Expression of GLOD4 Gene in the Testis of Qianbei Ma Goats and Its Effect on Testicular Leydig Cells

1. Introduction

In the testis, spermatogenesis begins with the mitotic division of spermatogonia in the seminiferous tubules. Spermatogonia undergoes two meiotic divisions to form spermatocytes, which subsequently metamorphose, continue to develop and mature in the seminiferous tubules, and ultimately undergo a complex series of changes to produce mature spermatozoa. In addition, it is regulated by many highly complex genes[1,2,3,4,5]. Spermatogenesis is a highly complex and organized process that occurs in the testis. Another important biological function of the testis, which is an important male reproductive organ, is androgen secretion. For example, testosterone increases the libido and sexual behavior, improves spermatogenesis and sperm quality in male animals, increases the success rate of fertilization[6], and can induce the testicular tubules to produce more sperm and support sperm maturation and motility[4,7]. In addition, estrogen and other hormones such as luteinizing hormone (LH) and follicle stimulating hormone (FSH) are indispensable throughout spermatogenesis[8,9].

The glyoxalase gene family is an important regulatory family consisting of six structurally and functionally distinct enzymes, including glyoxalase domain-containing protein 4 (GLOD4). GLOD4, also known as HC6-type or HC71-type, was originally cloned as the C17orf25 cDNA from human hepatocellular carcinoma[10,11]. GLO1 is an ancient analog of GLOD4 located at Chr 17p13.3 and consists of 13 exons[12]. The human GLOD4 gene region is approximately 23 kb long and contains 10 exons and 9 introns[13]. The full-length cDNA sequence of human GLOD4 is 1814 bp long and encodes an open reading frame (ORF) consisting of 313 amino acidsTreatment of mouse embryonic stem cells with cyclopiroxamine (CPX) results in the altered expression of multiple proteins, whereas downregulation of GLOD4 protein expression may inhibit cell proliferation and block the cell cycle[14]. In a canine model of retinal degeneration caused by mutations in the RPGRORF15 gene, researchers analyzed the expression profiles of mutant and normal retinas. In conjunction with other genes, GLOD4 was found to have an effect on cell-cycle progression and to potentially influence processes related to mitochondrial function and cellular energy metabolism[15]. In Blmh (Bleomycin Hydrolase) -/-mice, GLOD4 isoform 3 mRNA expression was decreased in the kidney but no effect on isoform 1 mRNA expression was observed. This suggests that GLOD4 protein expression may be subjected to Blmh+/+-mediated posttranscriptional regulation, which, in turn, has a detoxification effect[16]. Using the cryptic nematode Caenorhabditis elegans (C. elegans) as a model to explore the role of the TRPA1-Nrf signaling pathway in methylglyoxal detoxification revealed that it regulates the expression of GLOD4. This study also emphasized the potential role of GLOD4 in human diseases, especially in pathologies associated with α-diketone bodies (α-DCs) accumulation, such as diabetes and neurodegenerative diseases[17]. However, current GLOD4 research still focuses mainly on humans and rodents, with few reports on goats.

Testicular leydig cells are in the interstitial tissue of the testes and play an important role in the male reproductive system. One of the main functions of leydig cells is to secrete testosterone, a hormone that stimulates sperm production and development within the testes[18,19]. Since the peak of testosterone secretion mainly occurs during the reproductive and youthful stages of the animal, testosterone secretion also has an indispensable role in muscle protein synthesis, calcium uptake and bone density maintenance during these developmental stages. In addition to testosterone secretion, leydig cells also regulate the proper testis temperature and the Proliferation and function of different cell types within the testis, thus ensuring normal spermatogenesis[20].

As a domesticated animal with important economic value, the economic impact of goats can be increased by improving the fertility of breeding rams and the efficiency of goat breeding[21]. Recent studies have revealed that the GLOD4 gene is differentially expressed in leydig cells of Tibetan sheep at different developmental stages[22]. However, the mechanism by which GLOD4 gene expression affects leydig cells in goats has rarely been reported, especially within the context of testicular development. As a goat breed that is well adapted to the highland mountain environment, Qianbei Ma goats have a long history of breeding in Guizhouwith a medium body size, rough feeding resistance, strong disease resistance, good fur quality and strong fertility. Thus, Qianbei Ma goats are a good model animal for exploring the reproductive traits of plateau goats[23]. Therefore, the aim of this study was to investigate the profile of GLOD4 gene expression in the testes of goats at different ages and to analyze the regulatory effect of the GLOD4 gene on testicular leydig cells. We first silenced and overexpressed the GLOD4 gene in the testicular leydig cells of goats and investigated the effects of the GLOD4 gene on the cells including on the cell cycle and testosterone hormone secretion. By analyzing the effects of the GLOD4 gene expression on testicular leydig cells, we established a foundation for understanding the potential regulatory mechanism through which the GLOD4 gene functions in spermatogenesis and provided a theoretical basis for improving the reproductive traits of breeding rams.

2. Materials and Methods

The experimental protocol used in this study has been approved by the Animal Protection and Use Committee of Guizhou University, Guiyang, China (Approval number: EAE-GZU-2021-E021). The animal handling procedures were in line with the Chinese Animal Welfare Guidelines and were approved by the Animal Protection and Use Committee of Guizhou University, Guiyang, China (Approval number: EAE-GZU-2021-E021).

2.1. Tissue Collection

Experiment animals and sampling A total of 20 healthywere randomly selected from Xishui County, Guizhou Province, China, Fuxing Dairy Company Limited, and divided into four age groups (n = 5 for each age group): 0Y, 0 month old (before sexual maturity, body weight: 1.66 kg ± 0.5 kg); 6M, 6 months (after sexual maturity, body weight: 30.30 kg ± 1.0 kg); 12M, 12 months old (after physical maturity, body weight: 43.32 kg ± 1.5 kg); 18M, 18 months (sell on the market,body weight:47.53 kg±2.0 kg). The left testicular tissue was excised immediately after anesthesia, collected with sterile scissors, trimmed to approximately 5 mm testicular tissue, and washed in phosphate-buffered saline solution within 20 min, with three biological replicates per age. After clipping the testicles of the goats, the wounds were sterilized, and the goats were continued with subsequent feeding. After the experiment, the animals survived normally. One part of the sample was fixed in 4% paraformaldehyde solution for H&E staining, and the remainder was rapidly frozen in liquid nitrogen and subsequently transferred to 80C for storage in lab for other analyses such as extraction of total RNA and total protein.

2.2. Cell Culture and Transfection

Fine leydig cells was obtained from 12-month-old goat testes and cultured as previously described[24]. Briefly, testicular tissue from goats was immediately transferred to Dulbecco’s phosphate-buffered saline (DPBS) supplemented with 5% penicillin/streptomycin (Gibco, Grand Island, NY, USA) and incubated at 4°C. The white membrane on the surface of the testis was removed with eye clippers and then digested with 0.5 mg/mL collagenase IV (Sigma, St. Louis, MO, USA) in DMEM/F12 (Gibco, Grand Island, NY, USA) at 37°C for 20 min. The dispersed cells were then filtered using a 100 mm cell strainer (Biosharp, Shanghai, China), and the filtrate was centrifuged at 1200 × g for 5 min. The supernatant was discarded, and the cells were washed three times in DPBS and centrifuged at 1200 × g for 5 min and resuspended. The resuspended cells were purified by the differential applanation method. The harvested leydig cells (LCs) were cultured in DMEM/F12 containing 10% fetal bovine serum (Gibco, China, Beijing, China) and 1% penicillin/streptomycin and incubated in a cell culture incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2. Once the confluence reached 75-90%, the cells were evenly divided between 6 empty plates (NEST Biotechnology Co., Ltd., Wuxi, China). These cells were then transfected with GLOD4 overexpression and silencing plasmids using a Lipofectamine 2000 kit (Thermo Fisher Scientific Waltham, MA, USA). After 48 h, RNA luminescence was assessed as good and the cells were harvested to conduct all other experiments.

2.3. Pasmid Construction

After PCR amplification, amplicons were subjected to gel purification to obtain the desired target sequences, which were then ligated to pcDNA3.1 using T4 DNA ligase. Plasmids were amplified in transformed DH5α competent cells. Then, the desired plasmids were extracted from the cells by oscillation. The PCR reaction (25 µL) consisted of 1 µL of DNA template, 1 µL of each of the upstream and downstream primers (10 pmol/µL), 12.5 µL of Green Mix, and 9.5 µL of ddH2O. shRNA-GLOD4 interfering sequences were designed and synthesized by Shanghai Gemma Pharmaceuticals & Biologicals (see Table 1 for details).

2.4. Total RNA Was Extracted and Reverse-Transcribed

Total RNA was extracted from goat testicular tissue and testicular leydig cells using TRIzol reagent (Solarbio, Beijing, China). Subsequently, first-strand cDNA was performed using the StarScript II First-Strand cDNA Kit (GenStar, Beijing, China) according to the manufacturer’s instructions, and the reaction products were stored at -20°C.

2.5. Real-Time Polymerase Chain Reaction

The expression levels of the GLOD4 gene, and that of other genes, was measured in goat leydig cells on a CFX 9600 real-time PCR instrument (Bio-Rad, Hercules, CA, USA). The details of the real-time PCR primers used are summarized in Table 2. A 10 µL real-time PCR mixture (containing 5 µL of 2× Es-Taq Master mix, 3.2 µL of RNase-free ddH2O, 0.5 µL of cDNA, and 0.4 µL of forward and reverse primers (10 pmol/µL)) was prepared according to the manufacturer’s instructions and subjected to a three-step PCR program (95°C for 2 min, 95°C for 15 s, annealing temperature (see Table 2 for details) for 30 s, and 72°C for 30 s; the melting curve was generated automatically by the machine). The PCR assay was repeated three times for each test sample. The expression data were normalized to the expression of β-actin, which we used as an internal reference. The relative change in gene expression was then calculated using the relative quantification method 2-∆∆CT. β-actin primers were synthesized by Bioengineering (Shanghai) Co. (see Table 2 for details).

2.6. Western-Blot

Testicular tissues from different developmental stages were homogenized and lysed using a radioimmunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China) with the addition of phenylmethylsulfonyl fluoride (PMSF) (Solarbio, Beijing, China). Protein concentrations were quantified using a commercial bicinchoninic acid (BCA) protein assay (Solarbio, Beijing, China). Protein samples were separated on a 10% SDS‒PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane by wet transfer. After being blocked in 5% skim milk for 2 h, the membranes were incubated overnight with rabbit anti-GLOD4 polyclonal antibody (1:1000 dilution, Bioss, Beijing, China) at 4°C. The membranes were then washed three times in Tris-buffered saline containing Tween-20 (TBST), incubated with an HRP-conjugated secondary antibody (1:10,000 dilution) for 1 hr in TBST, washed again, and then incubated with an HRP-conjugated secondary antibody (1:10000 dilution, Bioss, Beijing, China) and visualized using an NcmECL Ultra kit (P10200, NCM, Suzhou, China) on a Universal Hood II instrument (Bio-Rad, CA, USA). The grayscale value of each band was analyzed using ImageJ software.

2.7. Immunohistochemistry

For immunohistochemistry, after the prepared paraffin sections of testis were dewaxed in water, they were boiled in 0.1 M citrate buffer (pH = 6.0) for 15 min in a microwave oven and then cooled to 37°C for antigen retrieval. The sections were subsequently washed with phosphate-buffered saline (PBS) and incubated in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. The sections were further incubated in blocking buffer (5% bovine serum albumin (BSA) in PBS) for 15 min at 37°C to block nonspecific binding. The membranes were then incubated with rabbit anti-GLOD4 antibody (1:1000; Bioss Biotechnology Co., Ltd., Beijing, China) in PBS overnight, washed with PBS, incubated with the biotinylated secondary antibody for 2 h at 37°C and with horseradish peroxidase (HRP)-streptavidin for 15 min, and then visualized using diaminobenzidine. Nonspecific rabbit immunoglobulin G (IgG) was used instead of the primary antibody as a negative control.

2.8. Subcellular Localization

For transfection, cell lysates were added before the cells were inoculated into six-well plates, and after 36 h of transfection, the cells were washed 3 times with PBS and fixed at room temperature for 20 min by the addition of precooled 4% paraformaldehyde (Solarbio, Shanghai, China); the cells were washed 3 times with PBS and fixed at room temperature for 5 min by the addition of 0.25% Triton X-100 (Biosharp Anhui, China); to conduct all other experiments, and the nuclei were stained with DAPI (Solarbio, Shanghai, China) to stain the cells; and the cells were washed 3 times with PBS. The fluorescence images were merged using Photoshop, and the subcellular localization of the fusion protein was assessed based on the overlap between the fluorescence of the fusion protein and the fluorescence of the cell nucleus.

2.9. Cell Proliferation Analysis

A CCK-8 assay was used to detect the effect of GLOD4 gene overexpression and silencing on the proliferative activity of leydig cells. Plasmids from the negative control and experimental groups were incubated at 37°C and 5% CO2 for 6, 12, 24, 48 and 72 h after transfection, as described in the instructions of the CCK-8 assay kit (Monmouth Junction, NJ, USA). The absorbance (OD) at 450 nm was subsequently measured using enzyme markers (Thermo Fisher Scientific, Waltham, MA, USA).

2.10. Steroid Assay

The culture medium was collected to measure the testosterone concentration using an ELISA kit (Kamels, Shanghai, China) according to the manufacturer’s instructions[25]; the absorbance of the samples was measured at 450 nm, and the concentration of testosterone was determined using a standard curve.

2.11. Flow Cytometry Analysis

Harvested cells were fixed in 70% ice-cold ethanol overnight at 20°C. After washing with PBS, the cells were incubated in 0.5 mg/mL RNase for 30 min at 35°C and stained with 0.025 mg/mL propidium iodide for 10 min. Finally, cells at different stages of the cell cycle were identified by flow cytometry analysis (BD Biosciences, Canto II plus, USA). The data from at least 20,000 cells were collected per sample.

2.12. Statistics

Statistical analyses were performed using Graphpad prism10. All trials were repeated at least three times, and the data were presented as means ± SD. Comparisons between two independent groups were determined by t-test; meanwhile, the differences among multiple groups were analyzed by one-way ANOVA or Two-way ANOVA. (significant, P < 0.05; highly significant, P < 0.01).

3. Results

3.1. Localisation of GLOD4 in the Testis of Qianbei Ma Goats

We detected GLOD4-positive protein signals by immunohistochemistry in testicular tissue sections from 12-month-old rams and found that the immunopositivity was confined to testicular leydig cells (Figure 1A). After transfection of the pcDNA3.1-NC vector containing GFP fluorescence, the subcellular localization results showed that GLOD4 was highly expressed in the nucleus and cytoplasm of leydig cells. (Figure 1B).

3.2. Expression Patterns of the GLOD4 Transcript at Different Developmental Stages of Qianbei Ma Goats Testes

Both the Western blot and qRT‒PCR showed that GLOD4 was expressed in the testes of Qianbei Ma goats at 0, 6, 12 and 18 months of age, and GLOD4 expression increased with age. GLOD4 protein expression was significantly higher at 18 months of age than at 0 months of age (p<0.01) and that at both 6 and 12 months of age (p<0.05). The GLOD4 mRNA expression was significantly higher at 6, 12 and 18 months of age than at 0 months of age (p<0.01; Figure 2A,B).

3.3. GLOD4 Promotes Qianbei Ma Goat LCs Proliferation In Vitro

To investigate how GLOD4 affects leydig cells, we transfected cells with GLOD4 silencing and overexpression vectors and then conducted mRNA and protein assays. After GLOD4 silencing and, both the mRNA and protein levels of GLOD4 were significantly downregulated and upregulated (p<0.01 each), respectively, compared to those in the control group (sh-NC, pcDNA3.1-NC) (Figure 2C–F). Furthermore, we performed a CCK-8 assay at 6, 12, 24, 48 and 72 h after transfection, which revealed that silencing of GLOD4 inhibited the proliferation of leydig cells, whereas the GLOD4 overexpression significantly promotes cell proliferation. (Figure 2H,I). In addition, we examined the expression of proliferating cell nuclear antigen (PCNA) and found that GLOD4 interference and overexpression resulted in the downregulation and upregulation of PCNA, respectively. (Figure 2G).

3.4. GLOD4 Promotes Cell Cycle Progress of Qianbei Ma Goat LCs In Vitro

After exploring the effect of the GLOD4 gene on the cells, we wanted to determine how GLOD4 regulates the cell cycle in leydig cells. We found that the percentage of cells in the G2/M phase was (p<0.05) lower than that in the control group after GLOD4 silencing and expression of the cell cycle-regulated protein kinase (CDK6) and Cyclin E (Cyclin E) was significantly reduced in these cells (p<0.01; Figure 3A,B). In contrast, overexpression of the GLOD4 gene significantly reduced the percentage of cells arrested in the G0/G1 phase and increased the percentage of cells in the G2/M phase compared to those in the control group, and highly significantly (p<0.01) upregulated the expression levels of CDK6 and Cyclin E (Figure 3B,C).

3.5. Promotion of Testosterone Hormone Secretion by GLOD4

After GLOD4 silencing, leydig cells secreted less testosterone (p<0.01) than that in the controls at 48 and 72 h, and the expression of 3β-HSD (p<0.05), CYP11A1 (p<0.01), and CYP17A1 (p<0.01) significantly decreased (Figure 4A,C). In addition, after overexpression of GLOD4, leydig cells secreted(p<0.01) more testosterone hormone than did the control group at 48 h and 72 h, significantly increased expression of 3β-HSD (p<0.01) and CYP17A1 (p<0.05) and had no effect on CYP11A1 expression (Figure 4B,D).

4. Discussion

Previous reports on GLOD4 have focused on its metabolic function and detoxification properties[17,26,27], although some have reported that GLOD4 may have certain effects on the cell cycle and other cellular functions[14,28]. However, few studies have explored the role of GLOD4 in reproduction. Therefore, by exploring GLOD4 expression in the testes of Qianbei Ma goats and its effect on leydig cells, we provided an in depth analysis of the function of GLOD4 and provided a theoretical basis for improving ram fertility.

Wang et al. noted that GLOD4 expression was significantly upregulated in the testis of Tibetan sheep wwith age, and GLOD4 was only expressed in leydig cells and that GLOD4 was mainly observed in the cytoplasm[23], our immunohistochemistry revealed that GLOD4 was expressed only in leydig cells, which is consistent with the findings of Wang et al. However, our subcellular localization analysis revealed that GLOD4 was widely expressed in both the nucleus and the cytoplasm of leydig cells. Interestingly, in a recent study, fluorescently tagged GLOD4 protein was widely distributed in the cytoplasm and nucleus of human retinal pigment epithelial cells (hTERT-RPE1)[28], which may result from differential gene expression in different species[29,30,31].

Based on the differential expression of GLOD4 at different testicular development stages and GLOD4 expression being confined to leydig cells, we hypothesized that the observed increase in the GLOD4 expression might relate to the continuous development of the testis. To date this, we isolated and cultured cells extracted from testes to further explore GLOD4 function. Previously, using a yeast two-hybrid method, Zhang et al. screened for NUDT9 gene interactors and reported that GLOD4 may inhibit cell growth by interacting with NUDT9[32]. In a recent report on GLOD4, GLOD4-deficient Hidradenitis elegans nematode worms were shown to exhibit increased α-DCs, as hortened lifespan, increased neuronal damage, and tactile sensitization, indicating that GLOD4 expression has a protective effect on the cells and ensures cellular proliferation[27]. After successfully constructing GLOD4 interference and overexpression models, and considering that GLOD4 may act with other genes to influence cell Proliferation[14,15], we performed a CCK-8 assay in leydig cells. We found that GLOD4 gene silencing significantly inhibited cell proliferation at two time points and downregulated the mRNA expression of the cellular nuclear antigen (PCNA), which is a proliferation marker. PCNA is widely distributed in a variety of cells, reflecting not only their proliferation rate but also affecting cellular Proliferation as a key gene[12,33]. In contrast, overexpression of GLOD4 had a significant effect on almost every time and significantly upregulated the expression of PCNA. Therefore, we speculate that GLOD4 may play an important regulatory role in cell function.

The cell cycle, which is regulated by the cell cycle proteins CDK and CKI, controls cell growth and plays a key role in cell proliferation[34,35,36]. Our results showed that both GLOD4 silencing and overexpression significantly inhibited and promoted the progression of the cell cycle from the S phase to the G2/M phase, respectively. These results side-step the assertion of Dihazi et al., in which they assert that downregulation of GLOD4 protein expression may inhibit cellular proliferation and block the cell cycle in mouse embryonic stem cells[14]. In a genome-wide study of Chlamydomonas reinhardtii cilia, Albee Alison et al. identified GLOD4 as one of the genes whose expression is upregulated during ciliogenesis. Moreover, knockdown of GLOD4 in human retinal pigment epithelial cells (hTERT-RPE1) results in shorter cellular cilia and cell cycle progression defects[28]. Cyclin-dependent kinase 6 (CDK6), a key gene in the CDK family, regulates cell cycle progression from G1 to S phase by phosphorylating retinoblastoma (RB) proteins when it is complexed with cyclin D[37]. In addition, Cyclin E performs a similar function via regulating the cellular transition from the G0/G1 phase to the S phase to promote cell cycle progression[38]. Thus, examining CDK6 and Cyclin E gene expression and found that silencing and overexpression GLOD4 significantly downregulated and upregulated both CDK6 and Cyclin E, respectively could be reasonable. In addition, overexpression of GLOD4 accelerated the progression from G0/G1, and we speculated that CDK6 and Cyclin E might play a role in this. Therefore, we speculate that GLOD4 may regulate cell proliferation by affecting the cell cycle.

Taken together, GLOD4 may be crucial for leydig cell proliferaiton, but how GLOD4 function in the regulation of testosterone secretion remains unknown. To address this, we examined testosterone secretion in cells cultured for different durations and the expression of genes involved in testosterone secretion. It is well known that in mammalian testes, the testicular cellular receptor (SCARB1) binds specifically to the raw material for androgen synthesis[39], and a series of cascade reactions stimulate the expression and phosphorylation of steroidogenic acute regulatory protein (STAR)[40]. In turn, STAR catalyzes the substrate of the cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1) reaction and converts it to pregnenolone, which is then catalyzed to progesterone by the micronutrient enzyme 3B-hydroxysteroid dehydrogenase 1 (3β-HSD1) in the smooth endoplasmic reticulum[41]. Progesterone is ultimately converted to testosterone by a series of steroid synthases[42,43,44]. These functional genes play a critical role in regulating testosterone secretion. 3β-HSD and CYP17A1 were significantly downregulated and upregulated, respectively, by GLOD4 suppression and overexpression. In addition, the suppression and overexpression of GLOD4 decreased and increased testosterone secretion in leydig cells at 48 and 72 h, respectively. These results suggest that GLOD4 influences testosterone secretion by affecting the expression of a range of genes involved in steroid synthesis.

5. Conclusions

In summary, GLOD4 was differentially expressed in the testes of goats at different ages. In addition, after the silencing and overexpression of GLOD4 in goats, the proliferation of leydig cells was decreased and increased, respectively; similar effects were observed on the cell cycle; and testosterone secretion was significantly decreased and increased respectively. Therefore, we speculate that GLOD4 may regulate testicular development and spermatogenesis by increasing the number of leydig cells and regulation of testosterone secretion, indicating that GLOD4 plays a key role in spermatogenesis by affecting the fate of LCs in goats. Although our study confirms the value of exploring the role of GLOD4 in reproduction, there remains a large gap in studies exploring the role of this gene in reproduction, and larger studies with longer follow-up periods are needed to explore the mechanism by which GLOD4 regulates testicular development in vitro and in vivo.