A convenient research strategy for functional verification of epigenetic regulators during spermatogenesis

Abstract

Spermatogenesis is a fundamental process that requires a tightly controlled epigenetic event in spermatogonial stem cells (SSCs). The mechanisms underlying the transition from SSCs to sperm are largely unknown. Most studies utilize gene knockout mice to explain the mechanisms. However, the production of genetically engineered mice is costly and time-consuming. In this study, we presented a convenient research strategy using an RNA interference (RNAi) and testicular transplantation approach. Histone H3 lysine 9 (H3K9) methylation was dynamically regulated during spermatogenesis. As Jumonji domain-containing protein 1A (JMJD1A) and Jumonji domain-containing protein 2C (JMJD2C) demethylases catalyze histone H3 lysine 9 dimethylation (H3K9me2), we firstly analyzed the expression profile of the two demethylases and then investigated their function. Using the convenient research strategy, we showed that normal spermatogenesis is disrupted due to the downregulated expression of both demethylases. These results suggest that this strategy might be a simple and alternative approach for analyzing spermatogenesis relative to the gene knockout mice strategy.

INTRODUCTION

Male reproductive function in mammals is dependent on normal spermatogenesis within the testicle sperm cell epithelium. Spermatogenesis involves a series of tightly controlled events in germ cells, from spermatogonia to sperm. Sperms are continuously produced since puberty due to the self-renewal division of spermatogonial stem cells (SSCs) and subsequent spermatogonia differentiation. Differentiated spermatogonia proliferate and transform into primary spermatocytes, which subsequently undergo meiosis.1,2 After the second meiosis, round spermatids are produced. Subsequently, these undergo significant morphological and molecular changes (spermiogenesis), forming elongated spermatids. Therefore, disruption of SSC self-renewal can cause spermatogenesis deficiency and, consequently, male infertility. This could be observed in a mouse model, where the transplantation of donor germline progenitor cells into a recipient germ cell-depleted testis could restore the spermatogenic cycle.3 In humans, this process remains incompletely understood; therefore, a comprehensive understanding of the mechanistic basis of SSC self-renewal requires additional investigation.

To date, several SSC self-renewal factors have been identified, including SRY-box transcription factor 2 (Sox2), octamer-binding transcription factor 4 (Oct4), and Nanog homeobox (Nanog).4 Spermatogenesis is strictly controlled through the systematic regulation of gene expression. Additionally, epigenetic regulation of orchestrating proper gene expression is the key mechanism governing spermatogenesis.5 Epigenetic regulation without alterations in the original DNA sequence contributes to the formation and maintenance of cell type-specific gene expression profiles. Dynamic epigenetic modification of histones is reportedly crucial for spermatogenesis, including gene expression regulation and sperm chromatin condensation.6 The methylation modification of lysine residues within histone tails is one of the epigenetic regulation events involved in these processes. Among them, H3K9 methylation patterns appear to be important and indispensable epigenetic modifications during different stages of spermatogenesis. It has been shown that the mono-, di-, and trimethylation levels of histone H3 lysine 9 (H3K9; H3K9me1/2/3) are modifiable and undergo dynamic restructuring during spermatogenesis.7,8 Consistent with these observations, some histone-modifying trimethyltransferases and dimethyltransferases exhibit consistent and specific expression in male germ cells. The histone H3K9 demethylase JMJD1A, which is preferentially expressed in the testis, is the key epigenetic regulator of spermatogenesis. Methyltransferase G9a and demethylase JMJD1A work together to maintain a H3K9 methylation balance.9,10 This equilibrium is critical for the regulation of spermatogenesis-specific gene expression, including promyelocytic leukemia zinc finger (Plzf), LIM homeobox 1 (Lhx1), and B-cell lymphoma 6 (Bcl6b). Furthermore, our assay displayed that transcript levels of Jmjd1a were elevated in the testes. Additional observations demonstrated that the methyltransferase, euchromatic histone lysine methyltransferase 2 (EHMT2), catalyzes H3K9me2/1 prior to the onset of meiosis. This process was shown to be essential for the inhibition of meiotic entrance, as evidenced by the observations in Ehmt2-deficient mice. Moreover, knockout mice of the H3K9 demethylase Jumonji domain-containing protein 1C (JMJD1C) cause age-dependent infertility11 due to the gradual decrease of germ cells after 3 months of age. These examples highlight the importance of a deep understanding of the fundamental role of histone modifications, given epigenetic mechanisms in spermatogenesis.

Although some studies revealed that appropriate histone modifications are crucial for germ cell development and sustainable spermatogenesis using gene knockout mice models,12,13,14 many laboratory conditions are limited regarding the construction of genetically engineered mice. Fortunately, SSC culture and transplantation technologies are easier to handle by most laboratories. These technologies have benefited multiple functional studies and promoted animal transgenesis. In this study, we presented a novel strategy to investigate spermatogenesis by lentivirus injection independent of gene-edited mice. Through this approach, we observed that JMJD1A is responsible for H3K9me2 hypomethylation, consistent with previous studies on mice.15,16 Additionally, we revealed that lower expression of JMJD1A together with JMJD2C resulted in the loss of male germ cells, including spermatocytes and spermatogonia. These results indicate that this novel strategy is feasible, simpler, and user-friendly, thereby facilitating the investigation of spermatogenesis regulatory mechanisms.

MATERIALS AND METHODS

Antibodies and animals

In this study, primary antibodies used were as follows: mouse anti-JMJD1A (demethylase; 12835; 1:500; Proteintech, Chicago, IL, USA), mouse anti-H3K9me2 (Ab1220; 1:500; Abcam, Cambridge, UK), and mouse anti-Thy-1 (small transmembrane glycoprotein; MA5-16671; 1:500; Invitrogen, Carlsbad, CA, USA). Secondary antibodies included Alexa 488-conjugated anti-mouse immunoglobulin G (IgG; A11001; 1:1000; Invitrogen) and Alexa 594-conjugated anti-mouse IgG (A11012; 1:1000; Invitrogen) for immunofluorescence. Mice used in this study had a C57BL/6J strain background. Mice were housed in a special animal room with 12-h light and 12-h dark cycle. They were provided with voluntary food and water. The day of birth was defined as day 0. All treatments were performed following the Animal Ethical Guidelines established by the Shandong University of Technology (Zibo, China; Approval No. SDUT20220520).

Lentivirus packaging

The designed small hairpin RNA (shRNA) targeting the Jmjd1a and Jmjd2c genes and the control shRNA were cloned into pCDH-U6-MCSEF1-green fluorescent protein (GFP)-puromycin. The lentiviral shuttle vector contains promoter of driving GFP expression. The lentiviral skeleton vectors pGag/Pol and pRev together with pVSV-G pCDH-U6-JMJD1A/JMJD2C/NC-shRNA plasmids were transfected into human embryonic kidney (HEK) 293T cells for packaging lentivirus based on described protocols.17

Spermatogonial stem cell isolation, culture, and transplantation

Mice SSC cultures were performed as described previously.18 Briefly, testicular tissues were stripped from 7-day-old mice (specify the stage of spermatogenesis), decapsulated, and then digested with collagenase B (2 mg ml⁻¹; Gibco, New York, NY, USA) and trypsin-ethylenediaminetetraacetic acid (EDTA; 0.25%; Gibco). Then, 20 ml of cell suspension was washed twice with phosphate-buffered saline (PBS), and the pellet (centrifugation conditions: 500g, 5 min; cence L600-A; Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China) was resuspended in sterile 20 ml of PBS with deoxyribonuclease (DNase; 50 mg ml⁻¹; Gibco). After washing (centrifugation conditions: 500g, 5 min) with 20 ml of PBS, 10 μl of the cell suspension was added to a magnetic-activated cell sorting (MACS) machine to sort SSCs using the Thy-1 marker, according to the published protocols.19 The sorted SSCs were cultured on the mouse embryonic fibroblast cell line STO feeder cells (ATCC 56-X) in serum-free medium (S0192; Sigma, St. Louis, MO, USA) supplemented with the self-renewal promoting factor glial cell line-derived neurotrophic factor (GDNF; SRP3230; Sigma). The culture medium was changed every 2 days. Lentivirus vectors (LVs) transduced SSC when the cell density reached 70%–80% to ensure high cell viability after transduction. Transduced positive cells were screened using puromycin (E607054; Sangon Biotech, Shanghai, China) for resistance of the transduction vector and then harvested with trypsin-EDTA (0.25%; Gibco). Isolated SSCs were then transplnted into recipient mice testes through efferent duct microinjection using a stereo microscope. Recipient mice were prepared as follows: male recipient mice aged 6 weeks old (specify the stage of spermatogenesis) were injected intraperitoneally with about 40 mg kg⁻¹ body weight busulfan (B2635; Sigma) for 2 months before transplantation. Based on the described protocols,20 10 μl of 1 × 10⁶ cells per ml was resuspended in PBS with trypan blue (0.04%) and transplanted into one of the testes. The other testicle received a control SSC, which was positively transduced by lentivirus expressing an empty vector. The testes underwent histology examination after 2 months of transplantation.

Histological examination and immunohistochemistry

Paraffin-embedded testicular tissue was fixed in immunostaining fixing solution (P0098; Beyotime, Shanghai, China) and cut into a thickness of 5 μm. For histological analysis, sections were stained with hematoxylin–eosin according to the published protocols.21 For immunohistochemistry, the sections were deparaffinized, hydrated, and then incubated in sodium citrate buffer (E673001; Sangon Biotech, Shanghai, China) for antigen retrieval. Next, the sections were treated with methanol (catalog No. 67-56-1) containing 3% H₂O₂ (catalog No. 7722-84-1) to quench endogenous peroxidase. Subsequently, the sections were incubated with PBS containing 2% skim milk (1.15363; Sigma), 10% goat serum (NS02L; Sigma), and 0.1% Triton X-100 (A110694; Sangon Biotech). Finally, the sections were stained with primary antibodies against JMJD1A or H3K9me2. For fluorescent staining, after incubation with primary JMJD1A or H3K9me2 antibodies, the expression status of JMJD1A and H3K9me2 was visualized using Alexa 488-conjugated (A-11001; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 568-conjugated (A-11004; Thermo Fisher Scientific) secondary antibodies, respectively. For each treatment, three sections sealed in glycerol (G5516; Sigma) were analyzed through microscopy (Leica SP8 confocal microscope, Wetzler, Hesse, Germany). Related quantitative numbers were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) in an image-threshold manner. Abnormal seminiferous tubule phenotypes were defined as follows: the appearance of voided areas in sections of the seminiferous tubules, Sertoli cell or other germ cells damage, and shortened seminiferous tubule diameter.

Semiquantitative real-time polymerase chain reaction (RT-PCR) and real-time quantitative PCR

Total RNA from the samples was extracted using Trizol reagent (B610409; Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Reverse transcribed complementary DNA (cDNA) was synthesized with a cDNA synthesis kit (D7170L; Beyotime) following the manufacturer’s instructions. For semiquantitative RT-PCR, the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene served as an internal control and was amplified with the following primers: forward, 5’-GTTCTTTGCTGACCTGCTGGA-3’; and reverse, 5’-GGCCACAGGACTAGAACACC-3’. SYBR premix Ex Taq II (Takara, Dalian, China) was used for real-time quantitative PCR with designed primers. The total volume for each PCR mixture was 10 μl (5 μl of 2× SYBR Green Master mix, 0.5 μl of cDNA, 0.25 μl of each primer at 10 μmol l⁻¹, and 4.0 μl of double-distilled water [ddH₂O]). The PCR cycle included initial denaturation, cycles of denaturation, annealing, and final extension (40 cycles). Each sample was assayed in three technical replicates. Relative mRNA concentrations were normalized using the endogenous levels of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) with the following primers: forward, 5’-CATCTTCTTGTGCAGTGCCA-3’; and reverse, 5’-CGTTGATGGCAACAATCTCC-3’. The primer pair for Jmjd1a expression analysis was: forward, 5’-AGCCAATTCTCCACCTAACA-3’; and reverse, 5’-TGACACCTGCTTTCACTTCT-3’; and for Jmjd2c expression analysis was: forward, 5’-AAGCCAAGACAGTGCTATGA-3’; and reverse, 5’-CTGGATGTTGTATTGCGTGA-3’.

Statistical analyses

Ten C57BL/6J strain background mice were analyzed in control or knockdown group. For the histological analysis, 15 seminiferous tubules were evaluated. Data were presented as mean and standard deviation. Statistical significance between the knockdown groups and control groups was determined by Student’s t-test. P < 0.05 was considered statistically significant. Statistical analysis was performed using the GraphPad Prism 9.0 program (GraphPad Software Company, San Diego, CA, USA).

RESULTS

Description of the convenient research strategy

We developed a research strategy to evaluate the function of a gene using an RNA interference (RNAi) and testicular transplantation approach. A brief description is as follows. Testicular tissues from 5-day-old to 10-day-old mouse were enzymatically digested to obtain single-cell suspension (Figure 1a and 1b). After differential plating several times, the preliminary sorted testicular cells were placed on STO (mouse embryo fibroblasts) feeders in a serum-free medium supplemented with self-renewal promoting factor GDNF for about a week. Following that, MACS assays were performed as described previously22 to further sort SSCs for a donor cell population.

Figure 1.

A schematic representation of the research strategy involved testicular tissue digestion, spermatogonial stem cell sorting, lentiviral-vector transduction, and cell transplantation. (a) Schematic diagram of spermatogonial stem cells sorting by MACS. (b) Bright-field images of testicular tissue digestion. SSCs were harvested from thirty pairs of testes and then cultured to approximately 90% density for lentiviral transduction. (c) Schematic diagram of lentiviral vector transduction and cell transplantation. (d) Immunostaining for Thy-1 (spermatogonial stem cell marker) in MACS sorted cells. Hoechst 33342 stained nuclei (blue; middle), anti-Thy-1 antibody (red; left), and a merged image (right) are shown. (e) Representative microscopic observation images of transplanted spermatogonial stem cells for recipient mice. The lentiviral vector of gene expression is the GFP tag-fused vector. Scale bars = 50 μm. GFP: green fluorescent protein; MACS: magnet-activated cell sorting; SSCs: spermatogonial stem cells; Thy-1: thymus cell antigen 1.

To confirm the regulatory function of SSCs through a gene, a plasmid interfering with the gene was constructed and packaged into a lentivirus using HEK 293T cells (Figure 1c). As Thy-1 has the functional properties of enriching germ stem cells,23 the spermatogonia-specific marker Thy-1 was used to isolate SSC by MACS (Figure 1d). Thy-1⁺ undifferentiated spermatogonia represent the SSCs used for transplantation. Successfully transduced positive SSC cells were screened by puromycin, which can induce death in nonpositive cells, and were washed away by changing the medium (Figure 1e). Subsequently, positive SSC cells were injected into the testes of germ cell-depleted mice (Figure 1c) through efferent duct injection to determine the role of the studied gene in the spermatogenesis process. As spermatogenesis develops from SSC for 2 months, it was evaluated 8 weeks after transplantation.

Jmjd1a and Jmjd2c genes as research objects to test the feasibility of the research strategy

To determine tissue expression distribution of the H3K9me2 demethylase JMJD1A, the testes, kidney, spleen, lung, heart, and liver tissues were isolated from the same mice for mRNA analysis. Semiquantitative RT-PCR analysis was first performed using RNA from multiple adult control mice tissues. Jmjd1a transcripts showed elevated levels in the testes, which decreased in the other tissues (Figure 2a and 2b). Similar results were observed in quantitative PCR experiments, which showed the exact expression of JMJD1A (Figure 2c). The expression of Jmjd1a started at postnatal day-14 (pachytene primary spermatocytes) and continued into adulthood in testis (Figure 2d), suggesting that JMJD1A is involved since middle (spermatocyte stage) spermatogenesis. To define the precise site of expression of the demethylase JMJD1A, immunohistochemistry was performed. However, in contrast to JMJD1A, tissue mRNA expression was comparatively lower in the liver and heart tissues and higher in the lung and spleen, as shown by semiquantitative (Figure 2e and 2f) and quantitative PCR experiments (Figure 2g). As for JMJD1A, the expression levels of Jmjd2c were highest at postnatal day-14 in the testis and continued into adulthood (Figure 2h), indicating a potential involvement since middle spermatogenesis. The results indicated that JMJD1A was expressed since day-1, where seminiferous tubules contain only spermatogonia (Figure 2i). To evaluate if the levels of H3K9me2 changed concomitantly with the expression of JMJD1A, immunohistochemical analyses were performed in testicular tissue sections at postnatal day-1, postnatal day-7, postnatal day-14, and adult mice. Sections were stained with antibodies against JMJD1A and H3K9me2. H3K9me2 signals were abundantly detected at postnatal day-1 and postnatal day-7, decreasing at postnatal day-14 and in adult tissue (Figure 2i and 2j). However, mRNA expression of the demethylase has only been high since day-14, while H3K9me2 decreased at the same stage. These results suggested that JMJD1A specifically demethylates H3K9me2 and regulates H3K9me2 hypomethylation since the pachytene primary spermatocyte stage. Therefore, the Jmjd1a gene was selected to analyze the feasibility of the proposed new research strategy.

Figure 2.

Spatiotemporal expression of JMJD1A and JMJD2C. (a) Semiquantitative RT-PCR examined transcripts of Jmjd1a in multiple tissue samples of adult mice. Hprt was used as an internal control. (b) Relative intensities of Jmjd1a expression in the indicated tissues of adult mice. Intensities were measured using ImageJ software. Relative intensity values were calculated by dividing the intensities of the Hprt gene. (c) Quantitative mRNA analysis for Jmjd1a in different tissues of adult mice. (d) Semiquantitative analysis for Jmjd1a mRNA during postnatal development of the testis. (e) Semiquantitative RT-PCR examined transcripts of Jmjd2c in multiple tissue samples of adult mice. Hprt was used as an internal control. (f) Relative intensities of Jmjd2c expression in the indicated tissues of adult mice. Intensities were measured using ImageJ software. Relative intensity values were calculated by dividing the intensities of the Hprt gene. (g) Quantitative mRNA analysis for Jmjd2c in different tissues of adult mice. (h) Semiquantitative analysis for Jmjd2c mRNA during postnatal development of the testis. (i) Quantitative mRNA analysis for Jmjd1a during postnatal development of the testis. (j) Quantitative mRNA analysis for Jmjd2c at the indicated time points. (k) GC-1 cells were transfected with a GFP tag fused, indicating a lentiviral vector. At 24 h, cells were observed by fluorescence imaging. DAPI stained nuclei (blue; right), GFP tag fused vector (green; middle), and a merged image (left) are shown. Scale bar = 10 μm. (l) mRNA analysis derived from Jmjd1a or Jmjd2c knock down-treated GC-1 cells. Data are displayed as mean ± standard deviation and were analyzed by unpaired Student’s t-test. ^**P < 0.01, indicates significant differences compared with control. Jmjd1a: Jumonji domain-containing protein 1a; Jmjd2c: Jumonji domain-containing protein 2c; Hprt: hypoxanthine-guanine phosphoribosyltransferase; DAPI: 4’,6-diamidino-2-phenylindole; GFP: green fluorescent protein; Gapdh: glyceraldehyde-3-phosphate dehydrogenase.

The tissue and temporal expression profiles of another H3K9me2 demethylase, JMJD2C, were simultaneously tested. Similar to the dynamics observed in Jmjd1a transcripts, testicular mRNA expression of Jmjd2c was highest in the testes. Therefore, the H3K9me2 demethylase JMJD2C was also selected to analyze the feasibility of the proposed new research strategy. To evaluate which siRNA sequence is effective, the interfering vectors of the two H3K9me2 demethylases were first transfected into GC-1 cell lines for expression analysis (Figure 2k). Then, the vectors with interfering effects were screened out by quantitative RT-PCR experiments (Figure 2l) and were transfected into HEK 293T cells together with skeleton vectors for retroviral production to transduce SSC.

Jmjd1a/Jmjd2c-deficient disturbs the normal process of spermatogenesis

After the interference vector was packaged as lentivirus, the sorted SSC was transduced with the appropriate titer. The successfully transduced positive cells were screened by puromycin (Figure 1e). The transplanted SSCs were adjusted to a density of 1 × 10⁷ ml⁻¹ and then injected into the seminiferous tubules of recipient mice treated with 40 mg kg⁻¹ busulfan to destroy germ cells. Testicular tissues were removed for examination 60 days after transplantation. The histological analyses revealed that the patterns of tubular abnormalities and the seminiferous tubule diameter differed between transplanted si-Jmjd2c-SSC and si-Jmjd1a-SSC. While si-Jmjd2c testes exhibited similar size to control (wild-type) testes, it was observed that reduced expression of Jmjd1a was correlated with smaller testis sizes than wild-type testes. These findings suggest that JMJD1A and JMJD2C have different roles and effects, and Jmjd1a acts as a key gene that regulates the proliferation and differentiation of SSC. Simultaneous deficient expression of two demethylases resulted in a more pronounced effect than the deletion of a single demethylase (Figure 3a and 3b). The results suggested that Jmjd1a and Jmjd2c have a synergistic effect in regulating the fate of SSC.

Figure 3.

JMJD1A/JMJD2C are required for spermatogenesis maintenance. (a) Gross phenotype of Jmjd1a/Jmjd2c-deficient testes and control littermates. Scale bar = 4 mm. (b) Testis weights divided by body weights of knockdown (purple bar) and control littermates (blue bar) at indicated genes (n ≥ 6 mice). (c) Histological analysis. Testes from the indicated experimental treatments are indicated in each figure above. Paraffin-embedded testis sections were stained with hematoxylin and eosin. Testis cross-sections show some abnormal tubules after Jmjd1a knockdown. Scale bars = 100 μm. (d) The histogram represents the proportion of abnormal tubules in each indicated slide. (e) The histogram represents the diameter of tubules in each indicated slide. More than 15 tubular sections were analyzed per sample. Data are presented as mean ± standard deviation and were analyzed by unpaired Student’s t-test. ^***P < 0.001, indicates significant differences compared with control. Jmjd1a: Jumonji domain-containing protein 1a; Jmjd2c: Jumonji domain-containing protein 2c.

To investigate the impact of Jmjd1a and Jmjd2c on spermatogenesis, we performed the histological analysis of the testicular tissue section (Figure 3c) and verified the morphological patterns of the seminiferous tubule. A slight difference was observed between the seminiferous tubule diameter of wild-type testes and the testes of single- or double-silencing demethylases (Figure 3d and 3e). The abnormal seminiferous tubule phenotypes were undetected in controls. Spermatogenesis proceeded normally in Jmjd2c knockdown SSC; however, the populations of spermatids were moderately decreased in Jmjd1a knockdown SSC, indicating that JMJD1A is required for spermiogenesis. These histological observations indicated that these Jmjd1a knockdown SSCs exhibited a reduced supply of undifferentiated spermatogonia, subsequently leading to disturbances in the process of spermatogenesis. Additionally, Jmjd2c and Jmjd1a double knockdown had a significant effect on spermatogenesis. In conclusion, these results suggest that silencing Jmjd1a significantly affected normal spermatogenesis, which is in line with the previous study. Therefore, our research strategy is feasible for evaluating the function of a gene using an RNAi and testicular transplantation approach instead of a gene knockout mice strategy.

DISCUSSION

We proposed a strategy for studying epigenetic regulation of spermatogenesis using successful culturing of enriched SSCs, lentiviral transduction, and transplant technology. Multiple studies have shown that H3K9 methylation levels are dynamically regulated during spermatogenesis. Previous studies have reported several classes of histone demethylases,24,25 particularly proteins that contain a Jumonji C-terminal (JmjC) domain. The various subgroups of JmjC domain-containing protein families harbor distinct histone demethylation activities.26 JMJD1A significantly contributes to the transcriptional regulation of SSCs maintenance genes through H3K9me2 demethylation. Additionally, H3K9me2 binds to the promoter region of the genes to regulate gene transcription and participates in spermatogenesis. The selection of JMJD1A to validate our research strategy was based on its regulatory function during spermiogenesis. Additionally, we performed a functional study of the JMJD1A homolog JMJD2C in spermatogenesis to compare their functional similarities and differences.

The single testicular cell suspension was obtained through the implementation of our methodology, which involved subjecting testicular tissues to physical filtration, enzymatic digestion, and mechanical separation. During the digestion of testicular tissue, all experiments were performed at 4°C. The SSCs were enriched through MACS using molecule marker Thy-1 from the testicular cell suspension. The primary rationale for selecting MACS is that it can sort approximately 1 × 10⁶ desired cell populations in 2 h. Silencing vectors of JMJD1A and JMJD2C were transfected into sorted SSCs using lentivirus because lentivirals can efficiently transduce primary cells with a high integrating efficiency. When transfected SSCs were transplanted, they were maintained at 4°C for high cell viability. Undoubtedly, quickly conducting all experiments is a critical aspect for success. Through the research strategy (Figure 1), we found that decreased expression of jmjd1a together with Jmjd2c or Jmjd1a testes was lower than that in control testes, decreased by 50% and 30%, respectively (Figure 3a and 3b). Moreover, we observed a loss of germ cells (Figure 3c) and an increase in the number of seminiferous tubules exhibiting abnormal spermatogenesis (Figure 3d). JMJD1A can be considered a candidate gene involved in male infertility, consistent with the known function of JMJD1A in spermatogenesis.27,28 Consequently, SSC transduction of LV can study the function of key genes during spermatogenesis. Lentiviral is an important technology that is currently in development for a number of clinical applications requiring the transfer of genetic material.29 LVs have gained significant attraction for clinical applications due to their superior transduction efficiency than retroviral vectors.30

Furthermore, LVs hold low immunogenic characteristics compared to adenoviruses. Advantages of LVs include low pathogenicity, transducing nondividing cells (SSC), and permanently integrating into the host genome over other gene transfer agents, which allow stable and long-term transgene expression. To ensure safety for utilization of the convenient strategy, we recommend safe third-generation LVs by splitting the viral genome into separate plasmids, which reduces the likelihood of recombinant virus production. Additionally, LVs are frequently used in the research setting to alter gene expression through RNAi. The majority of these approaches are still in the early stages of development. Additional research is required to determine the efficacy and safety of LVs (including nonhuman lentiviruses with a lack of pathogenicity31) as a viable platform for delivering these gene editing tools for therapeutic purposes. Innate antiviral host restriction responses in human cells pose a significant barrier to overcoming when LVs are utilized in gene therapy. Additional basic and clinical research is required to improve transduction efficiency and manufacturing. Testicular tissue-specific antiviral restriction will be beneficial for the development of recombinant viral vector variants specifically suitable for translational applications in reproductive medicine. If LVs were utilized in reproductive medicine, continued follow-up of patients who have already received LV-based gene therapies is required to understand the long-term safety and efficacy of LVs.

In the case of genes that are essential for the survival of spermatogonia, it would be both time-consuming and expensive to determine whether these genes play a role in spermatogenesis using a gene knockout mouse model. Our research provides a more convenient approach for comprehensively understanding the role of epigenetic regulation in spermatogonia development. SSCs are the target of transduction due to their capacity for in vitro culture and posttransplant sperm production.32,33 Biochemical features of some histone demethylases have been characterized; however, additional studies are required to investigate their physiological significance in SSC. In addition to the two H3K9me2 demethylases in this study, there are multiple other histone-modifying enzymes in mammals.34 Our research strategy can study them as summarized in Figure 1 because it is inevitable that other histone modifications are also dynamically regulated during spermatogenesis. Furthermore, researchers are recommended to choose other proteins of interest and investigate their physiological significance in regulating spermatogenesis using the strategy. Therefore, our research strategy is a considerable alternative approach for identifying critical genes involved in spermatogenesis. In summary, our work provides a practical and feasible research strategy sketched in Figure 1 for illustrating the complex regulatory and spatial landscape of epigenetic regulation. We hope the research strategy will contribute to the elucidation of the molecule mechanism of spermatogenesis.

CONCLUSIONS

Our experimental design may be viewed as a convenient research strategy to test the function of epigenetic regulators. We indicated that normal spermatogenesis is disrupted due to decreased JMJD1A expression based on the research strategy. Our work provides a practical and feasible research strategy to elucidate the complex regulatory and spatial landscape of epigenetic regulation during spermatogenesis. Therefore, our research strategy contributes to understanding the molecule mechanism of spermatogenesis.

AUTHOR CONTRIBUTIONS

JRL carried out the study design and drafted the manuscript. FTY revised the manuscript and provided the microscope. SL performed the most of the experiments. YY, YZK, and YDG performed a portion of the experiments. LTW, XYZ, SZ, QY, RZ, and JC performed data analysis. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.