The lncRNA Gm8097 is associated with hypospermatogenesis
Article information
Abstract
Objective
To investigate whether long non-coding RNA (lncRNA) Gm8097 (LncGm8097) is associated with male infertility.
Methods
The expression and bilogical role of LncGm8097 were investigated.
Results
LncGm8097 expression was down-regulated in the testis tissues with moderate and severe hypospermatogenesis compared with those with normal spermatogenesis and mild hypospermatogenesis (p<0.05). LncGm8097 down-regulation significantly promoted apoptosis and inhibited proliferation in GC1 and GC2 cells. In addition, LncGm8097 was significantly down-regulated in mouse model of hypospermatogenesis and correlated with cell apoptosis and proliferation. LncGm8097 was located immediately upstream of PRPS2, and correlated with Bcl-2/P53/caspase 6/caspase 9 signal pathway.
Conclusion
LncGm8097 down-regulation correlates with hypospermatogenesis, which may offer new insights into the pathogenesis of male infertility.
Introduction
Infertility is a significant public health issue worldwide [1]. Male factors are the primary cause of infertility, responsible for more than half of all cases [2,3]. Hypospermatogenesis is the most prevalent histopathological pattern observed in infertile men [4,5] and is characterized by reduced sperm production [6,7]. However, the molecular mechanisms underlying hypospermatogenesis remain unclear.
Long non-coding RNAs (lncRNAs) are a class of transcripts exceeding 200 nucleotides in length, distinguished by their lower expression levels, limited conservation, and more tissue-specific expression patterns [8,9]. Genome-wide transcriptome analyses have identified thousands of lncRNAs expressed in the testes across multiple species [10,11]. Furthermore, lncRNAs are involved in mammalian spermatogenesis, influencing cell proliferation, differentiation, and apoptosis [12,13]. For instance, Arun et al. [14] demonstrated that the lncRNA meiotic recombination hot spot locus (Mrhl) plays a crucial role in normal spermatogenesis by modulating the Wnt signaling pathway and SRY-box transcription factor 8 (Sox8) expression. Hu et al. [15] found that the testis-enriched lncRNA AK015322, which is highly expressed in spermatogonial stem cells, promotes their proliferation. Lei et al. [16] showed that the lncRNA H19 regulates the proliferation and apoptosis of male germline stem cells. Additionally, Liang et al. [17] reported that the lncRNA Gm2044, significantly upregulated in nonobstructive azoospermia with spermatogonial arrest, impedes male germ cell development by acting as the host gene for miR-202. However, despite these findings, only a small number of lncRNAs have been functionally characterized, and the relationship between lncRNAs and hypospermatogenesis remains unknown.
Recently, some researchers have hypothesized that an imbalance between cell proliferation and apoptosis may play a role in the development of hypospermatogenesis [18,19]. Consequently, it is crucial to determine the relationship between hypospermatogenesis and the apoptosis and proliferation of spermatogenic cells. In this study, lncRNA Gm8097 (LncGm8097) was identified as a lncRNA located near phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) and was found to positively regulate the expression of PRPS2. PRPS2 has been previously reported to be linked with male infertility and to play an essential role in regulating cell apoptosis [4,20,21]. The current study demonstrated that lncGm8097 expression was reduced in testicular tissues affected by moderate and severe hypospermatogenesis. The downregulation of lncGm8097 significantly increased apoptosis and decreased proliferation in GC1 and GC2 cells, which was associated with the B-cell lymphoma 2 (Bcl-2)/P53/caspase 6/caspase 9 signaling pathway. Therefore, these findings suggest that the downregulation of lncGm8097 influences the apoptosis and proliferation of spermatogenic cells and is related to hypospermatogenesis. This study sheds light on the connection between lncGm8097 and hypospermatogenesis, potentially offering novel insights into the mechanisms underlying male infertility.
Methods
1. Patients and tissue samples
Six patients with normal spermatogenesis and nine with hypospermatogenesis were enrolled in this study. Testicular biopsy tissues were obtained from the First Affiliated Hospital of Jinan University, Nanfang Hospital, and Guangdong Armed Police Hospital from 2010 to 2020. All patients were between 23 and 45 years old and had been unable to conceive after more than 1 year of unprotected intercourse. Patients were diagnosed with azoospermia through repeated semen analysis, with the exclusion of genetic diseases, trauma, and endocrinological defects. The pathological diagnoses were independently confirmed by two pathologists. This study received approval from the ethical committees of the First Affiliated Hospital of Jinan University, Nanfang Hospital, and Guangdong Armed Police Hospital (No. 21319203). All methods were carried out in accordance with the relevant guidelines and regulations. The patients were fully informed about the purpose, risks, and benefits of the study and provided signed consent for the use of their samples in our research.
2. Cell culture and transfection
The mouse-derived spermatogonial cell line (GC1), the spermatocyte cell line (GC2), and the TM4 mouse Sertoli cell line were obtained from the American Type Culture Collection. These cell lines were cultured in Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and maintained at 37 °C in a 5% CO2 incubator. Transfection of cells was carried out using Lipofectamine 3000 (L3000015; Invitrogen) when they reached 70% confluence. Murine lncGm8097-specific shRNA (shlncGm8097), an lncGm8097 overexpressing vector, sh-PRPS2, PRPS2 overexpressing vectors, and a negative control (NC) were synthesized by GeneChem Biomedical Biotechnology. Cells were harvested 72 hours post-transfection and utilized for subsequent experiments. All experimental methods were conducted following the manufacturer's instructions.
3. Animal models
All C57BL/6N male mice were obtained from the Jinan University Animal Center. The study received approval from the university's Institutional Animal Care and Use Committee (No. S3077). We established a mouse model of hypospermatogenesis following the method previously described [20]. Briefly, 10-week-old mice were randomly divided into a NC group (n=6) and an experimental group (n=6). Mice in the experimental group received an intraperitoneal injection of a busulfan and dimethyl sulfoxide (DMSO) solution (30 mg/kg), while those in the control group received an equivalent volume of DMSO alone. Two weeks later, testis tissues were harvested to assess histopathological changes.
4. Immunohistochemical analysis
The procedure was conducted as previously described [20]. In brief, antigen retrieval was performed using 0.01 M sodium citrate buffer (pH 6.0). The sections were then incubated with Ki-67 monoclonal antibody (0.5 mg/mL, dilution 1:100; Bioworld Technology) and caspase 3 polyclonal antibody (0.5 mg/mL, dilution 1:100; Bioworld Technology A) at room temperature for 2 hours. Subsequently, the sections were incubated with a secondary antibody and the signal was developed using diaminobenzidine. Following counterstaining with hematoxylin, staining for Ki-67 and caspase 3 was assessed under optical microscopy at ×40 magnification.
5. Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described [20]. Total RNA was extracted from human testis tissues, mouse testis, and cells using the TRIzol reagent (Takara). The RNA was then reverse-transcribed into cDNA using the Reverse Transcription System (Takara), and RT-PCR was carried out with a 7500-type quantitative fluorescence PCR system (ABI Company). The primer sequences for lncGm8097 in mice were 5’-TAACCTGTGGGACAGCAACA-3’ (reverse) and 5’-GCAGTGGATCGTAGACATGGA-3’ (forward). For lncGm8097 in humans, the sequences were 5’-TGAGGTCCACCACCCTGTTG-3’ (reverse) and 5’-CTGAACGGGAAGCTCACTGG-3’ (forward). The primer sequences for PRPS2 in mice were 5’-ACATCACCCACGAGAACCAT-3’ (reverse) and 5’-ATGCTGGAGGAGCCAAAAG-3’ (forward). For glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in humans, the sequences were 5’-TGAGGTCCACCACCCTGTTG-3’ (reverse) and 5’-CTGAACGGGAAGCTCACTGG-3’ (forward). Relative quantification was normalized to GAPDH levels. The relative expression levels of the target genes were calculated using the 2−ΔΔCt method.
6. Western blot
The procedure was conducted as previously described [20]. Total cellular protein was extracted using radioimmunoprecipitation assay (RIPA) buffer, and protein concentration was determined by the bicinchoninic acid assay (Biyuntian). Thirty micrograms of protein lysate were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4 °C with the following polyclonal antibodies: P53 (0.2 mg/mL, dilution 1:2,000, product No. LBP62662; Immunoway Corp.), PRPS2 (0.1 mg/mL, dilution 1:1,000, product No. PA5-42007; Invitrogen), Bcl-2 (0.2 mg/mL, dilution 1:500, product No. A11025; ABclonal Technology), caspase 6 (0.1 mg/mL, dilution 1:500, product No. BS90188; Bioworld Technology), caspase 9 (0.1 mg/mL, dilution 1:500, product No. BS1731; Bioworld), and GAPDH (0.1 mg/mL, dilution 1:2,000, product No. 92310; Cell Signaling Technology). The membranes were then incubated with a goat anti-rabbit immunoglobulin G-horseradish peroxidase-conjugated secondary antibody at room temperature for 30 minutes and the signal was detected using an enhanced chemiluminescence detection kit (Alpha Innotech). The intensity (gray value) of each band was quantified using ImageJ ver. 1.46 software (National Institutes of Health).
7. Cell apoptosis
The procedure was conducted as previously described [20]. Cells (2×105) were harvested and then resuspended in a binding buffer. Following this, the cells were incubated with propidium iodide and annexin fluorescein isothiocyanate labeled membrane associated protein V (BestBio) for 15 minutes at room temperature. Apoptotic cells were examined by flow cytometry.
8. Cell proliferation assay
Cell proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, transfected cells were seeded into 96-well plates at a density of 2×103 cells per well and incubated at 37 °C. At 0, 1, 2, 3, 4, and 5 days post-seeding, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for 4 hours, followed by the addition of 150 μL of DMSO for 10 minutes at 37 °C. The absorbance of each well was measured at 490 nm using a microplate reader (Bio-Rad). This procedure was repeated in triplicate for each experiment.
9. LncRNA microarray analysis
GC1 cells with PRPS2 overexpression and NCs were collected for lncRNA microarray analysis using the Agilent Mouse Gene Expression Microarray (Agilent Technology). GeneChip hybridization was carried out by KangChen Bio-tech Company, and the data were normalized using Agilent GeneSpring GX v12.1 software. Differentially expressed lncRNAs with statistical significance between the two groups were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis.
10. Dual luciferase reporter gene assay
The wild-type reporter of PRPS2 (PRPS2-WT) and the mutant reporter of PRPS2 (PRPS2-MUT) were amplified using PCR and subsequently cloned into the pGL3 vector (Promega). Following this, PRPS2 plasmids and lncGm8097 vectors were co-transfected into cells utilizing Lipofectamine 3000 (Invitrogen). At 48 hours post-transfection, luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega), following the manufacturer's instructions.
11. Statistical analysis
All data were analyzed using SPSS software ver. 18.0 (SPSS Inc.) and are presented as mean±standard deviation. The independent-samples t test was utilized for comparisons between two groups, while one-way analysis of variance was employed for comparisons among multiple groups. A p-value of less than 0.05 was considered statistically significant.
12. Ethics approval and consent to participate
All experimental animals were used in accordance with the guidelines and standards dictated by the Office of Laboratory Animal Welfare and approved by the Institutional Animal Use and Care Committee, as detailed in the Methods section. This study was approved by the ethical committees of the First Affiliated Hospital of Jinan University, Nanfang Hospital and Guangdong Armed Police Hospital (No.21319203). All patients signed consent and agreed to use the samples for our study.
Results
1. LncGm8097 is a nearby lncRNA associated with PRPS2 and linked to male infertility
An earlier study confirmed that PRPS2 is associated with male infertility [20]. To identify potential lncRNAs related to PRPS2, we performed an Agilent Mouse lncRNAs Expression Microarray in GC1 cells (Figure 1A). We analyzed the relationship between differentially expressed lncRNAs and nearby coding genes. Through this analysis, lncGm8097 emerged as a potential upstream lncRNA of PRPS2 (Figure 1A, 1B). To validate the findings from the microarray analysis, we measured the expression of lncGm8097 in human testis tissues and mouse germ cells using qRT-PCR. Figure 1C shows that lncGm8097 was significantly downregulated in human testis tissues with moderate and severe hypospermatogenesis compared to those with normal or mild hypospermatogenesis. Furthermore, lncGm8097 expression was notably lower in GC2 cells than in GC1 and TM4 cells (Figure 1D). These results suggest that lncGm8097 is a nearby lncRNA associated with PRPS2 and may be involved in hypospermatogenesis.
Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) was identified as a nearby lncRNA of phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) associated with male infertility. (A) An lncRNA expression microarray was performed in GC1 cells with PRPS2 overexpression and controls. (B) The expression of lncGm8097 is shown by a heat map. (C) Quantitative real-time polymerase chain reaction (qRT-PCR) indicated that lncGm8097 was downregulated in human testicular biopsy tissues with hypospermatogenesis. (D) The expression of lncGm8097 was detected in mouse germ cells by qRT-PCR. a)p<0.05 compared with normal spermatogenesis and mild hypospermatogenesis group; b)p<0.01 compared with normal spermatogenesis and mild hypospermatogenesis; c)p<0.05 compared with the GC1 group.
2. LncGm8097 regulates cell apoptosis and proliferation, and is correlated with hypospermatogenesis
To explore the biological role of lncGm8097, we evaluated cell apoptosis and proliferation in spermatogenic cells. We first successfully achieved downregulation and overexpression of lncGm8097 in GC1 and GC2 cells, as shown in Figure 2A. Subsequent analysis revealed that the apoptotic rates in GC1 cells were significantly increased following lncGm8097 downregulation and significantly decreased with lncGm8097 overexpression, compared to normal controls (p<0.01), as depicted in Figure 2B. This pattern was also confirmed in GC2 cells (Figure 2B). Furthermore, downregulation of lncGm8097 significantly inhibited cell proliferation, whereas overexpression of lncGm8097 significantly increased cell proliferation relative to normal controls (p<0.01) (Figure 2C).
Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) was found to regulate cell apoptosis and proliferation. (A) LncGm8097 knockdown and overexpression were validated in GC1 and GC2 cells by quantitative real-time polymerase chain reaction. (B) Flow cytometry assay showing that lncGm8097 downregulation promoted cell apoptosis. (C) 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay showing that lncGm8097 downregulation inhibited proliferation in GC1 and GC2 cells. NC, negative control; PI-A, propidium iodide-apoptosis; FITC-A, fluorescein isothiocyanate-apoptosis. a)p<0.05 compared with the NC group.
To further validate the correlation between lncGm8097 and hypospermatogenesis, we obtained a mouse model exhibiting this condition. Hematoxylin and eosin staining revealed a significant reduction in spermatogenic cells in the hypospermatogenic mice, as depicted in Figure 3A. The qRT-PCR analysis showed that lncGm8097 expression was significantly reduced in the hypospermatogenic mice compared to normal controls (Figure 3B). Additionally, we observed a marked increase in caspase 3 expression and a decrease in Ki-67 expression in the hypospermatogenic mice (Figures 3A, 3C). These findings further support the association between the downregulation of lncGm8097 and hypospermatogenesis, suggesting a potential role in the regulation of cell apoptosis and proliferation.
Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) downregulation was correlated with hypospermatogenesis. (A) Increased apoptosis and proliferation inhibition were observed in testis tissues with hypospermatogenesis. Caspase 3 and Ki-67 is immunohistochemical staining and each panel's magnification is ×40. (B) LncGm8097 downregulation was observed in a mouse model of hypospermatogenesis by quantitative real-time polymerase chain reaction. (C) Statistical analysis of the immune expression levels of caspase 3 and Ki-67 in testis tissues. a)p<0.05 compared with the normal spermatogenesis group; b)p<0.01 compared with the normal spermatogenesis group.
3. LncGm8097 regulates PRPS2 and activates the Bcl-2/P53/caspase 6/caspase 9 signaling pathway
For further validation of the relationship between lncGm8097 and PRPS2, experiments were conducted to knock down and overexpress PRPS2 in GC1 cells. As depicted in Figure 4A, the knockdown of lncGm8097 significantly induced the downregulation of PRPS2, while overexpression of lncGm8097 led to an upregulation of PRPS2. However, altering PRPS2 levels through knockdown or overexpression did not significantly affect the expression level of lncGm8097. Additionally, the luciferase activity of PRPS2 was significantly increased in GC1 cells with lncGm8097 overexpression and significantly decreased in cells with lncGm8097 downregulation, as shown in Figure 4B. These findings further confirmed that lncGm8097 is upstream of PRPS2 and positively regulates its expression.
Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) regulated phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) and was correlated with the B-cell lymphoma 2 (Bcl-2)/P53/caspase 6/caspase 9 signal pathway. (A) LncGm8097 positively regulated PRPS2 by quantitative real-time polymerase chain reaction. (B) The luciferase activity of PRPS2 was significantly higher in cells with lncGm8097 overexpression according to a dual luciferase reporter gene assay. (C) The expression of PRPS2, Bcl-2, P53, caspase 6, and caspase 9 was determined using Western blot assays. (D) Statistical analysis of the expression levels of PRPS2, Bcl-2, P53, caspase 6, and caspase 9. NC, negative control; WT, wild-type; MUT, mutant; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. a)p<0.05 compared with the NC group.
To investigate the potential mechanism of lncGm8097, we measured the expression levels of PRPS2, BCL-2, P53, caspase 6, and caspase 9. As depicted in Figure 4C, 4D, the downregulation of lncGm8097 significantly reduced the expression levels of PRPS2 and BCL-2, while it significantly increased the expression levels of P53, caspase 6, and caspase 9. Conversely, the overexpression of lncGm8097 significantly promoted the upregulation of PRPS2 and BCL-2, but markedly suppressed the expression of P53, caspase 6, and caspase 9.
Discussion
The global incidence of male infertility is reported to be on the rise; however, the underlying mechanisms of male infertility remain unclear [22,23]. Recent studies suggest that lncRNAs are associated with male infertility [15,16]. Although thousands of lncRNAs expressed in the testes have been observed in both mouse and human testes during the stages of spermatogenesis, only a few lncRNAs have been characterized. Therefore, it is essential to further explore the role of lncRNAs in male infertility.
In this study, lncGm8097 was identified as a nearby lncRNA to PRPS2 through lncRNA expression microarray analysis. Previous research reported that PRPS2 is significantly downregulated in testis tissues affected by hypospermatogenesis and is associated with the condition's development by regulating the apoptosis of spermatogenic cells [4,20,21]. Consequently, we examined the expression of lncGm8097 in human testis and mouse germ cells. The results revealed that lncGm8097 is expressed in both human testis and mouse germ cells. However, its expression was significantly reduced in human testis tissues with moderate and severe hypospermatogenesis compared to those with normal or mild hypospermatogenesis. These findings suggest that lncGm8097 may be a novel lncRNA associated with male infertility and could potentially regulate PRPS2 in the development of hypospermatogenesis.
Subsequently, we investigated the biological role of lncGm8097. Downregulation of lncGm8097 significantly promoted cell apoptosis and inhibited cell proliferation in vitro. To further explore the correlation between lncGm8097 and hypospermatogenesis, we created a mouse model of hypospermatogenesis. Results showed that lncGm8097 was significantly downregulated in the testis tissues of this model, consistent with observations in human testis tissues. Additionally, the expression levels of caspase 3 and Ki-67 in the mouse model suggested that hypospermatogenesis might be caused by increased apoptosis and inhibited proliferation. These findings further support the possibility that downregulation of lncGm8097 promotes cell apoptosis and inhibits cell proliferation, contributing to the development of hypospermatogenesis.
To investigate the mechanism of lncGm8097, the relationship between lncGm8097 and PRPS2 was further validated. lncGm8097 positively regulated the expression of PRPS2; however, PRPS2 did not regulate the expression of lncGm8097. Overexpression of lncGm8097 enhanced the luciferase activity of PRPS2. These data confirmed that lncGm8097 is upstream of PRPS2 and that PRPS2 is the adjacent coding gene. This finding is consistent with results obtained from microarray analysis. It is well-established that BCL-2 and P53 play crucial roles in the apoptosis and proliferation of spermatogonial cells [24-27]. Caspases are also involved in the apoptosis of these cells, with caspase 6 being the primary effector caspase that can be activated by the initiator caspase, caspase 9, to induce apoptosis [24,28,29]. Therefore, the expression levels of BCL-2, P53, caspase 6, and caspase 9 were evaluated. The results showed that downregulation of lncGm8097 significantly decreased the expression levels of PRPS2 and BCL-2, while significantly increasing the expression levels of P53, caspase 6, and caspase 9. Consequently, these data suggest that the downregulation of lncGm8097 is associated with the PRPS2/BCL-2/P53/caspase 6/caspase 9 signaling pathways.
These results demonstrate that lncGm8097 is associated with hypospermatogenesis by regulating the apoptosis and proliferation of spermatogenic cells. This suggests that it may be a novel lncRNA associated with male infertility.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Author contributions
Conceptualization: BL, LY, ZQ, SZ. Methodology: BL, LY, ZQ. Formal analysis: LY, ZQ. Data curation: BL, ZQ, SZ. Project administration: BL. Investigation: BL, SZ. Writing-original draft: BL, LY, ZQ. Writing-review & editing: BL, LY, ZQ, SZ. Approval of final manuscript: BL, LY, ZQ.
Acknowledgements
We are grateful for the ethical committees of the First Affiliated Hospital of Jinan University, Nanfang Hospital and Guangdong Armed Police Hospital. We are grateful to all patients for using their samples in our study.