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The glucose metabolism reprogramming of yak Sertoli cells under hypoxia is regulated by autophagy

BMC Genomics volume 26, Article number: 385 (2025) Cite this article

Abstract

Hypoxia often has negative effects on testis development and spermatogenesis of mammals. Plateau yaks have lived in the hypoxia environment for generations, but have ensured testicular function, which is closely related to their unique hypoxia response mechanism. Glucose metabolic reprogramming is an important way for cells to respond to stressful environments, especially the metabolite lactate, which is the energy basis for the development and differentiation of germ cells. In this study, hypoxia (5% O2) effectively promoted yak Sertoli cell proliferation and decreased autophagy and apoptosis. It was found that the cells showed good hypoxic adaptation. Metabolomics results showed that glucose metabolism was enhanced in yak Sertoli cells in response to hypoxia, and 13 glucose metabolites were increased, including the production and transport level of lactic acid (LA), which may have changed the pentose phosphate metabolic pathway of cells, these changes are conducive to support the glucose metabolism balance of cells under hypoxia. Crucially, when autophagy is activated under hypoxia, GLUT3, GLUT8, and MCT4 proteins are degraded, while GLUT1 and MCT1 are not affected, suggesting that autophagy may achieve glucose metabolic reprogramming by selectively regulating the expression of functional factors of glucose metabolism, which is conducive to energy intake and spermatogenesis in testis of yaks.

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Introduction

Hypoxia has a significant impact on the reproductive system of male mammals. Hypoxia in the spermatogenic environment caused by varicocele and testicular torsion reduces the number of undifferentiated spermatogonocytes and significantly increases the proportion of abnormal sperm, which can lead to oligospermia and low reproductive function [1,2,3]. After being treated with hypoxia, the mouse Leydig cells would undergo apoptosis, testosterone secretion decreased, and eventually infertility [2, 4]. Gene expression in the testis of hypoxic-exposed mice was altered, causing long-term damage to spermatogenesis [3, 5]. Therefore, hypoxia leads to changes in the internal environment of animal testis, which directly affects the development and differentiation of germ cells and spermatogenesis. However, the regulatory mechanism of hypoxia in plateau yaks seems to be different [6]. Yak has long lived on the Qinghai-Tibet Plateau at an altitude of 3000–5000 m and is a typical representative of plateau species [6, 7]. Compared with other breeds of cattle, yak has smaller, lighter testis, and more blood vessel distribution [8, 9]. These physiological characteristics ensure the normal development and spermatogenesis of yak testis [10]. Hypoxia often harms male reproduction, but yak shows excellent adaptability, so the molecular regulation mechanism of yak testis under hypoxia has also attracted the attention of humans.

Sertoli cells are important reproductive cells in male animals and produce lactate (LA) through glycolysis as the main energy source for spermatogenic cells [11,12,13]. The transmembrane transport of glucose into Sertoli cells is a rate-limiting step in the glycolytic pathway and is carried out by specific glucose transporters (GLUTs) [14,15,16]. Lactose dehydrogenase (LDH) also plays an important role in the process of lactate production. After LA is produced by Sertoli cells, it is transported from Sertoli cells to germ cells by monocarboxylate transporters (MCTs) [17, 18]. Studies have shown that LA effectively alleviates the apoptosis of testicular cells and contributes to the self-renewal of spermatogonocytes [19, 20]. Glucose metabolism is the basis of various physiological activities of cells in a hypoxia environment and is crucial for the maintenance of testis internal environmental homeostasis [21, 22]. Therefore, the precise regulations of glucose metabolism and the LA transport process are the key conditions for the normal maintenance of spermatogenesis [23, 24]. However, there is still a lack of data on how to achieve these regulations in yak testis.

Autophagy is an important physiological process for cells to maintain homeostasis when stimulated by external stimuli [25,26,27]. Studies have found that autophagy also plays an important role in the regulation of LA production and transport in Sertoli cells. Autophagy induced by heat stress promotes LA secretion in immature porcine Sertoli cells by driving the expression of GLUT3, LDHA, and MCT1 [28, 29]. Autophagy induced by ROS can promote the glycolysis process of Sertoli cells, to absorb more glucose to synthesize LA and promote LA secretion in Sertoli cells [23]. At present, it has been shown that hypoxia is closely related to glycolysis and LA transport in Sertoli cells, but its regulatory mechanism remains unclear, especially the role of autophagy in this process.

Many studies have shown that different culture conditions with low oxygen concentrations have different effects on the growth and proliferation of various animal cells. In the study of yak renal interstitial fibroblasts, it was found that the number of cell proliferation under 1% O2 was much lower than that under 20% and 10% O2, and the expression of apoptosis gene Caspase9 was significantly increased after 48 h treatment [30]. 5% O2 can temporarily promote the proliferation and HIF-1α expression of yak pulmonary artery smooth muscle cells, decrease the Bax/Bcl-2 ratio, and inhibit cell apoptosis [31]. Using oxygen at different concentrations (20%, 10%, 5%, and 1% O2) to mature yak oocytes and cultured yak parthenogenesis-activated embryos in vitro, it was found that the maturation rate of oocytes and the blastocyst rate of parthenogenesis-activated embryos were the highest under 5% O2 [32]. Under the culture condition of 2% O2, the apoptosis rate of type II alveolar epithelial cells in Tibetan pigs was significantly increased, resulting in cell damage [33]. These above studies have shown that 1% O2 is an extreme hypoxia condition, which is likely to cause a large number of cell apoptosis and a sharp reduction in cell number. The adaptation mechanism of hypoxia in cells may not be activated or is not sufficient to resist hypoxia damage, while a large number of cell apoptosis will not occur under 5% O2, and the proliferation level is not the same as the performance of hypoxia adaptation. A culture condition of 5% O2 is more suitable for studying the mechanism of cellular hypoxia adaptation.

Therefore, this study aimed to explore the effects of hypoxia (5% O2) on the proliferation, autophagy, and apoptosis of yak Sertoli cells, the changes of glucose metabolism in cells under hypoxia, and the regulation of autophagy on glucose metabolism and lactate transport in yak Sertoli cells under hypoxia. It provides new insights for analyzing the hypoxia adaptability of yak testis and improving the fertility of yaks.

Results

Culture and identification of yak Sertoli cells

When the primary yak Sertoli cells were cultured in vitro for about 1 week, it was observed under the microscope that the cells were irregularly fusiform, extended to all sides, and grew well (Fig. 1A-a). After 1 day of passage, the cells were laid out as monolayers and adjacent cells were cross-connected (Fig. 1A-b). After 2–3 days, adjacent cells were closely connected, and the cells were fused into sheets and spread into membrane-like monolayers (Fig. 1A-c, A-d).

Fig. 1
figure 1

Culture and Identification of yak Sertoli cells. A．Culture of yak Sertoli cells. a.12 h. b.24 h. c.48 h. d.72 h. B. Identification of yak Sertoli cells. a1-a3. Feulgen staining was observed in 100×, 200× and 400× field of view, respectively. b1-b3. Oil red O staining was observed in 100×, 200× and 400× field of view, respectively

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The second generation of purified yak Sertoli cells was identified by Feulgen staining and oil red O staining, respectively. Feulgen staining showed that the cytoplasm of the cells was light, the nucleus was dark, and purple particles were visible in the nucleus as special markers for Sertoli cells (Fig. 1B-a1, a2, a3). The results of oil red O staining showed the presence of red vacuole-like structures around the nucleus or at the cytoplasmic poles, indicating that Sertoli cells were successfully isolated and purified (Fig. 1B-b1, b2, b3).

Fig. 2
figure 2

Effects of hypoxia on the proliferation and characteristics of yak Sertoli cells. A. Cell proliferation was detected by the EdU method. Green fluorescence: EdU marks positive cells in a proliferative state. Blue fluorescence: Hoechst labeled cell nucleus. The scale is 200 μm. B. Cell proliferation rate calculation. Two-way ANOVA was used to analyze the differences in cell proliferation rates at 12, 24, 48 and 72 h under normoxia and hypoxia conditions. 4 replicates were set in each group, and the mean value was calculated for difference analysis. The intra-group errors were marked with the calculated SEM values as error bars. * on the bars indicates values that differ significantly (* P < 0.05), ** indicates values that differ extremely significantly (** P < 0.01). ns indicates no difference (ns P > 0.05). C. Morphology of yak Sertoli cells in normoxia group and hypoxia group. Normoxia: 21% O2. Hypoxia: 5% O2. The scale is 200 μm. D. Immunofluorescence staining of SOX9 in yak Sertoli cells under normoxia group and hypoxia group. Red fluorescence: Target proteins, respectively. Blue fluorescence: cell nucleus. The scale is 100 μm

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Fig. 3
figure 3

Effects of different time hypoxia treatments on autophagy and apoptosis levels of yak Sertoli cells. A. Effects of different time hypoxia treatment on autophagy and apoptosis genes expression in yak Sertoli cells. B. Effects of different time hypoxia treatment on autophagy and apoptosis proteins expression in yak Sertoli cells. 1, 3, 5, 7: Cell samples were cultured under normal oxygen (21% O2) for 12, 24, 48, and 72 h, respectively. 2, 4, 6, 8: Cell samples were cultured under hypoxia (5% O2) for 12, 24, 48, and 72 h, respectively. C. Relative expression of autophagy and apoptotic proteins

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Effects of hypoxia on the proliferation and characteristics of yak Sertoli cells

Yak Sertoli cells were cultured under normoxia (21% O2) and hypoxia (5% O2) conditions for 12, 24, 48, and 72 h, respectively, and the cell proliferation rate was detected by the EdU method. The results showed that the proliferation rate of cells in the normoxia group and the hypoxia group first increased and then decreased with time, and the proliferation rate was the fastest at 48 h, and the number and efficiency of cell proliferation in the hypoxia group were significantly higher than those in the normoxia group (Fig. 2A, B).

Yak Sertoli cells in the proper growth state of normoxia and hypoxia groups were fixed respectively. The results showed that the morphology of cells in the normoxia group and the hypoxia group was well under the bright field, and there was no obvious difference. There was also no difference in the expression sites of SOX9 protein in the normoxia group and the hypoxia group, and expression intensity was basically the same, indicating that hypoxia did not change the characteristics of yak Sertoli cells (Fig. 2C, D).

To sum up, hypoxia promoted the proliferation of yak Sertoli cells and did not change the morphology and characteristics of cells.

Effects of different time hypoxia treatment on autophagy and apoptosis level of yak Sertoli cells

Yak Sertoli cells were cultured under normoxia and hypoxia conditions for 12, 24, 48, and 72 h, and the expression of target genes was detected by the SYBR Green-based q-PCR method. The results showed that the expression levels of Atg5, Atg12, Beclin1, and LC3 in the two group cells changed with the culture time, and the increase was most significant between 48 and 72 h (P < 0.05). The expression levels of Atg5, Atg12, Beclin1, and LC3 genes decreased significantly after hypoxia treatment, and the differences were significant at 48 h (P < 0.05). The expressions of apoptotic genes Bax and Caspase3 in the hypoxia group were significantly decreased at 48 h (P < 0.05), and the expression of anti-apoptotic gene Bcl-2 was significantly increased (P < 0.01) (Fig. 3A).

WB results showed that the expression levels of ATG5, Beclin1, LC3, and P62 of cells in both the normoxia group and hypoxia group increased with culture time, and the difference was most significant between 48–72 h. The expression levels of ATG5, Beclin1, and LC3 proteins were significantly decreased after hypoxia treatment, while the expression levels of P62 were significantly increased, with significant differences at 48 h (P < 0.05). In the hypoxia group, the expression of apoptotic protein Bax was significantly decreased at 48 h, and the expression of anti-apoptotic protein Bcl-2 was significantly increased (P < 0.05) (Fig. 3B, C).

To sum up, hypoxia decreased the autophagy and apoptosis levels of yak Sertoli cells, and the effect was significant at 48 h.

In this figure, two-way ANOVA was used to analyze the differences in genes and proteins expression at 12, 24, 48 and 72 h under normoxia and hypoxia conditions. 4 replicates were set in each group, and the mean value was calculated for difference analysis. The intra-group errors were marked with the calculated SEM values as error bars. * on the bars indicates values that differ significantly (* P < 0.05), ** indicates values that differ extremely significantly (** P < 0.01). ns indicates no difference (ns P > 0.05).

Metabolomics analysis of the effects of hypoxia on yak Sertoli cells

Quality control and identification of metabolites of yak Sertoli cells

Total Ion Chromatogram (Fig. 4A-a) and metabolite m/z-rt distribution (Fig. 4A-b) were obtained by testing the metabolites of yak Sertoli cells in normoxia and hypoxia groups. The results showed that the machine ran stably and all metabolites were well separated by liquid chromatography. The PLS-DA model could better express and predict the difference between yak Sertoli cells in normoxia and hypoxia groups (Fig. 4A-c). The Intercept values of the regression line and Y-axis obtained by the permutation test plot are Intercepts. R2 and Intercept. Q2, Q2 < 0 indicates that there is no overfitting of the model, and the differential metabolite analysis is more accurate (Fig. 4A-d).

Fig. 4
figure 4

Metabolomics analysis of the effects of hypoxia on yak Sertoli cells. A: Quality detection of metabolites in all samples.a.Total Ion Chromatogram. b. Metabolite m/z-rt distribution. c. The PLS-DA model. d. Permutation test diagram. B: Statistics and analysis of all metabolites in yak Sertoli cells treated with hypoxia. a. Thermogram analysis of metabolic ions in individual cell samples. b. Enrichment of all metabolic ion pathways in yak Sertoli cells. c. TOP20 pathway of secondary metabolite enrichment. C: Differential metabolic ion count statistics. D: Statistics and analysis of differential metabolites in yak Sertoli cells treated with hypoxia. a. Differential metabolites up-regulated and down-regulated after hypoxia treatment of yak Sertoli cells. b. Heat maps of differential metabolites of yak Sertoli cells in normal and hypoxia groups. c. Volcanic maps of differential metabolites of yak Sertoli cells in normal and low oxygen groups. d. KEGG enrichment pathway of differential metabolites of yak Sertoli cells in normal and hypoxia groups

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By comparing and analyzing all the secondary metabolites detected, it was found that the metabolites of yak Sertoli cells mainly included alkaloids and their derivatives, benzene ring compounds, homogeneous nonmetallic compounds, hydrocarbons, lipid and lipid molecules, and nucleosides. Nucleotides and analogs, organic acids and their derivatives, organic nitrogen compounds, organic oxygen compounds, and other types (Fig. 4B-a). KEGG enrichment analysis was performed on all secondary metabolites to obtain TOP20 signaling pathways. It was found that metabolites were mainly concentrated in metabolic pathways, ABC transporters, central carbon metabolism, protein digestion and absorption, amino acid biosynthesis, aminoacyl-tRNA biosynthesis, mineral absorption, bile secretion, 2-oxo-carboxylic acid metabolism and glycerophospholipid metabolism (Fig. 4B-b, c). Metabolomics raw data results are included in the supplement (Table combine-norm-metaboAnalystInput).

Differential metabolite analysis of yak Sertoli cells under hypoxia treatment

After hypoxia treatment, 228 differentially metabolized ions were screened in positive ion mode, of which 91 were significantly up-regulated and 137 were significantly down-regulated. A total of 154 differentially metabolized ions were screened in the negative ion mode, with 43 up-regulated and 111 down-regulated ions (Fig. 4C, D-a, b, c). Specific analysis results are included in the supplementary materials (Table H_N.significant).

By analyzing the KEGG ID and MBROLE pathways of secondary annotated differential metabolites, the potential differential metabolic pathways of serine, glycine, and threonine metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolipid metabolism, carbon metabolism, glyoxylate and dicarboxylate metabolism, choline metabolism, arginine and proline metabolism in cancer, metabolic pathway in testicular hypoxic response were enriched. (Fig. 4D-d).

The key differential glucose metabolites in yak Sertoli cells under hypoxia were detected by targeted metabolism

Based on the results of metabolomics, we selected the energy metabolic pathway among the enriched signaling pathways for further targeted metabolic analysis. XIC (Extracted Ion Chromatogram) was obtained by the detection of metabolites from yak Sertoli cells in the normoxia and hypoxia groups. The results showed that the metabolites were well separated and their peaks were sharp and symmetrical. The metabolites could be quantified by mass spectrometry (Fig. 5A). All samples were mixed in equal amounts to prepare QC samples, and the RSD result of QC samples was less than 30%, which proved that the data of the object to be tested in QC samples was stable and reliable (Fig. 5B).

Fig. 5
figure 5

Effects of hypoxia on key energy metabolites of yak Sertoli cells. B. A: XIC (Extracted Ion Chromatogram). B: RSD value of the sample. C: Quantitative analysis of 15 differential cell metabolites between normal and hypoxic groups. * on the bars indicates values that differ significantly (* P <0.05), ** indicates values that differ extremely significantly (** P <0.01). ns indicates no difference (ns P > 0.05)

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Quantitative examination of the target metabolites contained in the samples showed that 15 energy-related metabolites in the cells changed significantly after hypoxic treatment. Among them, 13 metabolites were significantly upregulated, which were lactic acid, flavin mononucleotide, ADP, thiamine pyrophosphate, GDP, β−6-fructose phosphate, NAD + , cAMP, NADP + , 1, 6-fructose diphosphate, phosphoenolpyruvate, isocitric acid, and L-malic acid. And 2 metabolites were significantly down-regulated, namely aconite acid and reduced nicotinamide adenine dinucleotide phosphate (Fig. 5C a-o). Standard curve results for each differential metabolite are in the supplement (target metabolomics_linear). Quantitative analysis of metabolites is included in the supplement (Table Metabolite quantification).

Regulation of autophagy level in yak Sertoli cells under hypoxia by rapamycin (RAPA)

Rapamycin is a commonly used and effective autophagy activator. mTOR is a protein kinase that is involved in regulating many physiological processes such as cell growth, metabolism and autophagy. Rapamycin is able to bind to the intracellular FK506-binding protein 12 (FKBP12), forming a complex that inhibits mTOR activity and leads to initiation of autophagy. So we used it in cell culture to regulate autophagy level. q-PCR results showed that the expression of autophagy-related genes in the normoxia group (21% O2), hypoxia group (5% O2), and rapamycin group (5% O2 + RAPA) were significantly different. The expressions of ATG5, ATG12, Beclin1, and LC3 genes in the hypoxia group were significantly decreased (P < 0.05), while the expressions of autophagy genes ATG5, ATG12, Beclin1, and LC3 genes were significantly increased (P < 0.05) after the addition of autophagy activator rapamycin in cells compared with the hypoxia group (Fig. 6A).

Fig. 6
figure 6

Regulation of autophagy level in yak Sertoli cells under hypoxia by autophagy activator rapamycin (RAPA). A: Autophagy activator Rapamycin regulates mRNA levels of autophagy-related genes in yak Sertoli cells under hypoxia. B:Western-blot detection of mTOR, Atg5, Beclin1, LC3I, LC3II, P62 and β-actin proteins. C: Relative expression of autophagy-related proteins. D: Autophagy activator Rapamycin regulates autophagy-related protein levels in yak Sertoli cells under hypoxia. Red fluorescence: Target proteins, respectively. Blue fluorescence: cell nucleus. The scale is 100 μm. E: Fluorescence intensity analysis of proteins

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WB results showed that hypoxia increased the expression of mTOR protein, decreased the expression of autophagy related proteins ATG5, Beclin1, LC3I, LC3 II and LC3 II/LC3I (P < 0.05), and slightly increased the expression of P62 protein. In rapamycin group, the expression of mTOR protein was significantly decreased (P < 0.01), the expression of ATG5 and Beclin1 protein was significantly increased (P < 0.05), and the expression of P62 protein was significantly decreased (P < 0.05). The protein expressions of LC3I, LC3 II and LC3 II/LC3I were significantly increased (P < 0.05) (Fig. 6B, C).

The results of indirect immunofluorescence showed that ATG5, Beclin1, P62, and LC3 proteins were dotted in yak Sertoli cells and expressed strongly in the cytoplasm. The expression intensity of ATG5, Beclin1, and LC3 protein decreased significantly after hypoxia treatment, while the expression of P62 protein increased significantly, and the expression levels of ATG5, Beclin1, and LC3 protein increased significantly in the rapamycin group compared with that in the normal oxygen group. The expression of P62 protein was decreased, which was higher than that in the hypoxia treatment group (Fig. 6D, E).

The results showed that rapamycin successfully activated autophagy in yak Sertoli cells.

In this figure, unpaired t-test was used to analyze the differences between the normoxia group and the hypoxia group, and between the hypoxia group and the rapamycin group (21% O2 vs 5% O2, 5% O2 vs 5% O2 + RAPA). There were 4 replicates in each group, and the average value was taken for difference analysis. The calculated SEM value is used as the error bar to mark the intra-group error. * on the bars indicates values that differ significantly (* P < 0.05), ** indicates values that differ extremely significantly (** P < 0.01). ns indicates no difference (ns P > 0.05).

Regulation of glucose metabolism and LA transport by rapamycin in yak Sertoli cells under hypoxia

In the part of the experiment on the effect of hypoxia on autophagy of yak Sertoli cells, we found that hypoxia reduced the autophagy activity of yak Sertoli cells, which was different from the performance of increased autophagy in other mammalian cells under hypoxia conditions, which attracted our attention. At the same time, metabolomics techniques were used to detect the reprogramming of glucose metabolism and the production of LA and other glucose metabolites in yak Sertoli cells. It was found that glycolysis was enhanced to produce a large amount of LA. Glucose transporters (GLUTs) and lactate transporters (MCTs) may play important regulatory roles in this process. Then, does the decrease of autophagy in yak Sertoli cells promote glucose metabolism? In this section, we use rapamycin to activate autophagy and explore the regulatory relationship between autophagy and glucose metabolism. q-PCR results showed that after hypoxia treatment, the mRNA levels of glucose transporters GLUT1, GLUT3, GLUT8, and lactate transporters MCT1, MCT2, MCT4 were significantly higher than those of the normal oxygen group. The expression levels of GLUT1, GLUT3, GLUT8, MCT2 and MCT4 were significantly different (P < 0.01). However, GLUT1 did not change significantly after rapamycin treatment in the hypoxia group, and the gene expressions of MCT1 and MCT2 were decreased significantly (P < 0.05), and the gene expressions of GLUT3, GLUT8, and MCT4 were also decreased significantly (P < 0.01) (Fig. 7A).

Fig. 7
figure 7

Regulation of LA production and transport by autophagy activator rapamycin (RAPA) in yak Sertoli cells under hypoxia. A: Autophagy activator Rapamycin regulates lactate metabolism related genes in yak Sertoli cells under hypoxia. B: Western-blot detection of GLUT1, GLUT3, GLUT8, MCT1, MCT4 and β-actin proteins. C: Relative expression of lactate metabolism related proteins.D: Regulation of Rapamycin on lactate metabolism related proteins in yak Sertoli cells under hypoxia. Red fluorescence: Target proteins, respectively. Blue fluorescence: cell nucleus. The scale is 100 μm. E:Fluorescence intensity analysis of proteins. F:Regulation of Rapamycin on lactate secretion in yak Sertoli cells under hypoxia. a:The establishment of a standard curve of LA. b: Extracellular and intracellular LA content in yak Sertoli cells. c: Lactate transport rate in yak Sertoli cells. d: The establishment of a standard curve of LDH. e: Detection of LDH in yak sertoli cells

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The results of WB showed that GLUT1 and GLUT3 expression levels were significantly increased after hypoxia treatment compared with the normal oxygen group, but GLUT8 expression level had no significant change. The protein expressions of MCT1 and MCT4 were increased, and there was no significant difference in the expression of MCT1 (P > 0.05), the expression of MCT4 was significantly different (P < 0.01). After rapamycin treatment, the expression levels of GLUT3 and GLUT8 in the hypoxia group were decreased significantly (P < 0.05), the expression level of GLUT1 protein was not significantly changed, the expression level of MCT4 protein was decreased (P < 0.01), and the expression level of MCT1 protein was not significantly changed (Fig. 7B, C).

The results of indirect immunofluorescence showed that GLUT1 and GLUT8 proteins were positively expressed in the nucleus and cytoplasm of yak Sertoli cells, and GLUT3, MCT1, and MCT4 proteins were punctate expressed in the cytoplasm of yak Sertoli cells. After hypoxia treatment, the expression intensity of GLUT1, GLUT3, and MCT4 protein in the normal oxygen group, hypoxia group (5% O2), and rapamycin group were different. Compared with the normoxia group, the expression intensity of GLUT1, GLUT3, and MCT4 protein was significantly increased (P < 0.05) after hypoxia treatment. The expression levels of GLUT3, GLUT8, and MCT4 were significantly lower than those in the hypoxic treatment group (P < 0.05) (Fig. 7D, E).

At the same time, the lactate content and LDH activity of intracellular and cellular supernatants were determined by establishing the standard curve equations of LA and LDH, and the lactate transport rate was calculated. The results showed that after 48 h of hypoxia treatment, lactate content in intracellular and cellular supernatants increased significantly (P < 0.01), while LDH activity had no significant effect (P > 0.05). When rapamycin was added to hypoxia treatment, lactate content in both intracellular and cellular supernatant was significantly decreased (P < 0.05), while LDH activity was decreased (P < 0.05) (Fig. 7F).

To sum up, the results showed that glucose metabolism, LA secretion, and transport rate decreased significantly after autophagy activation in yak Sertoli cells.

In this figure, unpaired t-test was used to analyze the differences between the normoxia group and the hypoxia group, and between the hypoxia group and the rapamycin group (21% O2 vs 5% O2, 5% O2 vs 5% O2 + RAPA). There were 4 replicates in each group, and the average value was taken for difference analysis. The calculated SEM value is used as the error bar to mark the intra-group error. * on the bars indicates values that differ significantly (* P < 0.05), ** indicates values that differ extremely significantly (** P < 0.01). ns indicates no difference (ns P > 0.05).

Discussion

Studies have shown that hypoxia can affect the growth state of a variety of cells. Such as, hypoxia leads to apoptosis of testicular cells in mice and rats, and 5% O2 significantly increases the apoptosis rate of Sertoli cells in vitro [1, 34]. In studies of porcine granulosa cells, hypoxia was found to activate autophagy and apoptosis [35]. Yaks have different manifestations in the face of hypoxia. In the study of hypoxia adaptation of yak oocytes, using different concentrations of oxygen (20%, 10%, 5%, and 1% O2) to mature oocytes and in vitro culture of parthenogenic activated embryos, it was found that the maturity rate of oocytes under 5% O2 hypoxia was the highest, and the blastocyst rate of parthenogenic activated embryos was the highest [36]. Other studies have shown that autophagy can alleviate the quality decline of yak oocytes and early embryos caused by extreme hypoxia (1% O2), and promote oocyte maturation [37]. By hypoxia treatment (5% O2) of yak Sertoli cells, it was found that autophagy and apoptosis of cells were decreased. Different species of cells or different organ types of cells have different levels of tolerance to oxygen. And the demand for oxygen varies according to the nature, age, function, or environment of the cell. Cultured yak Sertoli cells with 21% O2 suitable for most mammalian cells seem to have superfluous oxygen in the cells, and 5% O2 is the best oxygen level for yak Sertoli cells. 5% O2 under laboratory conditions may represent normal oxygenation of yak Sertoli cells, while 21% O2 is in a hyperoxic state, resulting in increased cell death, reduced proliferation and activation of autophagy. The results were similar to those found in heart muscle cells. In human cardiomyocytes, it was found that the in vitro proliferation of neonatal cardiomyocytes could be increased by reducing the oxygen concentration from the "conventional" (20% O2) level to the "physiological" (3% O2) level, indicating that high oxygen concentration has an inhibitory effect on the proliferation of cardiomyocytes in vitro [38]. These results indicated that the yak Sertoli cells grow well under hypoxia, showing good hypoxia adaptability, and the enhancement of anti-apoptosis ability of cells was conducive to the development of yak testis and spermatogenesis. In further research, we found that this may be related to its particular metabolic pattern.

Then, through the establishment of metabolic profiles and the identification and comparison of metabolites of yak Sertoli cells in the normoxia group and the hypoxia group. It was found that after hypoxia treatment, LA, fructose 6-phosphate, ADP, and other substances were significantly increased, and NADPH was significantly decreased. In addition, we found that hypoxia has a significant effect on the pentose phosphate pathway (PPP), and NADPH is a key product of PPP. Studies have shown that glucose metabolic reprogramming is a common adaptive response of tumor cells under hypoxic conditions that regulate the growth and metastasis of osteosarcoma [39,40,41]. In glioblastoma, AMPK-HIF-1α signaling was found to enhance glucose-derived serine biosynthesis and promote cell growth [42, 43]. In studies of porcine Sertoli cells, melatonin was found to reduce heat stress-induced damage by reprogramming glucose metabolism [44]. Therefore, in this study, hypoxic reprogramming of glucose metabolism in yak Sertoli cells may be the key to their adaptation to hypoxia, which not only meets the energy requirements of germ cell development but also releases nutrients such as LA, which is conducive to maintaining the microenvironment of sperm tubules of yaks. Other studies have shown that PPP mainly produces important molecules such as 5-phosphoribosyl synthesis nucleotides, ATP, CoA, NAD + , and NADPH as a hydrogen donor to participate in various metabolic reactions, which is extremely important for cell reoxidation [45, 46]. The non-oxidizing branch of PPP consists of a series of reversible reactions to produce several important metabolites such as glucose-6-phosphate (F6P), glyceraldehyde-3-phosphate (G3P), and pentose phosphate, which supplement the needs of glycolysis and anabolism and also provide conditions for the mutual conversion of various monosaccharides [46, 47]. Studies have shown that when LA accumulates, PPP inhibits glycolysis and avoids tissue superacid [48, 49]. These results suggest that yak Sertoli cells have strong glycolytic ability, and the change of PPP assisted the glycolytic process to maintain glucose metabolism and cell homeostasis under hypoxia condition. The decrease of NADPH indicated that lactate of yak Sertoli cells did not accumulate too much, and thus adapted well to the hypoxia environment by adjusting metabolic pattern. Autophagy and reprogramming of glucose metabolism are important ways for cells to cope with hypoxia [50,51,52]. Yaks have a good ability to cope with the hypoxia environment, but whether autophagy plays a regulatory role in the glucose metabolism of yak Sertoli cells under hypoxia is still unclear. Whether the decreased autophagy level of yak Sertoli cells induced by hypoxia is involved in the regulation of glucose metabolism reprogramming and LA secretion is worthy of further study.

Studies have shown that ROS-induced autophagy promotes the increase of LA synthesis in TM4 cells [53]. In liver cancer cells, studies have found that inhibition of autophagy can significantly reduce glucose uptake and promote glycolysis by upregulating MCT1 [54]. Heat stress-induced autophagy promotes lactate secretion by activation of GLUT3, LDHA, and MCT1 [55]. In hypoxia-induced glucose metabolic reprogramming, inhibition of autophagy may promote the expression of glucose transporters GLUT1, GLUT3, and GLUT8, thereby accelerating cellular glycolysis to lactate. However, the lactate transporter MCT4 is sensitive to hypoxia, and increased expression suggests that hypoxia may benefit germ cell energy uptake. On this basis, rapamycin was applied to hypoxic yak Sertoli cells. Rapamycin can activate autophagy by inhibiting mTOR, which is commonly used as an autophagy activator [56]. The results showed that the expression of autophagy genes and proteins was significantly increased, and the expression of P62 protein was significantly decreased after the addition of rapamycin. When LC3 II increased, P62 decreased at the same time, indicating that autophagy flow was stable. Rapamycin effectively activated autophagy in yak Sertoli cells, and it was found that autophagy activation had no significant effect on GLUT1 and MCT1. Interestingly, the expression of GLUT3 and GLUT8 was significantly reduced, the production of lactic acid in the intracellular and culture supernatant was reduced, and the expression of MCT4 was significantly reduced. Cells remove metabolic waste and excess proteins through autophagy [57], and the reduction of GLUT3, GLUT8, and MCT4 may be related to this. LA is the source of energy for germ cells. Although MCT1 and MCT4 belong to the same family of proteins, they have different transport functions for lactate. MCT1 controls the intake and inflow of LA, while MCT4 is involved in the excretion and outflow of LA [58, 59]. LA content and transport rate are necessary conditions for spermatogenesis, and LA has been found to inhibit the apoptosis of human testicular germ cells. This study confirmed that under hypoxia conditions, the decrease in the autophagy level of yak Sertoli cells has a positive regulatory effect on LA secretion, which is more conducive to the energy uptake and utilization of germ cells. Therefore, it plays an important role in maintaining the homeostasis of the testis.

In conclusion, yak Sertoli cells have a good hypoxia adaptation. Under hypoxia conditions, glycolytic activity increases, and PPP changes significantly, which contributes to cell proliferation. Glucose metabolic reprogramming may be the key to adaptation to hypoxia. Moreover, hypoxia can reduce the autophagy level of yak Sertoli cells, increase the expression of GLUT3, GLUT8, and MCT4 proteins, promote glucose transport and metabolism, and increase the production and transport level of LA. These changes in Sertoli cells provided the necessary conditions for yak spermatogenesis(Fig. 8).

Fig. 8
figure 8

Regulatory relationship between autophagy and LA transport in yak testis Sertoli cells under hypoxia

Full size image

Materials and methods

Main reagents and instruments

DMEM F12 culture medium, fetal bovine serum, penicillin, and streptomycin were purchased from Gibco (USA). MicroRNA extraction kit and two-step reverse transcription Kit were purchased from Promega (USA). SYBR Green II Fluorescent Quantitative PCR Kit was purchased from Accurate Biotechnology Company (China). Lactate content detection kit, lactate dehydrogenase activity detection kit, immunofluorescence detection reagents, and Western blot (WB) reagents used were purchased from Nanjing Beyotime Biological Company and Beijing Solarbio Company.

Culture and identification of primary yak Sertoli cells

Five pairs of adolescent yak testicles were collected from Tianzhu, Gansu Province, China, placed in sterile saline at 35- 37 °C, and brought back to the laboratory within 4 h. In vitro isolation, culture and identification of yak Sertoli cells refer to the operation methods of Zomer et al. [60] and H. Zhang et al. [61]. The cells were separated by the combined digestion method of collagenase and trypsin pancreatic enzyme, hypotonic treatment with 20 mmol·L−1 Tris–Hcl, and starvation culture to purify the cells. The cells were then cultured in DMEM F12 medium containing 10% FBS under the conditions of 37 ℃, 5% CO2, and 21% O2. After cell growth was stable, Feulgen staining and oil red O staining were used for identification.

Hypoxia and the autophagy activator rapamycin (RAPA) treated the cells

First, yak Sertoli cells were cultured under normoxia (21% O2) and hypoxia (5% O2) conditions for 12 h, 24 h, 48 h, and 72 h, respectively, and samples were collected at each time point for follow-up detection. Then, to further explore the regulatory effects of autophagy on glucose metabolism and lactate transport of yak Sertoli cells, we set up cells in normoxia, hypoxia, and RAPA groups. In the RA group, cells in the hypoxia (5% O2) group were added with 25 nM rapamycin for 24 h, and then cells were cultured for 24 h with the culture medium without rapamycin.

Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay

Yak Sertoli cells cultured for 12 h, 24 h, 48 h and 72 h in normoxia group and hypoxia group were taken, preheated 2 × EdU (20 μM) at 37 ℃, and added EdU to cells in the same volume as the culture medium 1:1, then return in the incubator for further culture for 2 h. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Removed fixing solution and washed 3 times with PBS for 3–5 min each time. Used 0.3% Triton X-100 to penetrate for 10–15 min. The liquid was removed and the cells were washed with PBS 3 times per well for 3–5 min each time. Added Click reaction solution and incubated cells for 30 min. The reaction solution was removed and the cells were washed 3 times with PBS for 3–5 min each time. Nuclear staining was performed by incubation with Hoechst at room temperature for 10 min, sheltered from light. The cells were observed under a fluorescence microscope. Positive cell counts were performed using Image J.

Extracted RNA, synthesized cDNA, and used quantitative real-time PCR (qPCR) to detect the relative expression of the target genes

After 48 h of cell culture, the cells were cleaned 3 times with Phosphate Buffer Saline (PBS), total RNA was extracted with a microRNA extraction kit, and cDNA was synthesized with a two-step reverse transcription kit. The primers were designed according to the bovine mRNA reference sequence provided by GenBank (Table 1), and the expressions of Atg5, Atg12, Beclin1, LC3, Bax, Bcl-2, Caspase3, GLUT1, GLUT3, GLUT8, MCT1, MCT2 and MCT4 genes were detected by qPCR. The reaction system was 20 μL: cDNA (100 ng/μL) 2 μL, forward and reverse primes (0.2 μmol/mL) 0.8 μL, 2 × SYBR Premix Ex Taq II 10 μL, Nuclease-Free Water 6.4 μL. Reaction conditions: predenaturation at 95 ℃ for 10 s, denaturation at 95 ℃ for 10 s, annealing for 10 s (specific temperature was shown in Table 1), extension at 72 ℃ for 10 s, 40 cycles, 4 repeat groups were set for each sample, and the reference gene was β-actin. The 2−ΔΔCt formula was used to calculate the relative expression of target genes.

Table 1 Primer sequence information
Full size table

The effect of hypoxia on the metabolism of yak Sertoli cells was detected by Liquid Chromatography-Mass Spectrometry (LC–MS)

The cells were treated according to the metabolomics sample request provided by Lianchuan Biological Technology Co., Ltd. (Hangzhou, China). XCMS software was used to process the original data files generated by LC–MS/MS, and the collected MS data were pre-processed. The LC–MS raw data files are converted to mzXML format and then processed by R software-based XCMS, CAMERA, and metaX tools. Each ion is identified in combination with the retention time (RT) and m/z data, the intensity of each peak is recorded, and a three-dimensional matrix is generated containing any specified peak index (retention time-m/z pairs), sample name (observations), and ion strength information (variables). Using KEGG, HMDB online database, the samples were matched with the accurate molecular mass data (m/z) in the database, and the metabolites were annotated. Meta X was used to further preprocess the intensity of peak data. The features detected in 50% QC samples or 80% biological samples are eliminated, and the peaks of the remaining missing values are added by the K-nearest neighbor algorithm to further improve the data quality. The pre-processed data were used for principal component analysis to detect outliers and evaluate batch processing effects, and the relative standard deviations of metabolic characteristics of all QC samples were calculated. Remove components with large deviations. T-test was used to detect the difference in metabolite concentration between the two phenotypes. The P value was adjusted several times using FDR (Benjamini-Hochberg). PLS-DA is monitored by metaX, with different variables between the groups. Calculate the VIP value with a VIP critical value of 1.0 for selecting important features.

Targeted energy metabolomics analysis based on the MRM method

Sample separation was performed using the Agilent 1290 Infinity LC ultra-high-performance liquid chromatography system. The samples were placed in an automatic injector at 4 ℃, the column temperature was 35 ℃. The mobile phase A: 50 mM ammonium acetate aqueous solution + 1.2% ammonium hydroxide. The mobile phase B: 1% acetyl acetone acetonitrile solution, the flow rate was 300 μL/min, and the sample size was 2 μL. The relevant liquid phase gradient was as follows: 0–1 min, liquid B 70%, 1–10 min, liquid B from 70% linear change to 60%. In 10–12 min, liquid B changed linearly from 60 to 30%. At 12.1–15 min, liquid B remained at 30%, at 15–15.5 min, liquid B changed linearly from 30 to 7%, at 15.1–22 min, liquid B remained at 70%. A QC sample was set up for every certain number of experimental samples in the sample queue to detect and evaluate the stability and repeatability of the system. The sample cohort was set to a standard mixture of target substances for correction of chromatographic retention time. The 5500 QTRAP mass spectrometer (SCIEX) was used for mass spectrometry in negative ion mode. The 5500 QTRAP ESI source conditions: source temperature 450 ℃, ion Source Gas1 (Gas1): 45, Ion Source Gas2 (Gas2): 45, Curtain gas (CUR): 30, ionSapary Voltage Floating (ISVF) −4500 V. Detect the ion pair under test in MRM mode. The chromatogram peak area and retention time were extracted by Multiquant 3.0.2, and the retention time was corrected by the standard of the target substance to identify the metabolites.

The expression and localization of target proteins were detected by indirect immunofluorescence staining (IF)

When the cells covered about 80% of the slide, fixed in 4% paraformaldehyde at room temperature for 1 h, washed 3 times with PBS, incubated with 0.5% Triton X-100 at room temperature for 30 min, and washed 3 times with PBS. Next, 1% BSA was mixed in the cell for 2 h. And cells were separately incubated with anti-Sox9 (1:250 dilution, Bioss, Beijing, China), anti-Atg5 (1:500 dilution, NOVUS, Littleton, US), anti-Beclin1 (1:500 dilution, Prepared by our laboratory, China), anti-P62 (1:500 dilution, Prepared by our laboratory, China), anti-LC3 (1:500 dilution, Prepared by our laboratory, China), anti-GLUT1 (1:250 dilution, Proteintech, Wuhan, China), anti-GLUT3 (1:250 dilution, Bioss, Beijing, China), anti-GLUT8 (1:250 dilution, Bioss, Beijing, China), anti-MCT1 (1:250 dilution, Bioss, Beijing, China), and anti-MCT4 (1:250 dilution, Bioss, Beijing, China) overnight at 4 ℃. Subsequently, cells were paired with secondary antibody Alexa Fluor 594 labeled Goat anti-rabbit IgG (1:500 diluted; Bioss, Beijing, China) incubated for 1 h. Washed each step with PBS 3 times before restaining with DAPI (4' −6-diamidine-2-phenylindole) for 3 min. The cells were examined with a fluorescence microscope and photographed.

Western blot was used to detect the relative expression of target proteins

The cells were fully lysed with protein lysis buffer for 30 min, centrifuged at 10, 000 × g for 15 min, and the supernatant was collected and stored in the refrigerator at −80 ℃. Sodium dodecyl sulfate (SDS) loading buffer was added and the protein was denatured in a metal bath at 100 ℃ for 15 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was taken with 10 μL loading amount, and the proteins were transferred to the PVDF membrane after electrophoresis. Blocked at room temperature for 2 h, anti-mTOR (1:1000 dilution, Affinity, Jiangsu, China), anti-Atg5 (1:500 dilution, NOVUS, Littleton, US), anti-Beclin1 (1:1000 dilution, Prepared by our laboratory, China), anti-P62 (1:1000 dilution, Prepared by our laboratory, China), anti-LC3 (1:1000 dilution, Prepared by our laboratory, China), anti-Bax (1:1000 dilution, Affinity, Jiangsu, China), anti-Bcl-2 (1:1000 dilution, Affinity, Jiangsu, China), anti-GLUT1 (1:1000 dilution, Proteintech, Wuhan, China), anti-GLUT3 (1:500 dilution, Bioss, Beijing, China), anti-GLUT8 (1:500 dilution, Bioss, Beijing, China), anti-MCT1 (1:500 dilution, Bioss, Beijing, China), and anti-MCT4 (1:500 dilution, Bioss, Beijing, China) incubated cells at 4 ℃ overnight, respectively, and cleaned with PBST. Then the secondary antibody was incubated at room temperature for 1 h, and cleaned with PBST. The electrochemical luminescence (ECL) kit was used to expose the protein for 1–5 min. And the protein bands were photographed and analyzed when they were clear. Image Pro Plus software was used to measure the IOD value of the protein strip, and the relative protein expression was analyzed according to the IOD value (protein expression levels = target IOD/ internal reference IOD).

Determination of LA content and LDH activity

According to the instructions provided by Solarbio company, the LA content in the cells was measured with the ratio of cell number (104) and extraction solution (mL) 500–1000:1, and the cells were broken by ultrasonic in an ice bath (power 300 w, ultrasonic 3 s, interval 7 s, total time 3 min). Centrifuged at 12,000 g for 10 min (4 °C), took 0.8 mL supernatant, added 0.15 mL extract solution II, centrifuged at 12,000 g for 10 min (4 °C), and took supernatant to be measured. Dilution of standard solution: Dilute 100 μmol/mL standard solution with distilled water to 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.078 μmol/mL standard solution to be measured. Drawing the standard curve: taking the concentration of each standard solution as the X-axis and its corresponding absorption value (ΔA standard) as the Y-axis, drawing the standard curve to obtain the standard equation y = kx + b, and bringing the determination of ΔA into the formula to obtain x (μmol/mL). LA content (μmol/106 cell) = x × (V supernatant + V extract 2) ÷ (5 × V supernatant ÷ V extract 1) = 0.2375 × x.

Tested cell lactate dehydrogenase activity according to the instructions provided by Solarbio: Collected the cells into the centrifuge tube, discard the supernatant after centrifugation, according to the number of cells (104): extracted the liquid volume (mL) of 500–1000: 1 in proportion to ultrasonic crushing cells, 8000 g, centrifuged at 4 ℃ for 10 min, taken supernatant, put on ice to be measured. Preparation of standard substance: Took 100 μL standard liquid and dilute it with distilled water to 1, 0.5, 0.25, 0.125, 0 μmol/mL, and made a standard curve with 2, 1, 0.5, 0.25, 0.125, 0 μmol/mL standard liquid. LDH (U/104 cell) = y × V sample ÷ (500 ÷ total V sample × V sample) ÷ T × 103 = 0.133 × y.

Data analysis

All data involving multiple comparisons in this study were calculated using ANOVA and T test. Statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA), and each group was repeated at least 3 times. The analysis results were presented using GraphPad Prism 8.0. All data were expressed as mean ± SD, and P < 0.05 was considered significant, while values of P < 0.01 were considered extremely significant.

Data availability

Data is provided within the manuscript or supplementary information files.

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Acknowledgements

Data curation: Rui Ma, Yaying Wang, Jinglei Wang. Formal analysis: Junfeng He, Qian Zhang. Funding acquisition: Yan Cui, Sijiu Yu, Yangyang Pan. Investigation: Rui Ma, Xiaoyan Wang, Xuefeng Bai, Hui Zhang, Shanshan Yang, Qian Zhang. Methodology: Yangyang Pan, Rui Ma, Jinglei Wang, Hui Zhang. Supervision: Yan Cui, Sijiu Yu. Writing – original draft: Rui Ma, Yangyang Pan. Writing – review & editing: Yangyang Pan, Yan Cui, Sijiu Yu.

Funding

This work was supported by the fund of the National Natural Science Foundation of China (32473110), the Gansu Province key talent project (2022RCXM016) and the Gansu seed industry public relations project (GZGG-2021–1).

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Authors and Affiliations

  1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China

    Rui MA, Yan CUI, Si-Jiu YU, Yang-Yang PAN, Jun-feng HE, Ya-ying WANG, Jing-lei WANG, Xiao-yan WANG, Xue-feng BAI, Hui ZHANG, Shan-shan YANG & Qian ZHANG

  2. Gansu Province Livestock Embryo Engineering Research Center, Lanzhou, 730070, China

    Rui MA, Yan CUI, Si-Jiu YU, Yang-Yang PAN, Jun-feng HE, Ya-ying WANG, Jing-lei WANG, Xiao-yan WANG, Xue-feng BAI, Hui ZHANG, Shan-shan YANG & Qian ZHANG

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  1. Rui MA
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Contributions

Data curation, R.M., Y.W, and J.W.; methodology, Y.P., R.M., J.W., and H.Z.; software, J.H. and Q.Z.; validation, J.H. and Y.W.; formal analysis, J.H. and Q.Z.; investigation, R.M., X.W. and X.B.; resources, H.Z., S.Y., J.W. and Q.Z.; writing original draft preparation, R.M.; writing review and editing, Y.P., Y.C. and S.Y; supervision, S.Y.; project administration, S.Y. and R.M.; funding acquisition, S.Y.,Y.C., Y.P. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Yan CUI or Si-Jiu YU.

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Ethics approval and consent to participate

All experiments in this study have been approved by the Animal Ethics Committee of Gansu Agricultural University (Ethic approval file No. GSAU-Eth-VMC-2023–004). And all operations are carried out in strict accordance with ARRIVE guidelines and regulations of the Ethics Committee of Gansu Agricultural University.

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MA, R., CUI, Y., YU, SJ. et al. The glucose metabolism reprogramming of yak Sertoli cells under hypoxia is regulated by autophagy. BMC Genomics 26, 385 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11497-x

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Keywords

BMC Genomics

ISSN: 1471-2164

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