, Shanghai, China) according to the manufacturer's instructions. Optical density (OD value) was measured at 450 nm using a microplate reader (Sunrise, Tecan, Switzerland).

Statistical analysis

Statistical analyses were carried out using the GraphPad Prism software version 6.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed using an unpaired two-tailed Student's t-test. One-way analysis of variance was used to compare more than two groups. Data were shown as means ± standard deviations unless otherwise indicated. P values of 0.05 or less were considered to be statistically significant.

  Results Top

Identification of extracellular vesicles in the mouse testes

All eukaryotic cells can secrete a range of EVs, which include exosomes and microvesicles.[10] To investigate the role of EVs in spermatogenesis, we used transmission electron microscopy (TEM) to track the EVs in adult mouse testes. There are numerous small vesicles in the interstitium and the basal compartments of seminiferous tubules [Figure 1]Aa and [Figure 1]b. The diameter of these vesicles ranges from 30 nm to 150 nm. To enrich and characterize these EVs, we utilized collagenase Type IV and hyaluronidase to dissociate cells and EVs from adult mouse testes. Following digestion, the EVs were isolated by differential centrifugation [Figure 1]B. Next, the EVs were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. The TEM images revealed that the vesicles are a heterogeneous population between 30 nm and 200 nm in diameter that present a cup shape [Figure 1]C. The diameter was also confirmed by NTA analysis [Figure 1]D. Further, exosomal markers, including Thy1, Gfra1, CD81, and CD9, were present in EVs [Figure 1]E. These observations and analysis results revealed obvious characteristics of EVs, which proved the existence of EVs in mouse testes.
Figure 1: Identification of extracellular vesicles in the mouse testis. (A) TEM images of testicular EVs in cross-sections of seminiferous tubule from adult mice. (B) Flowchart for the EVs purification procedure based on differential ultracentrifugation. (C) Representative TEM images of isolated testicular EVs. (D) Size distribution of the testicular EVs determined by NTA analysis. (E) Western blot analysis for CD81, Thy1, Gfra1, and CD9 using extracts from testicular EVs and supernatant. TEM: Transmission electron microscopy; EVs: Extracellular vesicles; NTA: Nanoparticle tracking analysis.

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Testicular extracellular vesicles elevate the serum testosterone in mice

Recent evidence suggests that EVs could carry biological signals to distant destinations through the peripheral circulation, and the EVs can also cross the blood–brain barrier.[11],[12] We assume that the testicular EVs may be involved in the hypothalamic–pituitary–testicular axis, as the intratesticular testosterone is essential for spermatogenesis. We first measured the testosterone level in the testis after 1 h, 4 h, 8 h, 12 h, and 16 h when the mice were injected with testicular EVs via the tail vein. The intratesticular testosterone level first began to rise but then returned to the original level, which probably implied that testicular EVs only temporarily increased the amount of testosterone in the testes [Figure 2]a. Next, we measured the serum testosterone levels after different numbers of EV injections, finding that testicular EVs have concentration-dependent effects on testicular testosterone [Figure 2]b. Then, LH and FSH were assessed after 8 and 16 h following the injection of testicular EVs into the tail vein: serum concentrations of LH were significantly increased, while those of FSH remained unchanged [Figure 2]c and [Figure 2]d. Testosterone is critical to spermatogenesis. It is well known that testosterone is primarily produced in Leydig cells so, to investigate whether testicular EVs directly promote Leydig cell to produce testosterone, we exposed different numbers of EVs to TM3 Leydig cells. Ultimately, enzyme-linked immunosorbent assay results revealed that testicular EVs significantly promote TM3 to secrete testosterone and have concentration-dependent effects [Figure 2]e. These results indicate that EVs from the testes increase the circulation of testosterone in the blood.
Figure 2: Testicular EVs elevate the serum testosterone in mouse. (a) Intratesticular testosterone levels in adult mice injected with PBS, serum EVs, or testicular EVs after 1 h, 4 h, 8 h, 12 h, and, 16 h. (b) Serum testosterone levels in adult mice injected with serum EVs or testicular EVs at different concentration after 8 h. (c and d) Serum LH or FSH levels in adult mice injected with serum EVs or testicular EVs after 8 and 16 h. (e) Testosterone production in TM3 Leydig cells exposure to various concentrations of testicular EVs. (n = 6, *P < 0.05, **P < 0.001). EVs: Extracellular vesicles; PBS: Phosphate-buffered saline; LH: Luteinizing hormone; FSH: Follicle-stimulating hormone.

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Extracellular vesicles from C18-4 cells promote the production of testosterone in TM3 Leydig cells

The EVs in the seminiferous tubules were close to the basal membrane [Figure 1]Aa, which prompted the thought that the EVs may be produced by spermatogonia or Sertoli cells. However, it has been reported that the serum testosterone concentration was significantly decreased when germ cells were lost, while the serum testosterone concentration was not affected with Sertoli cell ablation.[8] Similarly, the serum testosterone of an infertile man with Sertoli cell-only syndrome was significantly lower than that of healthy controls.[13] We speculated that the spermatogonia cells might regulate the production of testosterone by EVs. To test this hypothesis, the spermatogonial stem/progenitor cell line C18-4 and TM3 Leydig cells were used. The EVs isolated from C18-4 significantly stimulated TM3 Leydig cells to secrete testosterone [Figure 3]a. On the other hand, the control exosomes failed to show the same effects. Next, the exosome biogenesis/release inhibitor GW4869 was then employed.[14] We performed coculture studies to assess whether GW4869 treatment of C18-4 cells would affect the nature of testosterone production in recipient TM3 Leydig cells. C18-4 cells were plated on permeable inserts above TM3 Leydig cells, and we found that blocking C18-4 cell exosome secretion using GW4869 treatment significantly reduced the testosterone production in TM3 Leydig cells, while the control exosomes failed to show the same effects [Figure 3]b. In conclusion, the EVs from C18-4 cells promoted the production of testosterone in TM3 Leydig cells.
Figure 3: The EVs from C18-4 cells promote the production of testosterone in TM3 Leydig cell. (a) Testosterone production in TM3 Leydig cells exposure to PBS, 293T EVs, or C18-4 EVs; (b) TM3 cells were cocultured with C18-4 cells, GW4869-treated C18-4, DMEM alone (Blank), or GW4869 in DMEM (Blank + GW4869) for 3 days. Testosterone production at the bottom of the plate was measured. (Results are expressed as mean ± SEM from three independent experiments. ns, P > 0.05, *P < 0.05, **P < 0.001). EVs: Extracellular vesicles; PBS: Phosphate-buffered saline; DMEM: Dulbecco's Modified Eagle Medium.

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  Discussion Top

Spermatogenesis is the culmination of a complex process of autocrine, paracrine, and endocrine interactions between multiple cell types. In the past few decades, a third mechanism for intercellular communication has emerged in the literature that involves the intercellular transfer of EVs, both in prokaryotes and eukaryotes.[15] This is due to their capacity to transfer proteins, lipids, and nucleic acids, thereby influencing various physiological and pathological functions of the recipient cells. It has been reported that EVs in the epididymis have multiple functions in sperm maturation and storage,[16] while the seminal EVs are also involved in the pathogenesis of asthenozoospermia.[17] However, whether or not there are EVs in the male testes has remained unanswered. In particular, how EVs are involved in the cell communication of spermatogenesis is unclear. In this report, we showed that the EVs are detected both inside and outside the seminiferous tubule and found that these EVs can elevate the serum testosterone when injected into the tail vein, which provides new clues to illuminate the regulation of testosterone.

In our experiment, the testicular EVs were separated from the whole adult testes, so the EVs we isolated likely could have originated from any cell in the testes. Even though we found that EVs elevated the serum testosterone when injected through the tail vein, we cannot identify which cells were directly involved in this process. Further research should strive to identify the source of testicular EVs by proteomics, and then the EVs can be purified by molecular markers of specific cells.

Leydig cells produce testosterone, the male sex hormone responsible for the growth and maintenance of the cells of the germinal epithelium and the development of secondary sex characteristics. EVs are one of the critical forms of intercellular communication between Leydig cell and spermatogonia. Testosterone is one of the essential factors that introduce spermatogenesis. As we known, EVs contain a variety of protein and small noncoding RNA cargo, which may influence Leydig cell development and the secretion of testosterone. In this study, we speculated that the spermatogonia cells might regulate the production of testosterone by EVs. The spermatogonial stem/progenitor cell line C18-4 and TM3 Leydig cells were used. The EVs isolated from C18-4 significantly stimulated TM3 Leydig cells to secrete testosterone [Figure 3], while the EVs from spermatogonia stimulated the production of testosterone. Whether any other types of cells are involved in the testosterone secretion requires further elucidation.

In conclusion, our study proved the existence of EVs in mouse testes, which suggests that EVs may constitute an intercellular communication means in the testes and could be used as a potential drug delivery platform to treat testicular diseases. The testicular EVs involved in testosterone secretion may provide new clues to illuminate the regulation of testosterone.

Financial support and sponsorship

Financial support was received from the National Key Research and Development Program of China (No. 2018YFC1003500 to F.S) and the National Natural Science Foundation of China (Nos. 81430027 and 81671510 to F.S).

Conflicts of interest

There are no conflicts of interest.

  References Top

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[PUBMED]  [Full text]  
Lin Y, Liang A, He Y, Li Z, Li Z, Wang G, et al. Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ. Mol Reprod Dev 2019;86:1094-105. doi: 10.1002/mrd.23224.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3]


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