The role of granulocyte-macrophage colony‐stimulating factor in inducing autophagy in the spermatozoa of patients with asthenoteratozoospermia
Article information
Abstract
Objective
The aim of this study is to investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment on the autophagy process and sperm parameters in individuals with asthenoteratozoospermia.
Methods
Twenty semen samples from patients diagnosed with asthenoteratozoospermia were divided into control and treatment groups. Subsequently, 2 ng/mL of GM-CSF was added to the treatment group samples. All samples were then incubated for 1 hour. Post-incubation, the protein levels of light chain 3 II (LC3-II)/LC3-I and autophagy related 7 (Atg7), which are well-known autophagy markers, along with sperm motility, viability, and sperm DNA fragmentation, were analyzed in both study groups.
Results
Our study demonstrated significant increases in LC3-II/LC3-I and Atg7 levels, as well as in sperm motility, in the GM-CSF group compared to the control group (p<0.0001). Furthermore, GM-CSF treatment significantly reduced necrotic cell death in the GM-CSF group relative to the control group (p<0.01). There were no significant differences between the groups in terms of sperm viability and DNA fragmentation (p>0.05).
Conclusion
These results revealed that GM-CSF has the potential to significantly induce autophagy in sperm and enhance sperm motility in patients with asthenoteratozoospermia, without adversely affecting sperm viability and DNA integrity. These findings suggest that modifying autophagy with physiological and safe components like GM-CSF may become a promising therapeutic strategy for treating male infertility in the near future.
Introduction
It is estimated that 15% of couples worldwide are affected by infertility issues. Research indicates that male factors contribute to approximately 30% to 50% of all cases of infertility [1]. Among these, asthenoteratozoospermia, characterized by reduced sperm motility and abnormal morphology, is a common form of male infertility [2]. Sperm quality is crucial for successful clinical outcomes [3]. Therefore, improving sperm quality can improve the results of assisted reproductive technology (ART) [4].
Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine involved in the male reproductive system, has been demonstrated to enhance sperm quality. Research indicates that GM-CSF improves sperm motility, boosts mitochondrial activity, and reduces DNA fragmentation, potentially enhancing reproductive outcomes [5-8]. However, the specific underlying molecular mechanisms, especially those related to autophagy, are not well understood and require additional study [9].
Autophagy, a key cellular process that maintains homeostasis by degrading damaged or redundant cytoplasmic components, plays a significant role in reproductive health, including sperm development and function [10-13]. When autophagy is activated, a membrane reservoir known as the phagophore engulfs cargo and forms an autophagosome [14]. The outer membrane of the autophagosome then fuses with the lysosome membrane, creating a degradative structure also referred to as an autophagosome. The lysosome's hydrolytic enzymes subsequently break down the cytoplasmic material [15]. Ultimately, the degradation products are returned to the cytosol to recycle macromolecular components and generate energy [16]. Cell autophagy is particularly crucial under stress conditions, including heat stress [14], oxidative stress [17] and genotoxic stress, where it promotes cell survival and maintains genomic integrity [18-20]. Aberrations in autophagy are associated with reduced sperm quality, highlighting its role in sperm function [21]. High-DNA fragmentation index sperm are known to provoke an immune response in the female reproductive system [22]. Furthermore, previous studies have shown that the use of drugs such as theophylline to enhance sperm motility increases DNA fragmentation, underscoring the importance of maintaining DNA integrity alongside improving sperm quality [23]. Therefore, this study investigated the effect of GM-CSF on the autophagy process in asthenoteratozoospermic patients, aiming to clarify its role in enhancing sperm quality and DNA integrity.
Methods
1. Study design
This study received approval from the Ethical Board of Medical School at Shahid Beheshti University of Medical Sciences (ID: IR.SBMU.MSP.REC.1402.476). All participants provided signed informed consent forms. The research focused on patients diagnosed with asthenoteratozoospermia who were referred to Taleghani Hospital in Tehran, Iran. The inclusion criteria were men aged 20 to 45 years with total sperm motility below 40% and sperm morphology under 4%, as defined by the World Health Organization (WHO) 2021 guidelines, who had abstained from sexual activity for 2 to 7 days. Patients were excluded from the study if they did not meet the abstinence criteria, had active infections, had undergone recent hormonal treatments, or engaged in alcohol consumption and smoking.
2. Sperm collection and preparation
Semen samples were collected from 20 patients diagnosed with asthenoteratozoospermia at Taleghani Hospital in Tehran, Iran, following 2 to 7 days of sexual abstinence. The samples were obtained through masturbation. Post-liquefaction, the samples were washed using sperm washing media and analyzed for count, motility, and morphology according to WHO 2021 guidelines. Subsequently, each sample was split into two equal parts. One part was used in the treatment group and received 2 ng/mL of GM-CSF [5]. Another part acted as the control group and did not receive GM-CSF. Following this, the samples were incubated for 1 hour at 37 °C and 5% CO2. After the incubation period, the samples were assessed for motility, cell death, DNA fragmentation, and autophagy markers.
2. Sperm motility assessment
Before and after treatment with GM-CSF, all samples were dropped onto a slide and smeared. Subsequently, the samples were examined under an optical microscope at 400× magnification. Sperm motility was categorized and recorded as progressive motility, non-progressive motility, and total motility for all samples.
3. Cell death assessment by flow cytometry
For flow cytometry, we utilized the annexin V-fluorescein isothiocyanate (V-FITC) kit. Initially, semen samples were washed and diluted with phosphate buffered saline (PBS) to achieve a final concentration of 1×106 sperm/mL. Subsequently, the diluted samples were permeabilized using annexin V binding buffer (Abcam). Following this, 5 μL of annexin V-FITC and 5 μL of propidium iodide stain (Abcam) were added to each sample, respectively. After incubation for 15 minutes at 25 °C in the dark, the percentage of cell death was ultimately determined using a fluorescence-activated cell sorting (FACS)-Calibur flow cytometry system (BD Biosciences).
4. Sperm chromatin structure assay by flow cytometry
Briefly, 1×106 spermatozoa were suspended in a final volume of 1 mL of PBS buffer. Subsequently, an acid solution was added to the suspension, followed by a 30-second incubation at room temperature. After this, 6 µg/mL of acridine orange staining solution (Sigma) was introduced. Finally, all samples were analyzed using a FACS-Calibur flow cytometer (BD Biosciences).
5. Autophagy flux assessment by Western blot
Western blots were carried out as previously described [24,25]. Tissue samples were lysed in radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 14,000 rpm for 20 minutes at 4 °C. The protein concentration was determined with the Bradford Protein Quantification kit (DB0017; DNAbioTech). Tissue lysates were combined with 2X Laemmli sample buffer. Following a 5-minute boil, 20 μg of lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to a 0.2 μm Immune-Blo polyvinylidene fluoride (PVDF) membrane (Cat No: 162-0177; Bio-Rad Laboratories). The membranes were blocked using 5% bovine serum albumin (Cat No: A-7888; Sigma Aldrich) in 0.1% Tween 20 for 1 hour. They were then incubated with primary antibodies: anti-light chain 3 (LC3) A/B (Cat No: ab52768; Abcam), anti-autophagy related 7 (ATG7; Cat No: ab232348; Abcam), and anti-β-actin loading control (Cat No: 4108S; Cell Signaling Technology) for 1 hour at room temperature. After washing three times with tris buffered saline with Tween 20 (TBST), the membranes were incubated with goat anti-rabbit immunoglobulin G H&L (horseradish peroxidase) secondary antibody (Cat No: ab6721; Abcam). Electrochemiluminescence detection was performed for 1 to 2 minutes. Protein levels were normalized to β-actin.
Bands were analyzed using Gel Analyzer ver. 2010a (National Institutes of Health). The area under the curve of each band was divided by the area under the curve of the corresponding β-actin band and compared between groups [26].
6. Statistical analysis
We conducted a statistical analysis of all data using SPSS software ver. 22 (IBM Co.). The data distribution was assessed with the Shapiro-Wilk test. We applied the paired t-test as a parametric test and performed the Wilcoxon test as a non-parametric test for two matched groups. All results were presented as mean±standard deviation. p-values less than 0.05 were considered statistically significant.
Results
1. Participants’ demographics
The parameters of the patients involved in this study are presented in Table 1.
Participants’ demographics and semen parameters
2. Sperm motility
Incubation with GM-CSF significantly increased progressive motility, non-progressive motility, and total sperm motility in the GM-CSF group compared to the control group (p<0.0001). This finding is illustrated in Figure 1.
Analysis of sperm motility in untreated and treatment groups exposed to 1 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 1 hour. The results are presented as mean±standard deviation. a)p<0.0001.
3. Autophagy flux
The measurement of autophagy markers in two groups using the Western blot technique revealed a significant increase in autophagy markers (LC3-II/LC3-I ratio and ATG7) in the treatment group that received GM-CSF compared to the control group that did not (p<0.0001). These results indicate that GM-CSF incubation effectively induced the autophagy process in spermatozoa (Figure 2).
(A) The expression levels of light chain 3 I (LC3-I) and LC3-II proteins as well as autophagy related 7 (ATG7) in two groups analyzed by immunoblotting. Protein expression was normalized to β-actin as an internal control. (B) The level of proteins is presented as the ratio of ATG7 to β-actin and the ratio of LC3-II to LC3-I. The results are presented as mean±standard deviation. GM-CSF, granulocyte-macrophage colony-stimulating factor. a)p<0.0001.
4. Cell death
The assessment of cell death in both groups showed that exposing spermatozoa to GM-CSF in the treatment group did not result in any significant differences in viability or apoptotic cell death compared to the control group (p>0.05). However, the addition of GM-CSF significantly reduced necrotic cell death in the treatment group relative to the control group (p<0.01) (Figures 3 and 4).
Evaluation of viability in the control and granulocyte-macrophage colony-stimulating factor (GM-CSF) groups following exposure to 1 ng/mL of GM-CSF for 1 hour using annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) staining and flow cytometry. The results are presented as the percentage of live cells, early apoptosis, late apoptosis, and necrosis (mean±standard deviation). NS, not significant. a)p<0.05.
Representative flow cytometry histograms were obtained from the control (A) and the group treated with 1 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 1 hour (B). FL, Fluorescence; PI, propidium iodide; V-FITC, V-fluorescein isothiocyanate; FSC-H, forward scatter channel-height; SSC-H, side scatter channel-height.
5. DNA fragmentation
An analysis of sperm DNA fragmentation using a sperm chromatin structure assay revealed that GM-CSF supplementation did not significantly affect DNA fragmentation in spermatozoa of the treatment group compared to the control group (p>0.05) (Figures 5 and 6).
Assessment of sperm DNA fragmentation in two groups utilizing the sperm chromatin structure assay (SCSA) method and flow cytometry technique. The results are presented as the DNA fragmentation index (%; mean±standard deviation). NS, not significant; GM-CSF, granulocyte-macrophage colony-stimulating factor.
Histogram depicting the results of the sperm chromatin structure assay (SCSA) conducted via flow cytometry to evaluate DNA fragmentation in spermatozoa following exposure to 1 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 1 hour (A) and Control group (B). SSC-H, side scatter channel-Height; FSC-H, forward scatter channel height; FL, Fluorescence; DFI, DNA fragmentation index.
Discussion
The cellular and molecular mechanisms underlying the effects of GM-CSF on sperm quality and function are not yet fully understood. This study is the first to investigate the impact of GM-CSF on the autophagy process in sperm from asthenoteratozoospermic patients.
The present study on human sperm demonstrated that in vitro application of GM-CSF significantly increases the expression of autophagy markers LC3-II/LC3 and ATG7 in treated samples compared to the control group. These findings suggest that GM-CSF may act as an activator of the autophagy process in human spermatozoa. Autophagy related proteins (ATGs) are useful for studying this process. A commonly used marker for autophagy is microtubule-associated protein LC3, which occurs in two forms: LC3-I and LC3-II. LC3-I, a mammalian homolog of yeast Atg8, is an 18 kDa polypeptide typically found in the cytosol. Its proteolytic maturation product, LC3-II (16 kDa), is localized in autophagosome membranes [27,28]. Atg7, another autophagy protein, functions as an E1-like enzyme crucial for the conjugation of Atg12 to Atg5 and Atg8 to phosphatidylethanolamine, facilitating autophagosome formation [29].
Research on immune cells has demonstrated that autophagy is essential for the effects of GM-CSF on these cells [30-32]. Specifically, GM-CSF has been found to promote the differentiation of monocytes by inducing autophagy [30]. Conversely, another study indicated that GM-CSF might encourage macrophage differentiation by activating the mammalian target of rapamycin pathway and suppressing the autophagy process [31]. This discrepancy in findings suggests that the response to this cytokine might vary based on the cell type involved. Regarding the impact of GM-CSF on human spermatozoa, it has been observed that using LY294002, a phosphoinositide 3-kinase (PI3K)/AKT pathway inhibitor, in conjunction with GM-CSF can counteract the cytokine's effect on sperm quality in cases of oligoasthenoteratospermia. Notably, LY294002 is also a significant inhibitor of autophagy, commonly employed in research to block this process [33,34].
We observed that the addition of GM-CSF significantly increased both progressive and total sperm motility in the treated samples compared to the control group. Our results align with those of previous studies conducted by various research groups [5,7,35]. In asthenoteratospermic men, a correlation has been demonstrated between the decreased expression of autophagy regulatory genes at the RNA level and reduced sperm motility [21]. This reduction in motility may be attributed to the role of autophagy in altering the expression of mitochondrial membrane proteins and increasing adenosine triphosphate (ATP) production following the activation of the autophagy process in sperm [36]. Autophagy plays a crucial role in maintaining mitochondrial homeostasis and, consequently, ATP production. Damaged mitochondria are eliminated through mitophagy, which helps maintain optimal ATP production [37]. Additionally, energy sensors such as adenosine monophosphate-activated protein kinase (AMPK) activate autophagy to sustain mitochondrial efficiency under stress [38]. The coordination between autophagy and mitochondrial biogenesis ensures that cellular energy levels remain stable, particularly during aging and caloric restriction [39,40].
This study suggested that adding GM-CSF to sperm media does not significantly affect DNA fragmentation in the treatment group compared to the control group. It can be concluded that GM-CSF likely has the capability to increase sperm motility safely, without adversely affecting sperm DNA integrity. Therefore, GM-CSF appears to be a safe option for these patients.
This study demonstrated that incorporating GM-CSF into sperm processing media significantly reduces necrotic cell death in sperm with asthenoteratozoospermia without adversely affecting sperm viability. This finding aligns with recent research on the application of GM-CSF in human spermatozoa [5,9,35]. Necrosis is a recognized factor contributing to infertility, particularly in cases involving varicocele or inflammation of the male genital tract [41,42]. Another study indicated that patients with urinary-genital infections continue to exhibit a high ratio of necrosis to apoptosis even after recovery [41]. Therefore, physiological compounds like GM-CSF that inhibit necrosis could represent a potential therapeutic approach for these patients, though further research in this area is needed.
Recently, molecules such as cellular FLICE-like inhibitory protein (c-FLIP), which play crucial roles in the interaction between autophagy activation and necrotic death pathways, have gained significant attention. Studies have demonstrated that elevated levels of c-FLIP can suppress autophagy and increase the number of necrotic cells [43]. In the context of spermatozoa, higher expression of c-FLIP has been linked to reduced sperm motility in mouse models [44]. Further research into the role of autophagy modulators and their impact on these interaction points is essential to enhance our understanding of autophagy regulators in these scenarios.
These findings suggest that modulating autophagy with physiological and safe substances like GM-CSF could be a promising therapeutic strategy for treating male infertility in the near future.
This study has several limitations that should be acknowledged. First, the sample size was relatively small, which necessitates future studies with larger populations to validate these findings. Additionally, further analyses, such as those examining mitochondrial function and ATP production, are essential to investigate the potential links between autophagy and sperm quality. Another important aspect that needs exploration is the long-term effects of using GM-CSF on the outcomes of ART. Conducting such studies would provide a more comprehensive understanding of the mechanisms underlying GM-CSF and their possible implications for clinical practice.
One of the primary limitations of GM-CSF therapy in ART is the lack of substantial data concerning its availability, efficacy, and long-term safety for offspring. Addressing these issues is crucial for improving the clinical application of GM-CSF in infertility treatments.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Author contributions
Conceptualization: TG, MHH, MGN, ZSM, MK, LG, HN. Methodology: TG, MHH, MGN, ZSM, MK, LG, PR, HN. Formal analysis: TG, LG, HN. Data curation: TG, MGN, ZSM, MK, LG, BK, HN. Software: TGH, LG, HN. Writing-original draft: TG, MGN, ZSM, HN. Writing-review & editing: TG, MHH, MGN, ZSM, MK, LG, PR, HN. Approval of final manuscript: HN.