miR-1285-3p targets TPI1 to regulate the glycolysis metabolism signaling pathway of Tibetan sheep Sertoli cells.

Glycolysis in Sertoli cells (SCs) can provide energy substrates for the development of spermatogenic cells. Triose phosphate isomerase 1 (TPI1) is one of the key catalytic enzymes involved in glycolysis. However, the biological function of TPI1 in SCs and its role in glycolytic metabolic pathways are poorly understood. On the basis of a previous research, we isolated primary SCs from Tibetan sheep, and overexpressed TPI1 gene to determine its effect on the proliferation, glycolysis, and apoptosis of SCs. Secondly, we investigated the relationship between TPI1 and miR-1285-3p, and whether miR-1285-3p regulates the proliferation and apoptosis of SCs, and participates in glycolysis by targeting TPI1. Results showed that overexpression of TPI1 increased the proliferation rate and decreased apoptosis of SCs. In addition, overexpression of TPI1 altered glycolysis and metabolism signaling pathways and significantly increased amount of the final product lactic acid. Further analysis showed that miR-1285-3p inhibited TPI1 by directly targeting its 3'untranslated region. Overexpression of miR-1285-3p suppressed the proliferation of SCs, and this effect was partially reversed by restoration of TPI1 expression. In summary, this study shows that the miR-1285-3p/TPI1 axis regulates glycolysis in SCs. These findings add to our understanding on the regulation of spermatogenesis in sheep and other mammals.

1. Introduction

Tibetan sheep (Ovis aries), one of the three major coarse-haired sheep breeds in China, is the most populous domestic animal on the Qinghai-Tibet Plateau and its adjacent areas located 3000 m above sea level [1]. It provides extremely important living materials and economic income for local farmers and herdsmen. However, its population is severely restricted largely due to the reproductive physiological characteristics of Tibetan sheep, such as long development cycle, low fecundity, and late sexual maturity. At present, the molecular biology involving testis development and spermatogenesis of male Tibetan sheep has not been fully elucidated. As the most critical organ in maintaining the reproductive ability of male animals, the main biological function of the testis is to produce sperm [2]. Studies have revealed that the process of spermatogenesis is regulated by many genes at multiple levels, such as epigenetics, transcription, and translation [3]. Therefore, exploring the expression characteristics and regulation of related genes in Tibetan sheep testicular development and spermatogenesis has important scientific significance for understanding the molecular mechanism through which maintenance of testicular function in sheep and other plateau animals is regulated.

In the process of spermatogenesis, Sertoli cells (SCs) can not only provide physical structure scaffolds and the microenvironment required for survival of spermatogenic cells with different degrees of differentiation in the seminiferous tubules, but also provide various levels of spermatogenic cells for energy production [4]. A previous study reported that SCs can be closely connected with adjacent seminiferous tubules to form a blood-testis barrier which prevents invasion by certain exogenous toxic substances, thereby playing an extremely important role in spermatogenesis [5]. Simultaneously, the lactic acid produced by SCs during glycolytic metabolism pathways can be used as an energy substrate for the development of spermatogenic cells [6]. It could also promote RNA in spermatogenic cells and protein synthesis, thereby reducing apoptosis of spermatogenic cells [7]. Glycolytic metabolism in SCs means that, under the action of glucose transporters on the cell membrane of SCs, sugars such as starch and sucrose are passively transported into the cytoplasm of SCs by the glucose produced by catabolism. In addition, acetone is produced through catalysis of a series of enzymes. The enzymes are then converted into lactic acid, which ultimately enters the spermatogenic cells [8-10]. Among them, triosephosphate isomerase 1 (TPI1), one of the key enzyme molecules in the glycolytic pathway, can catalyze the conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate [11].

It is worth noting that TPI1 was formed before eukaryotic/prokaryotic differentiation, and it was one of the older enzyme families [12]. The lack of TPI1 enzyme in mammals might cause hemolytic anemia, and even death of individual organisms in severe cases [13]. In higher animals, mutation of the TPI1 gene can cause a rare autosomal recessive genetic disease referred to as TPI1 deficiency [14]. Studies in mice found that the TPI1 gene is expressed in spermatogenic cells in the testis and the main segment of the sperm flagella in the epididymis [15]. On the other hand, studies in humans have revealed that anti-sperm antibodies can target TPI1, thereby preventing sperm acrosome reaction and secondary binding of sperm to the zona pellucida [16]. Moreover, in vitro experiments found that the addition of TPI1 inhibitors can significantly reduce sperm motility in rats [17]. The above findings suggest that the TPI1 gene might have the function of positively regulating the development of spermatogenic cells. In our earlier studies [18], we explored the proteomics of sheep testis tissue and found that the TPI1 protein was expressed in sheep testis tissue. After conducting further functional annotations, we found that the TPI1 protein participates in the glycolytic metabolism pathway. At the same time, we cloned the TPI1 gene in the testis tissue of Tibetan sheep at different developmental stages (before pre-puberty, sexual maturity, and adult), and explored its expression and location in the testis and epididymis tissues. Results showed that the Tibetan sheep TPI1 gene CDS 833th Synonymous mutations (C→T) occur in the bases, their mRNA and protein expressions increase with age, and it was mainly located in SCs [19]. However, the specific role of the TPI1 gene in the glycolytic metabolism of SCs, and even in testicular development and spermatogenesis has not yet been reported in mammals, especially domestic animals.

MicroRNAs (miRs, miRNAs) are small noncoding single-stranded RNAs that lack protein-encoding functions. It has been reported that miRNAs can bind to the 3′ untranslated regions (3′-UTRs) of their target mRNAs to suppress protein expression [20]. To date, many studies have found that miRNAs modulate the progression of most human cancers [21]. MiR-1285-3p, with a length of 22 nt and located on chromosome 7, was the first miRNA to be discovered in human embryonic stem cells. One study found that it could specifically bind to p53 and exert an inhibitory effect on tumor cells by regulating the expression level of p53 [22]. Notably, miR-1285-3p is a highly conserved miRNA, which is aberrantly expressed in a variety of tumor cells. A recent study reported that it mainly regulates downstream genes, and inhibits proliferation, migration, and invasion of tumor cells, thereby exerting a tumor suppressor effect [23]. In addition, studies in breast cancer have predicted that nine miRNAs, including miR-1285-3p, target TPI1 (the key enzyme in glycolysis), and produce anti-glycolysis and anti-proliferation effects [24]. Herein, we used the miRDB prediction software (http://www.mirdb.org) to predict the target miRNAs of the TPI1 gene. Results indicated that there were six miRNAs that had a targeting relationship with TPI1. Furthermore, their expression in Tibetan sheep SCs was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Among them, the expression level of miR-1285-3p was the highest. However, only a handful of studies have explored the role of miR-1285-3p in glycolysis metabolism, and it is not yet clear whether it targets TPI1 and plays a role in the glycolysis of SCs.

In the present study, we isolated Tibetan sheep primary SCs, and effectively transfected them with TPI1 overexpression vector and siRNA silencing vector, with the overarching goal of exploring the effects of TPI1 gene overexpression and silencing on the proliferation and apoptosis of SCs, as well as the influence of glycolytic metabolic pathways. Based on the obtained results, we discovered that TPI1 was inhibited by miR-1285-3p and could reverse the inhibitory effect of miR-1285-3p on the glycolytic metabolism of SCs. Elucidation of the underlying mechanism found that the glycolytic metabolism signaling pathway was regulated by the miR-1258-3p/TPI1 axis, and further verified that miR-1285-3p inhibits the proliferation of SCs by targeting TPI1. Collectively, our findings reveal the key role of miR-1285-3p/TPI1 axis in mediating the activation of the SCs glycolytic metabolism signaling pathway.

2. Materials and methods

2.1. Isolation and culture of primary sheep SCs

All animal experiments were performed in accordance with the animal care and experimental procedure guidelines approved by the Animal Committee of Gansu Agricultural University (GSAU-AEW-2020-0057). Three well-grown 3-month-old Tibetan sheep were selected and brought back to the laboratory for intravenous injection of sodium pentobarbital, no heartbeat, continuous involuntary breathing for 2–3 min, no blink reflex, and then bilateral testes tissues were collected. SCs cells were isolated according to a protocol described by Xuejiao et al. [25]. Briefly, three well-grown 3-month-old Tibetan sheep were selected and brought back to the laboratory. The animals were first anaesthetized with intravenous injection of sodium pentobarbital to ensure that there was no heartbeat, no blink reflex, and there was continuous involuntary breathing for 2–3 min. Next, bilateral testes tissues were isolated, sterilized using 75% alcohol, and then the surface envelope was cut (the aseptic part should be taken out with scissors as much as possible). The tissues were digested with trypsin and collagenase, followed by addition of serum-containing DMEM/F12 (Gibco, New York, USA) to stop the digestion and screening with different mesh cells. Cells were then cultured in 15% fetal bovine serum (FBS, Gibco, New York, USA) supplemented with 1% penicillin streptomycin sol in DMEM/F12 (FBS, Gibco, New York, USA). After the cells were adhered and differentiated, they were collected, and discontinuous density gradient centrifugation was performed to purify the cells using Percoll cell separation solution (Solarbio, Beijing, China) with volume fractions of 11%, 19%, 27%, 35%, and 43%, respectively. The differential adhesion method was then used to purify cells several times in order to obtain relatively pure SCs. Notably, the specific GATA4 antibody was used to identify SCs by immunofluorescence staining. When the concentration reached more than 90%, the cells could be used for subsequent tests.

2.2. Construction of TPI1 gene silencing or overexpression vectors

The TPI1-siRNA interference sequence was designed according to our earlier cloned Tibetan sheep TPI1 gene sequence (MN847717), and the negative control (NC-siRNA) was used as the control group (S1 Table). Notably, both the TPI1-siRNA and NC-siRNA vectors were synthesized by Genepharma Biological. On the other hand, the TPI1 overexpression vector (pc-DNA-3.1(+)-TPI1) was constructed by Genewiz Biological, and the empty vector (pc-DNA-3.1(+) was used as the control group. The plasmid small extraction kit (Tiangen, Beijing, China) was used to extract the plasmid according to the manufacturer’s instructions, and then the recombinant plasmid was double digested with NheI and NotI restriction enzymes. Finally, the digestion products were identified by 2% agarose gel electrophoresis.

2.3 The dual-luciferase reporter gene

The Target Scanprediction software (https://www.targetscan.org) was used to predict the miRNA targeted by the TPI1 gene. It was found that the 56–61 region of the TPI1 3’UTR had a binding site with miR-1285-3p. Primers were then designed according to the 3’-UTR sequence of TPI1, followed by synthesis of TPI1 3’-UTR wild-type gene fragments and mutant gene fragments. It should be noted that the mutant gene fragment is a sequence that mutates the binding point sequence of TPI1 to the reverse complementary sequence of the original target binding point. Next, pmiRGLO was used as a vector to synthesize plasmids of TPI1 3’-UTR wild-type gene fragment (TPI1 3’UTR WT) and mutant gene fragments (TPI1 3’UTR MUT). MiR-1285-3p mimics, NC mimics, miR-1285-3p inhibitor, and NC inhibitor were also designed and synthesized (Genepharma Biological, Shanghai, China). The synthetic sequences are shown in S2 Table. The TPI1 3’UTR WT and TPI1 3’UTR MUT plasmids were combined with miR-1285-3p mimics, NC mimics, miR-1285-3p inhibitor, and NC inhibitor NC, followed by transfection into HEK293T cells. Finally, the luciferase activity was measured 48 h after transfection using a dual-luciferase assay system (Promega, Beijing, China).

2.4. Cell transfection

SCs were selected of the F5 generation after isolated, purified and identified, the transfection only began when the cells reached 60%-70% confluence. On one hand, 2500 ng of the extracted TPI1 overexpression plasmid DNA was diluted with 250μL serum-free medium Opti-MEM (Gibco, New York, USA), and gently mixed, whereas on the other hand, 5μL Lipofectamine 2000 (Invitrogen, California, USA) was diluted with 250μL serum-free medium Opti-MEM and gently mixed gently, followed by incubation at room temperature for 5 min. The two liquids were then mixed and incubated at room temperature for 20 min to form a DNA-Lipofectamine 2000 complex. Next, 500μL of the complex was taken and added to 1.5 mL of Opti-MEM in a basic six-well plate. The two liquids were gently mixed, placed in a 37°C incubator for 4 h, and then replaced with DMEM/F12 medium supplemented with 15% FBS for culture. Notably, the dosage of siRNA for transfection was 200nM/well, the dosage of miR-1285-3p mimics and NC mimics was 50nM/well, the dosage of miR-1285-3p inhibitor and NC inhibitor was 100nM/well, and the lipofectamine 2000 dosage was 5μL/well. The rest of the steps were similar to the steps involved in the transfection of plasmids.

2.5. Cell counting kit-8 (CCK-8) proliferation assay

CCK-8 reagent (MedChen Express, Shanghai China) was used to determine cell viability. Briefly, SCs growing in the logarithmic phase were selected, digested with trypsin, and counted. Cells were then seeded in a 96-well plate at a density of 1×104 cells/well for 24 h, followed by transfection for 24, 48, and 72 h. Next, 10μL of CCK-8 reagent was added to each well and incubated for 1 h at 37°C. Finally, the OD value of each well was measured at 450 nm using enzyme-linked immunosorbent assay (ELISA) and the growth curve was plotted.

2.6. Flow cytometry

Flow cytometry was used to detect Annexin V-FITC/PI double-labeled necrotic and apoptotic cells (Q2 + Q3). Briefly, cells were digested with EDTA-free trypsin to make a single cell suspension, and washed with pre-cooled PBS. Next, 300μL of the binding buffer was added to the cells at a density of about 5 × 105 cells/tube, followed by addition of 5μL of Annexin V-FITC in each tube, and thorough mixing. 5μL of PI solution was then added to the tube, followed by thorough mixing and protection from light for 10 min. Finally, the liquid in the tube was detected using flow cytometry.

2.7. Total RNA extraction, cDNA synthesis, and RT-qPCR analysis

The total RNA of SCs after transfection were extracted using TransZol (TransGen Biotech, Beijing, China) according to manufacturer’s instructions. The integrity of RNA was determined by 1% agarose gel electrophoresis, whereas the concentrations and quality of RNA samples were examined by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA)) and Agilent 2100 (Agilent, Santa Clara, CA, USA), respectively. Next, Evo M-MLV RT Kit with gDNA Clean for RT-qPCR (Accurate Biotech, Hunan, China) was used to reverse transcribe the RNA into cDNA, and SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotech, Hunan, China) was used to perform qPCR on the Roche LightCycler96 in accordance with the manufacturer’s protocols. Relative mRNA expression was normalized to β-actin (a housekeeping gene) mRNA and calculated using the 2–ΔΔCt method [26]. Reverse transcription of miRNA using mir-XTM miRNA first-strand Synthesis Kit (Takara, Japan), the qPCR method was the same as that of mRNA. S3 and S4 Tables shows the primers used for RT-qPCR analysis.

2.8. Western blot analysis

After transfection of SCs, they were homogenized and lysed using a radioimmunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Protein concentrations were then quantified using a commercial bicinchoninic acid (BCA) protein assay (Solarbio, Beijing, China). Next, the extracted proteins were denatured with 4x protein loading buffer (DTT, Solarbio, Beijing, China), resolved using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). Membranes were first blocked with phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk for 2 h at room temperature, followed by incubation with either rabbit anti-TPI1 polyclonal antibody (1:600; Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:600; Bioss, Beijing, China) at 4°C overnight. On the next day, membranes were washed with PBST, and then incubated with goat anti-rabbit IgG/HRP antibody (1:5000; Bioss, Beijing, China) for 2 h at 37°C. Finally, they were washed with PBST and the protein signals were visualized using NcmECL Ultra reagents (New Cell & Molecular Biotech Co.LTD, Suzhou, China) in an X-ray room.

2.9. Detection of the final products and content of key enzymes of the glycolytic metabolic signal pathway

After transfection, the cell fluid was collected and the lactic acid extraction kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China) was used to measure the content of lactic acid production during glycolysis process. In addition, the SCs were collected after transfection, and then the ATP kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China), lactate dehydrogenase (LDH) kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China), and pyruvate extraction kit (Nanjing Jiancheng Bioengineer Institute, Nanjing, China) were used to measure the change level of key enzymes, energy, and pyruvate content in the glycolysis process. Notably, all the experiments were conducted following the manufacturer’s instructions.

2.10. Statistical analyses

All statistical analyses were performed using SPSS 21.0 software and all data were expressed as mean ± standard error (Mean±SE). The gray value of the band obtained after western blot analysis was scanned and determined using AlphaEaseFC image analysis software. The relative expression of the target protein was the ratio of the gray value of the target protein to the gray value of β-actin. One-way analysis of variance (ANOVA) was used to compare differences among multiple groups. P<0.05 was considered statistically significant.

3. Results

3.1 Identification of Tibetan sheep primary SCs

Immunofluorescence staining of SCs with specific antibody GATA4 and TPI1 antibody showed that purified SCs could synthesize and express SCs-specific binding protein GATA4, and staining with TPI1 antibody showed that TPI1 protein was expressed in SCs. The results could rule out contamination by other testis cell types, and the purified SCs could be used in subsequent experiments (Fig 1).

Fig 1

Immunofluorescence staining of primary SCs of Tibetan sheep (20×) [25].

3.2 Recombinant plasmid double digestion and determination of transfection efficiency

The contents obtained after double digestion of pcDNA3.1(+)-PGAM1 were electrophoresed on a 2% agarose gel (Fig 1F). Results showed that the size of the digestion product was consistent with the theoretical value. QRT-PCR was then performed to determine the expression level of TPI1 mRNA in the transfected sheep SCs. The results revealed that the expression of TPI1 gene in SCs of the si-TPI1-1, si-TPI1-2, and si-TPI1-3 transfection groups were all downregulated after transfection for different time periods compared to the control group, but the silencing effect was most obvious after transfection for 48 h (Fig 2A–2C). The transfection efficiency of the three siRNAs was then compared after transfection for 48 h. It was found that the silencing effect of si-TPI1-1 was significantly higher than the others (P<0.05) (Fig 2D), indicating that transfection of si-TPI1-1 for 48 h had the best silencing effect. Compared to the empty vector group, the expression of TPI1 gene in the pcDNA3.1(+)-TPI1 transfection group was upregulated after transfection for different time periods. However, the upregulation was the most obvious after 48 h, indicating that the overexpression efficiency was the best after transfection for 48 h (Fig 2E).

Fig 2

Evaluation of recombinant plasmid and detection of transfection efficiency.

(A-E) TPI1 gene silencing or overexpression efficiency. (F) Evaluation of recombination plasmids. “*” indicate significant difference (P<0.05), the same below.

3.3 Western blot analysis of TPI1 protein expression

The si-TPI1 plasmid with the highest interference efficiency and its control NC, as well as the overexpression plasmid pcDNA3.1(+)-TPI1 and the empty vector were transfected into sheep SCs for 48 h. Western blot analysis was then performed to determine the expression of TPI1 protein. Results showed that pcDNA3.1(+)-TPI1 could significantly increase the expression of TPI1 compared to the control group (P<0.05), and si-TPI1 could significantly reduce the expression of TPI1 compared to the control group (P<0.05) (Fig 3). These results suggest that pcDNA3.1(+)-TPI1 could effectively promote the expression of TPI1 protein in sheep SCs, whereas si-TPI1 could effectively inhibit the expression of TPI1 protein in sheep SCs.

Fig 3

Expression patterns of TPI1 protein in SCs after silencing or overexpression.

3.4 Effect of TPI1 gene silencing or overexpression on the proliferation, apoptosis, and cycle of SCs

The proliferation of SCs after transfection was determined by CCK-8 assay and flow cytometry. The CCK-8 test results showed that there was no significant difference in overexpression or silencing after 24h of transfection (P>0.05). However, the number of cells in the pcDNA3.1(+)-TPI1 group was significantly higher than that in the empty vector group after 48 h and 72 h (P<0.05). In addition, the number of cells in the si-TPI1 group was significantly lower than that in the NC group (P<0.05) (Fig 4A and 4C). Flow cytometry was used to determine the changes in cell apoptosis after 48 h of transfection. Results showed that the apoptosis rate of the pcDNA3.1(+)-TPI1 group was significantly lower than that of the empty vector group whether in early or late apoptosis (P<0.05). The apoptosis rate of the si-TPI1 group was higher than that of the NC group (P<0.05) (Fig 4B). Furthermore, RT-qPCR was used to determine the changes in the expression of proliferation-, cycle-, and apoptosis-related genes at the mRNA level. The results demonstrated that the relative expression of pro-apoptotic genes (Caspase3 and Bax) was significantly downregulated in the overexpression group compared to the empty vector group (P<0.05), whereas the expressions of proliferation-related genes (PCNA and Bcl2) and cell cycle-related gene (CyclinB1) were significantly upregulated in the overexpression group compared to the empty vector group (P<0.05). After silencing the TPI1 gene, the results were in contrast to the overexpression results (Fig 4D and 4E). Collectively, these results suggest that the overexpression of TPI1 inhibited cell apoptosis, whereas silencing of TPI1 promoted cell apoptosis.

Fig 4

Effect of TP1 gene silencing and overexpression on the proliferation, cycle and apoptosis of SCs.

A, C: CCK-8 detects the cell proliferation rate after overexpression and silencing of TPI1; B: Flow cytometry was used to detect the apoptosis of SCs after overexpression and silencing of TPI1; D, E: The effect of overexpression and silencing of TPI1 gene on the expression of proliferation, cycle and apoptosis related genes.

3.5 Effect of silencing or overexpression of TPI1 on the glycolytic metabolism pathway

After 48 h of transfection, the expression levels of the eight genes downstream of the TPI1 gene in the glycolytic metabolism pathway in the SCs was determined by qRT-PCR. The results showed that the expression levels of ENO1, PKM, LDHA, LDHB, MCT1, PGAM1, and PGK genes were significantly upregulated in the pcDNA3.1(+)-TPI1 group compared to the empty vector group (P<0.05), whereas the expressions of ENO1, PKM, LDHA, LDHB, MCT1, GAPDH, and PGK genes were significantly downregulated in the si-TPI1 group compared to the NC group (P<0.05). This suggested that overexpression of the TPI1 gene could increase the expression of downstream genes, whereas silencing of the TPI1 gene could reduce the expression of downstream genes (Fig 5A and 5C). The pyruvate, lactic acid, and ATP kits were then used to determine the content of pyruvate and ATP in SCs, and the activity of LDH after 48 h of transfection. Results revealed that the LDH activity, lactic acid production, and contents of pyruvate and ATP in the pcDNA3.1(+) group were significantly higher than those in the pcDNA3.1(+)-TPI1 group (P<0.05). After silencing the TPI1 gene, the result was the opposite of overexpression (Fig 5B, 5D, 5E and 5F).

Fig 5

Effect of TPI1 gene silencing and overexpression on glycolytic metabolic pathways.

A, C: The expression levels of downstream genes in the glycolytic metabolic pathway after silencing and overexpression of the TPI1 gene. B, D, E and F: The content of glycolytic metabolic pathway products, energy and key enzymes after silencing and overexpression of TPI1 gene.

3.6 TPI1 is a direct target of miR-1285-3p

We studied the expression of 6 miRNAs predicted to be targeted to TPI1 in SCs and found that the expression level of miR-1285-3p in SCs was significantly higher than that of other miRNAs(P<0.05) (Fig 6F). We first confirmed the interaction between miR-1285-3p and TPI1 with the predicted sites (Fig 6A). The dual-luciferase assay system indicated that the miR-1285-3p mimics significantly decreased the luciferase activity of TPI1-WT, but not TPI1-MUT (Fig 6B). Moreover, it was found that knockdown of miR-1285-3p significantly increased TPI1 expression, whereas enforced expression of miR-1285-3p significantly decreased TPI1 expression in SCs (Fig 6D and 6H). These results suggest that miR-1285-3p can inhibit the expression of TPI1 by directly binding to the 3’-UTR of TPI1. In addition, the results of CCK8 assay, flow cytometry, and the expression level of apoptosis-related and proliferation-related genes showed that the overexpression of miR-1285-3p could inhibit the proliferation of SCs (Fig 6C, 6E and 6G). The expression abundance of the eight genes downstream of the TPI1 gene in the glycolytic metabolism pathway was determined by qRT-PCR after transfection of the SCs. Results showed that the expression of these genes was significantly reduced in miR-1285-3p mimics compared to the negative control (P<0.05), whereas their expression was significantly increased in the miR-1285-3p inhibitor group compared to the negative control (P<0.05) (Fig 6J). Next, ELISA kits were used to detect LDH activity, lactic acid production, and content of pyruvate and ATP during glycolysis in SCs. It was found that their contents were significantly lower in the miR-3614-5p mimics compared to the negative control group (P<0.05), and significantly increased in the miR-1285-3p inhibitor group compared to the negative control group (P<0.05) (Fig 6I). Overall, these results suggest that TPI1 is a direct functional target of miR-1285-3p in the glycolytic metabolic pathway of SCs.

Fig 6

TPI1 expression is downregulated by miR-1285-3p directly targeting of the 3’-UTR of TPI1.

A, TPI1 was found for the potential regulatory targets of miR-1285-3p using prediction tool. B, Dual-luciferase reporter assay analysis of the target relationship of miR-1285-3p with TPI1. F, Expression of miRNA in SCs. The TPI1 expression levels were determined by real-time PCR analysis (D) western blot (H) after transfection with the miR-1285-3p mimics or negative control or after transfection with the miR-1285-3p inhibitor or negative control in SCs. CCK-8 (E), Flow cytometry (G) and the expression of proliferation, cycle and apoptosis related genes (C) was used to detect the apoptosis of SCs after transfection miR-1285-3p mimics, negative control, miR-1285-3p inhibitor or negative control. The expression levels of downstream genes (J) and the content of glycolytic metabolic pathway products, energy and key enzymes (I) of SCs after transfection miR-1285-3p mimics, negative control, miR-1285-3p inhibitor or negative control.

3.7 Rescue experiment verified that miR-1285-3p negatively regulates SCs glycolytic metabolism by targeting TPI1

Given the potential suppressive role of miR-1285-3p in SCs glycolytic metabolism, we further explored whether TPI1 mediated the suppressive role of miR-1285-5p in SCs. QRT-PCR and western blot results confirmed that co-transfection of miR-1285-3p with the TPI1 overexpression plasmid mildly rescued the TPI1 expression in SCs (Fig 7A and 7B). Further functional experiments showed that restoring the expression of TPI1 can effectively reverse the decline in the proliferation of SCs caused by overexpression of miR-1285-3p (Fig 7C, 7D and 7F). Furthermore, the expression of glycolytic metabolism signaling pathway-related molecules was decreased after enforced expression of miR-1285-3p in SCs, whereas reintroduction of TPI1 abolished the suppressive effect of miR-1285-3p mimics on the glycolytic metabolism signaling pathway (Fig 7A, 7E and 7G). In addition, given that TPI1 is an important enzyme in the aerobic glycolysis process, we further confirmed the effect of miR-1285-3p/TPI1 axis on SCs glucose metabolism. Altogether, these findings suggest that the miR-1285-3p/TPI1 axis promotes SCs progression, at least in part, via the glycolytic metabolism signaling pathway.

Fig 7

Effect of miR-1285-3p on SCs glycolytic metabolism by targeting TPI1 in rescue experiment.

A, B: RNA and protein level of TPI1 were detected by qRT-PCR and western blot, respectively. C, D and F: SCs proliferation and apoptosis detected by CCK-8, Flow cytometry and the expression of proliferation, cycle and apoptosis related genes. E, G: The expression levels of downstream genes and the content of glycolytic metabolic pathway products, energy and key enzymes of SCs.

4. Discussion

As one of the key enzyme molecules in the glycolysis pathway, TPI1 catalyzes conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate [11]. Then, under the action of GADPH, PGK, PGAM1, ENO, PKs, etc., it was converted into pyruvate and generates ATP, and then finally lactate was generated under the action of lactate dehydrogenase. [9] Studies have revealed that the lactic acid produced by glucose through glycolytic metabolism in the process of cancer occurrence and development could provide energy for the proliferation, spread, and metastasis of cancer cells [27, 28], thereby inhibiting or blocking certain aspects of cancer cell glycolytic metabolism. Therefore, the key link, cutting off the energy supply required for cell proliferation and migration, has become a hotspot in clinical research on tumor prognosis. For example, Jiang et al. [11] found that the TPI1 gene could inhibit the occurrence of liver cancer, which suggested that TPI1 can be used as a potential target for the treatment of liver cancer. Over the years, there have been many reports on the glycolytic metabolism pathway in the development of the reproductive system of male mammals, especially in the development of the testis and epididymis [7, 29, 30]. Studies in humans have found that the TPI1 gene is expressed in the head of sperm, and anti-sperm antibodies can target TPI1, thereby preventing the sperm from undergoing acrosome reaction and preventing secondary binding of sperm to the zona pellucida [16]. Studies in adult bulls demonstrated that TPI1 was expressed on the plasma membrane of the sperm acrosome [31]. Moreover, studies in mice have reported that TPI1 was mainly expressed in the main segment of the sperm flagella, which is located in the tail of the sperm [32]. It is well known that oxidative phosphorylation provides energy for sperm movement [33]. Therefore, the TPI1 gene may be mainly involved in the regulation of energy metabolism, so as to provide energy for normal movement of the sperm stored in the epididymis.

A previous study found that when the glycolytic metabolic process is abnormal, it could cause obstacles in sperm production, which can ultimately lead to male sterility [34]. Given that TPI1 could participate in the glycolytic metabolic pathway and the main metabolic mode of the SCs of the testis was glycolysis, the final product (lactic acid) produced through glycolysis provides nutrition for all levels of spermatogenic cells to ensure normal operation of the sperm formation process [6]. Studies have reported that dibromophenyl ether inhibits the glycolytic metabolic pathways in mouse testes and disrupts glucose metabolism homeostasis, thereby affecting the survival of germ cells [35]. Exogenous injection of 6-propyl-2-thiouracil could significantly reduce the glucose intake, LDH content, and lactic acid concentration in the testis of mice, ultimately increasing the apoptotic rate of germ cells and reducing the growth rate [36]. At the same time, when the expression of another important enzyme (PGAM1) in the glycolytic metabolic pathway was downregulated, the apoptosis rate of mouse SCs was significantly increased, the proliferation and migration abilities were restricted, and the proliferation of spermatogonia significantly inhibited cell apoptosis [37]. Herein, functional experiments confirmed that TPI1 knockdown significantly inhibited activation of the glycolytic metabolism signaling pathways. Notably, previous studies have reported that the glycolytic metabolism of SCs has an important regulatory effect on the proliferation and apoptosis of spermatogenic cells [38, 39]. The results of this study found that overexpression of the TPI1 gene could lead to increased pyruvate content, lactate production, ATP production, and LDH activity in SCs, whereas the expression of downstream genes in the glycolytic pathway was significantly upregulated. Consistently, this study showed that TPI1 enhanced the synthesis of lactic acid during glycolytic metabolism of SCs, thereby enhancing the development of spermatogonial stem cells. These findings imply that TPI1 may be involved in spermatogenesis, at least in part, by activating glycolytic metabolism signaling pathways to provide nutrients for spermatogenesis. The results also suggested that TPI1 might participate in regulating the development and functional maintenance of sheep SCs through glycolytic metabolism to produce the energy substrate required for the development of spermatogenic cells-lactic acid. In addition, it was evident that TPI1 participated in regulating the development of male sheep spermatogonial stem cells.

It has been reported that the miRNA regulatory network is effective and complex in physiological and pathological conditions [40]. The biological function of miRNAs is mainly mediated through pairing with a complementary site in the 3’UTR of the target mRNA, which results in post-transcriptional regulation by translational inhibition [41]. Accumulating evidence suggests that certain miRNAs participate in cancer progression by targeting distinct mRNAs [20]. For example, miR-133b and miR-511 could regulate the expression of tumor suppressor genes to inhibit tumor growth [42]. MiR-200 could downregulate the expression of ZEB1 and ZEB2 in liver cancer, thereby inhibiting cell invasion and cancer progression [43]. Moreover, miR-1285-3p could inhibit the migration and invasion of cancer cells in liver cancer and pancreatic cancer [44, 45]. Numerous studies have shown that miRNAs participate in the pathogenesis of numerous diseases by regulating glycolytic metabolism and mitochondrial energy production [46, 47]. MiR-1285-3p inhibitor significantly improved the mitochondrial respiratory function and respiratory chain complex activity, thereby increasing ATP production, and reducing the number of cells with low MMP and mtROS to protect jejunal epithelial cells against Cu-induced toxic injury. Besides, miR-1285 mediated the deficiency in ATP generation by targeting AMPKα2 in immature boar SCs [45, 48]. The above results suggest that in addition to the role of miR-1285-3p in the occurrence of cancer, it has a certain regulatory effect on the growth and development of SCs.

To date, only a handful of reports have explored the role of miR-1285-3p in SCs glycolytic metabolism. In this study, analysis of multiple prediction websites identified TPI1 as a functional target of miR-1285-3p (miR-1285-3p could bind to TPI1 in a targeted manner). In addition, our dual luciferase reporter assay demonstrated that elevated expression of miR-1285-3p could significantly reduce the luciferase activity driven by the luciferase gene containing the 3’UTR of TPI1, which confirmed that miR-1285-3p can inhibit the expression of TPI1 by directly targeting its 3’UTR. Furthermore, it was found that overexpression of TPI1 weakened the inhibitory effect of miR-1285-3p on SCs apoptosis and the glycolytic metabolism signaling pathway. In summary, these findings identify miR-1285-3p as a new determinant of TPI1 expression and establish a new miR-1285—3p/TPI1/glycolytic metabolism signaling pathway axis for spermatogenesis during testicular development in male animals.

5. Conclusion

In conclusion, our results further confirm that the TPI1 gene regulates the glycolytic metabolic pathway of sheep SCs to provide energy substrates for the development of spermatogenic cells, thereby ensuring smooth progress of spermatogenesis. Mechanism investigations revealed that TPI1 was the functional target of miR-1285-3p, and miR-1285-3p could reduce the expression of TPI1 in Sertoli cells. In addition, the miR-1285-3p/TPI1 axis regulated the energy substrate required by spermatogenic cells, at least in part, by activating the glycolytic metabolism signaling pathway in Sertoli cells. Overall, these findings suggest that the miR-1285-3p/TPI1 axis provides a scientific basis for studying spermatogenesis and reproductive disorders in male mammals such as sheep.

Information of si-TPI1 sequence.

(DOCX)

Click here for additional data file.

Information of miRNA sequence.

(DOCX)

Click here for additional data file.

mRNA information of primer sequence.

(DOCX)

Click here for additional data file.

miRNA information of primer sequence.

(DOCX)

Click here for additional data file.

The original data and supplementary materials were stored in Datadryad at: https://datadryad.org/stash/share/dg60cOPjYmG406-dwNzI2JdkOwleeknqaQI0nSR2oKA (doi:10.5061/dryad.5dv41ns7k).

(TXT)

Click here for additional data file.

22 Dec 2021

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Reviewer #2: Yes

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Reviewer #1: Comments:

An et al. studied the role of Triose phosphate isomerase 1 (TPI1) in testicular Sertoli cells (Sc) isolated from Tibetan sheep. The authors demonstrate that TPI1 regulates Sc proliferation, apoptosis, and gene expression. Authors show that mir-1285-3p directly targets TPI1, and manipulating the levels of the microRNA has a similar effect that is observed upon knockdown/over-expression of TPI1 in Sc. This is an interesting manuscript; however, some aspects of the study need to be clarified, and more experiments are necessary to describe the manuscript's findings. Also, serious issues with the writing must be addressed. Overall, the paper feels premature and would benefit from some major rewriting. Throughout the article, including abstract, many vocabulary, grammar, and pose errors make it very hard to follow and interpret.

Major comments:

The abstract is poorly written.

All the figure legends should be revised—for example, Fig2: Changing of TPI1 protein in SCs after silence or overexpression.

What about the images or graphs in the figure? Don’t they need any explanation? What about the statistical significance? What methods were used? Fig 2 is cited at the end of the paragraph. Very hard to understand.

In the text, it was mentioned si-PGAM1, whereas in the figure TPI1.

In the 3rd figure and 3.3, what is CCK8? How can first-time readers understand?

Difference in overexpression or silence after 24 h of transection: What is transection?

The number of cells in the si-PTPI1 group was significantly lower than that in the NC group.

What is si-PTTPI1?

Further qRT-PCR was used to detect the changes in the expression of proliferation, cycle and apoptosis-related genes at the mRNA level.

What is cycle?

The above typos are from a single paragraph. I haven’t included the English corrections! Similarly, every paragraph has typos and English corrections.

1. Materials and methods (Isolation and culture of Sertoli cells): The authors are suggested to provide the culture purity data (GATA4/SOX9 immunostaining) and the expression of TPI1 (immunostaining, immunoblotting) in cultured Sc in the main figure. This is important as contamination by other testicular cell types (also known to express TPI1) needs to be ruled out.

2. For how many days were the cells cultured in vitro? After how many days were the cells transfected, and what was the transfection efficiency? The authors should provide information regarding the same in the material and methods section.

3. The authors have studied the effect of TPI1 on Sc proliferation and apoptosis. There is a dogma that Sc proliferates during the neonatal infantile period and stops proliferating onset of puberty. Thus authors are suggested to check the expression of TPI1 changes during pubertal maturation of Sc. Along the same lines, does the expression of mi-1285-3p change during the functional maturation of Sc at puberty? Incorporation of this data would improve the quality of the manuscript.

4. How does knock-down/over-expression of TPI1 regulate the mRNA levels of glycolytic pathway genes? Does TPI1 regulate the expression of these genes directly, or is the change in gene expression indirect (due to a shift in metabolic flux as a consequence of TPI1 knockdown/over-expression)? The authors should comment on this in the discussion section.

5. The authors are suggested to provide separate figures for the TPI1 knockdown and over-expression experiments along with the consequent effect(s) of knockdown/over-expression of TPI1 on Sc function (proliferation, apoptosis, gene expression etc..).

6. Which gene was used to normalize the gene expression data? This needs to be mentioned in the materials and methods section.

7. Results section 3.4- the authors state that “the expression of ENO1, PKM, LDHA, LDHB, MCT1, GAPDH, and PGK genes were significantly down-regulated in the si-PGAM1 group (P<0.05), indicating the over-expression of the PGAM1 gene could increase the expression of downstream genes while silencing the PGAM1 gene could reduce the expression of downstream genes”. PGAM1 needs to be replaced with TPI1 in the text.

8. There are major grammatical errors and awkward sentences in the manuscript which need to be corrected.

Minor:

• Line 2. Repetition of sentences. breeds in China; Tibetan sheep (Ovis aries) is one of the three major coarse-haired sheep breeds in China. breeds in China.

• Page no-8 In the initial introduction, the importance of spermatogenesis should be brief and crisp. It covers half of the introduction with repeated sense.

• The authors should provide details of Ab used for isolation, polyclonal or monoclonal?

• Would the authors mind providing precise details of the sample collection procedure (collected from a slaughterhouse or by sacrifice) and the number of testicles collected for each experiment?

• No need to rewrite the material method in the result section.

• The author should provide good-quality images. Fig 5 and 6 captions are not readable.

Reviewer #2: In this study, the authors suggest a regulatory mechanism of Sertoli Cell metabolism and function based on the control of TPI1 expression via the miRNA miR-1285-3p. To support their hypothesis, the authors have designed an in vitro model based in primary Sertoli cells of Tibetan sheep. The manuscript is fairly well-written, although some methodological aspects are not easy the follow. The authors provide evidence of this regulatory mechanism at both gene and protein expression levels. Despite that, I have some reserve about the causal relationship between miR-1285-3p, TPI1 and the effects on Sertoli cell proliferation and downstream gene expression observed by the authors, due to the multiple targets of the miR-1285-3p.

Major issues:

1) It was not possible to confirm the existence of a binding site to miR-1285-3p in the TPI1 gene using miRanda, because the website is no longer available. However, using miRDB (http://mirdb.org) it is possible to estimate two binding sites between TPI1 and miR-1285-3p in 3’ UTR, in humans. However, according to this database, there are 41 miRNAs targeting the TPI1 gene, and the miR-1285-3p is not the miRNA with higher “score”. The authors state in the introduction that this miRNA was selected because it is the most expressed in the Tibetan Sheep among the miRNAs with high affinity for the TPI1 gene. Yet, miR-1285-3p has 895 targets and TPI1 is not within the top 50 targets. Among the miR-1285-3p targets with higher score than TPI1 are several genes that could be involved in cell proliferation and metabolic regulation. Therefore, it is unlikely that the action of miR-1285-3p in Sertoli Cells is restricted to the regulation of TPI1 gene. To claim that, the authors would have to characterise the transcriptome of the Sertoli Cells transfected with the TPI1 inhibitor.

2) Related to the previous issue, the authors do not refer the parameters to predict miRNAs targeting TPI1 using miRanda, especially which animal database was used. Although the primers were specific to Tibetan Sheep, the target prediction was likely performed using a database for another species. This limitation must be discussed.

3) It is not clear to me how the miR-1285-3p mimic is expressed in Sertoli Cells by means of a plasmid, and the supplementary files are not available. How can the authors guarantee that the sequence will mimic the behaviour of the original miR-1285-3p? If the mimic sequence is expressed along the luciferase gene, then it must be processed post-transcriptionally. This must also be discussed by the authors.

4) In section 3.5 and Figure 5, the authors mention the miR-3614-5p. Did the authors mean miR-1285-3p?

Minor issues:

1) Please replace the term “testicles” by the term “testes” (singular “testis”).

2) QRT-PCR -> qRT-PCR

3) Multipanel figures must be labelled uniformly. In some figures the panels are ordered from left to right, top to bottom, but in other figures the authors label from top to bottom and only then from left to right. Although I understand the authors have a lot of information to show, please consider relegating some figure panels to supplementary data to improve the visualization of main manuscript figures.

**********

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Reviewer #1: No

Reviewer #2: Yes: Luís Crisóstomo

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1 Apr 2022

Response to Reviewer 1 Comments

We thank the reviewer for the time in closely viewing our manuscript. Most of the suggestions have been adopted in the revision.

Major comments:

Point 1: The abstract is poorly written.

Response 1: Thanks for your comments, we have revised the abstract section, and the specific revision results are as follows: Glycolysis in Sertoli cells (SCs) can provide energy substrates for the development of spermatogenic cells. Triose phosphate isomerase 1 (TPI1) is one of the key catalytic enzymes involved in glycolysis. However, the biological function of TPI1 in SCs and its role in glycolytic metabolic pathways are poorly understood. On the basis of a previous research, we isolated primary SCs from Tibetan sheep, and overexpressed TPI1 gene to determine its effect on the proliferation, glycolysis, and apoptosis of SCs. Secondly, we investigated the relationship between TPI1 and miR-1285-3p, and whether miR-1285-3p regulates the proliferation and apoptosis of SCs, and participates in glycolysis by targeting TPI1. Results showed that overexpression of TPI1 increased the proliferation rate and decreased apoptosis of SCs. In addition, overexpression of TPI1 altered glycolysis and metabolism signaling pathways and significantly increased amount of the final product lactic acid. Further analysis showed that miR-1285-3p inhibited TPI1 by directly targeting its 3'untranslated region. Overexpression of miR-1285-3p suppressed the proliferation of SCs, and this effect was partially reversed by restoration of TPI1 expression. In summary, this study shows that the miR-1285-3p/TPI1 axis regulates glycolysis in SCs. These findings add to our understanding on the regulation of spermatogenesis in sheep and other mammals.

Point 2: All the figure legends should be revised—for example, Fig2: Changing of TPI1 protein in SCs after silence or overexpression. What about the images or graphs in the figure? Don’t they need any explanation? What about the statistical significance? What methods were used? Fig 2 is cited at the end of the paragraph. Very hard to understand.

Response 2: Thanks for your comments, I have modified it in the text.

Point 3: In the text, it was mentioned si-PGAM1, whereas in the figure TPI1.

Response 3: Thanks for your comments, this is a mistake in my negligent text, it should be si-TPI1, I have made changes in the text.

Point 4: In the 3rd figure and 3.3, what is CCK8? How can first-time readers understand?

Response 4: Thanks for your comments, CCK8 is Cell counting kit-8, I have modified it in the text.

Point 5: Difference in overexpression or silence after 24 h of transection: What is transection? The number of cells in the si-PTPI1 group was significantly lower than that in the NC group. What is si-PTTPI1? Further qRT-PCR was used to detect the changes in the expression of proliferation, cycle and apoptosis-related genes at the mRNA level. What is cycle? The above typos are from a single paragraph. I haven’t included the English corrections! Similarly, every paragraph has typos and English corrections.

Response 5: Thanks for your comments, I have double checked the article and corrected the mistakes.

Point 6: Materials and methods (Isolation and culture of Sertoli cells): The authors are suggested to provide the culture purity data (GATA4/SOX9 immunostaining) and the expression of TPI1 (immunostaining, immunoblotting) in cultured Sc in the main figure. This is important as contamination by other testicular cell types (also known to express TPI1) needs to be ruled out.

Response 6: Thanks for your comments, I have modified the results section, and the specific modification results are as follows:

3.1 Identification of Tibetan sheep primary SCs

Immunofluorescence staining of SCs with specific antibody GATA4 and TPI1 antibody showed that purified SCs could synthesize and express SCs-specific binding protein GATA4, and staining with TPI1 antibody showed that TPI1 protein was expressed in SCs. The results could rule out contamination by other testis cell types, and the purified SCs could be used in subsequent experiments (Fig. 1).

Fig. 1 Immunofluorescence staining of primary SCs of Tibetan sheep (20×).

Point 7: For how many days were the cells cultured in vitro? After how many days were the cells transfected, and what was the transfection efficiency? The authors should provide information regarding the same in the material and methods section.

Response 7: Thanks for your comments, SCs were cultured in vitro and purified to the F5 generation for transfection, and the cells at 24, 48 and 72 after transfection were collected, and the transfection efficiency was detected by fluorescence qRT-PCR. It was found that the transfection efficiency at 48h was the highest. Therefore, other follow-up experiments were selected 48h after transfection. I have modified it in the article.

Point 8: The authors have studied the effect of TPI1 on Sc proliferation and apoptosis. There is a dogma that Sc proliferates during the neonatal infantile period and stops proliferating onset of puberty. Thus authors are suggested to check the expression of TPI1 changes during pubertal maturation of Sc. Along the same lines, does the expression of mi-1285-3p change during the functional maturation of Sc at puberty? Incorporation of this data would improve the quality of the manuscript.

Response 8: Thanks for your comments, our research group tested the expression of TPI1 in the testis of Tibetan sheep at different developmental stages, and found that the expression of its protein and mRNA before sexual maturity (3 mouth) was significantly lower than that of sexual maturity (1 year) and somatic maturity (3 years), and stabilized after sexual maturity.

Point 9: How does knock-down/over-expression of TPI1 regulate the mRNA levels of glycolytic pathway genes? Does TPI1 regulate the expression of these genes directly, or is the change in gene expression indirect (due to a shift in metabolic flux as a consequence of TPI1 knockdown/over-expression)? The authors should comment on this in the discussion section.

Response 9: Thanks for your comments, as one of the key enzymatic molecules in the glycolytic pathway, TPI1 catalyzes the conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate [11]. Then, under the action of GADPH, PGK, PGAM1, ENO, PKs, etc., it is converted into pyruvate and generates ATP, and then finally lactate is generated under the action of lactate dehydrogenase [9]. Therefore, changes in TPI1 gene expression can directly change the changes in the downstream genes of the glycolytic pathway, and through these genes regulate the changes of enzymes in the glycolytic metabolic pathway, and finally change the content of its final product, lactate.

Point 10: The authors are suggested to provide separate figures for the TPI1 knockdown and over-expression experiments along with the consequent effect(s) of knockdown/over-expression of TPI1 on Sc function (proliferation, apoptosis, gene expression etc..).

Response 10: Thanks for your comments, the first half of the article is the test of TPI1 gene knockdown or overexpression, and also explores the effect of TPI1 gene knockdown or overexpression on SCs proliferation, apoptosis and glycolysis metabolic pathways.

Point 11: Which gene was used to normalize the gene expression data? This needs to be mentioned in the materials and methods section.

Response 11: Thanks for your comments, β-actin gene was used to normalize the gene expression data, I have added in the methods section of the text.

Point 12: Results section 3.4- the authors state that “the expression of ENO1, PKM, LDHA, LDHB, MCT1, GAPDH, and PGK genes were significantly down-regulated in the si-PGAM1 group (P<0.05), indicating the over-expression of the PGAM1 gene could increase the expression of downstream genes while silencing the PGAM1 gene could reduce the expression of downstream genes”. PGAM1 needs to be replaced with TPI1 in the text.

Response 12: Thanks for your comments, I have changed “PGAM1” to “TPI1”in the text.

Point 13: There are major grammatical errors and awkward sentences in the manuscript which need to be corrected.

Response 13: Thanks for your comments, I have found native English speakers to revise the grammar and sentences.

Minor:

Point 1: Line 2. Repetition of sentences. breeds in China; Tibetan sheep (Ovis aries) is one of the three major coarse-haired sheep breeds in China. breeds in China.

Response 1: Thanks for your comments, I have modified it in the text.

Point 2: Page no-8 In the initial introduction, the importance of spermatogenesis should be brief and crisp. It covers half of the introduction with repeated sense.

Response 2: Thanks for your comments, I have modified it in the text.

Point 3: The authors should provide details of Ab used for isolation, polyclonal or monoclonal?

Response 3: Thanks for your comments, the construction of the vector was completed by Genewiz Biological. We just verified the double-enzyme digestion of the overexpression vector synthesized by them, so the single-clonal and polyclonal information was not described in detail in the method.

Point 4: Would the authors mind providing precise details of the sample collection procedure (collected from a slaughterhouse or by sacrifice) and the number of testicles collected for each experiment?

Response 4: Thanks for your comments, three well-grown 3-month-old Tibetan sheep were selected and brought back to the laboratory for intravenous injection of sodium pentobarbital, no heartbeat, continuous involuntary breathing for 2-3 min, no blink reflex, and then bilateral testes tissues were collected. I have modified it in methods section.

Point 5: No need to rewrite the material method in the result section.

Response 5: Thanks for your comments, I have modified the results section.

Point 6: The author should provide good-quality images. Fig 5 and 6 captions are not readable.

Response 6: Thanks for your comments, I have modified the Fig 5 and 6.

Response to Reviewer 2 Comments

We thank the reviewer for the time in closely viewing our manuscript. Most of the suggestions have been adopted in the revision.

Major issues:

Point 1: It was not possible to confirm the existence of a binding site to miR-1285-3p in the TPI1 gene using miRanda, because the website is no longer available. However, using miRDB (http://mirdb.org) it is possible to estimate two binding sites between TPI1 and miR-1285-3p in 3’ UTR, in humans. However, according to this database, there are 41 miRNAs targeting the TPI1 gene, and the miR-1285-3p is not the miRNA with higher “score”. The authors state in the introduction that this miRNA was selected because it is the most expressed in the Tibetan Sheep among the miRNAs with high affinity for the TPI1 gene. Yet, miR-1285-3p has 895 targets and TPI1 is not within the top 50 targets. Among the miR-1285-3p targets with higher score than TPI1 are several genes that could be involved in cell proliferation and metabolic regulation. Therefore, it is unlikely that the action of miR-1285-3p in Sertoli Cells is restricted to the regulation of TPI1 gene. To claim that, the authors would have to characterise the transcriptome of the Sertoli Cells transfected with the TPI1 inhibitor.

Response 1: Thanks for your comments, in our earlier studies, we explored the proteomics of sheep testis tissue and found that the TPI1 protein was expressed in sheep testis tissue. After conducting further functional annotations, we found that the TPI1 protein participates in the glycolytic metabolism pathway. In order to further verify its role in testicular development of Tibetan sheep, we further explored the expression and localization of TPI1 in testis tissue of Tibetan sheep at different developmental stages (prepubertal, sexual maturity and adulthood), and found that its mRNA and protein expression varied with the It increases with age and is mainly located in SCs. However, its role in SCs and its regulation of glycolytic metabolism have not been reported yet. This study mainly explored the role of TPI1 in the glycolysis process of Tibetan sheep SCs on the basis of previous studies. Since most of the previous research on TPI1 was in cancer, some reports on the regulation of miRNA on TPI1 were found in the literature when reviewing the literature, so combined with previous reports and prediction software (miRDB(http://www.mirdb.org) and Targetscan (http://www.targetscan.org), screened out several miRNAs that have a targeting relationship with TPI1, and found that miR-1285-3p had the highest expression in Sertoli cells, and the dual luciferase reporter gene assay was used to detect the high expression of miR-1285-3p. The targeting relationship between the three miRNAs and TPI1 was verified, and it was found that only miR-1285-3p had a targeting relationship, so this study directly selected miR-1285-3p for research.

Point 2: Related to the previous issue, the authors do not refer the parameters to predict miRNAs targeting TPI1 using miRanda, especially which animal database was used. Although the primers were specific to Tibetan Sheep, the target prediction was likely performed using a database for another species. This limitation must be discussed.

Response 2: Thanks for your comments, in this study, the first prediction was based on the human database through miRDB. After predicting that miR-1285-3p has a targeting relationship with TPI1, the sequence of sheep miR-1285-3p was downloaded through miRNAbase. The seed region of miR-1285-3p was aligned with the 3'UTR region of the ovine TPI1 gene, and it was found that it was completely complementary. Therefore, it was shown that TPI1 also has a targeting relationship with miR-1285-3p in sheep, so follow-up experiments were carried out.

Point 3: It is not clear to me how the miR-1285-3p mimic is expressed in Sertoli Cells by means of a plasmid, and the supplementary files are not available. How can the authors guarantee that the sequence will mimic the behaviour of the original miR-1285-3p? If the mimic sequence is expressed along the luciferase gene, then it must be processed post-transcriptionally. This must also be discussed by the authors.

Response 3: Thanks for your comments, the mimics and inhibitors are designed according to the gene sequence on the miRNAbase. The sense strand of the mimics sequence is the miRNA sequence, and the antisense strand is the sequence of the sense strand after removing the reverse complement of the last two bases and adding UU.The inhibitor sequence is the sequence after the complete reverse complementation of the base sequence. Synthetic miRNA mimics are chemical compounds of more than 20 bases, which simulate endogenous gene sequences through transfection with transfection reagents and then perform gene expression or knockdown.

Point 4: In section 3.5 and Figure 5, the authors mention the miR-3614-5p. Did the authors mean miR-1285-3p?

Response 4: Thanks for your comments, I made a typo, it should be miR-1285-3p, I have made changes in the text.

Minor issues:

Point 1: Please replace the term “testicles” by the term “testes” (singular “testis”).

Response 1: Thanks for your comments, I have modified it in the article.

Point 2: QRT-PCR -> qRT-PCR

Response 2: Thanks for your comments, I have modified it in the article.

Point 3: Multipanel figures must be labelled uniformly. In some figures the panels are ordered from left to right, top to bottom, but in other figures the authors label from top to bottom and only then from left to right. Although I understand the authors have a lot of information to show, please consider relegating some figure panels to supplementary data to improve the visualization of main manuscript figures.

Response 3: Thanks for your comments, I have modified the figures in the article.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

20 Apr 2022

15 May 2022

Response to Reviewer Comments

We thank the reviewer for the time in closely viewing our manuscript. Most of the suggestions have been adopted in the revision.

Point 1: Particularly, it is important to state in the "Methods" the parameters of the miRNA target prediction tool, especially the species database. For instance, miRDB does not have a sheep database, so I guess the authors have relied on another database. Then, this issue must be discussed as a limitation of the study.

Response 1: Thanks for your comments, in this study, the first prediction was based on the human database through miRDB. It was found that 6 miRNAs had a targeting relationship with TPI1. Then the miRNA sequences of sheep were downloaded by miRNAbase software, and the sequences were compared, and it was found that their seed regions were the same. Then primers were designed to detect the expression of these 6 miRNAs in Tibetan sheep SCs. It was found that the expression level of miR-1285-3p was the highest in Tibetan sheep, and then the targeting relationship between miR-1285-3p and TPI1 was further predicted. It was found that the seed region of miR-1285-3p and the 3'UTR of TPI1 gene could be completely complementary. Therefore, it showed that miR-1285-3p had a targeting relationship with TPI1, and we further verified the targeting relationship with the double luciferase reporter gene.

Point 2: Also, I cannot find in the text any reference about the author's previous work, notably the claim that miR-1285-3p is expressed the most in testis of Tibetan sheep. This previous work is important to support the rationale for the present study, especially due to the lack of information and databases in the species.

Response 2: Thanks for your comments, the result that miR-1285-3p was most expressed in Tibetan sheep SCs was originally included in Figure S1 as supplementary information. According to expert advice, I have put this part of the results in 3.5 and put the picture in Fig. 6F. As shown below:

We studied the expression of 6 miRNAs predicted to be targeted to TPI1 in SCs and found that the expression level of miR-1285-3p in SCs was significantly higher than that of other miRNAs(P<0.05) (Fig. 6F).

Fig. 6 TPI1 expression is downregulated by miR-1285-3p directly targeting of the 3’-UTR of TPI1. A, TPI1 was found for the potential regulatory targets of miR-1285-3p using prediction tool. B, Dual-luciferase reporter assay analysis of the target relationship of miR-1285-3p with TPI1. F, Expression of miRNA in SCs. The TPI1 expression levels were determined by real-time PCR analysis (D) western blot (H) after transfection with the miR-1285-3p mimics or negative control or after transfection with the miR-1285-3p inhibitor or negative control in SCs. CCK-8 (E), Flow cytometry (G) and the expression of proliferation, cycle and apoptosis related genes (C) was used to detect the apoptosis of SCs after transfection miR-1285-3p mimics, negative control, miR-1285-3p inhibitor or negative control. The expression levels of downstream genes (J) and the content of glycolytic metabolic pathway products, energy and key enzymes (I) of SCs after transfection miR-1285-3p mimics, negative control, miR-1285-3p inhibitor or negative control.

Point 3: There is a erroneous reference to Figure 5 in line 335.

Response 3: Thanks for your comments, I have modified it in the text.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

9 Jun 2022

miR-1285-3p targets TPI1 to regulate the glycolysis metabolism signaling pathway of Tibetan sheep Sertoli cells

PONE-D-21-36704R2

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14 Sep 2022

PONE-D-21-36704R2

miR-1285-3p targets TPI1 to regulate the glycolysis metabolism signaling pathway of Tibetan sheep Sertoli cells

Dear Dr. Ma:

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