Dysfunction in Sertoli cells participates in glucocorticoid‐ induced impairment of spermatogenesis

Li Ren1 | Yanwen Zhang1 | Yining Xin1 | Guo Chen1 | Xiaoxiao Sun1 |
Yingqi Chen1 | Bin He1,2

Abstract
The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corti- costerone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage‐specific marker antigen F4/80 as well as inflammation‐related genes suggested that corticosterone induced inflammation in the testis. Lactate content
was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism‐related genes were downregulated.

In vitro phagocytosis assays showed that the phagocytic activity in corticosterone‐treated
Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase‐4 inhibitor supple- mentation restored this process. Taken together, our results demonstrated that
dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells partici- pates in corticosterone‐induced impairment of spermatogenesis.

KEYWORDS

corticosterone, lactate, phagocytosis, Sertoli cell, spermatogenesis
1Key Laboratory of Animal Physiology & Biochemistry, Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
2MOE Joint International Research Laboratory of Animal Health & Food Safety, Nanjing Agricultural University, Nanjing, China

Correspondence
Bin He, Key Laboratory of Animal Physiology & Biochemistry, Ministry of Agriculture and Rural Affairs, Nanjing Agricultural University. No. 1 Weigang Rd, Nanjing, 210095 Jiangsu, China.
Email: [email protected]

Funding information
National Natural Science Foundation of China, Grant/Award Numbers: 31872436, 32072807; Natural Science Foundation of Jiangsu Province, Grant/Award Number: BK20181323; Priority Academic Program Development of Jiangsu Higher Education Institutions

INTRODUCTION

Infertility is a widespread public health issue, and nearly half of all infertility cases are attributed to male factors (Ding et al., 2020). Many negative effects due to environmental factors (e.g., exposure to radiation, heavy metals, or endocrine disrupters) and lifestyle
factors (e.g., high‐fat diet and alcohol, caffeine, or nicotine use) on
male reproductive health have been reported (El Hajj et al., 2014; Guerrero‐Bosagna & Skinner, 2014; Lane et al., 2014; Soubry et al., 2014; Soubry, 2015; Stuppia et al., 2015).

Stress is a widespread condition, and stressors are increasingly present in modern society, leading to damaged gonadal function and fertility. A recent cross‐sectional study of 1215 Danish men showed that sub-
jects with high levels of self‐reported stress had worse semen parameters than subjects with low levels of self‐reported stress (Nordkap et al., 2016). It is widely accepted that hyperactivation of the hypothalamus‐pituitary‐adrenal axis due to stress and excessive
glucocorticoid levels are the main causes of damaged gonadal func- tion and fertility (Ren et al., 2010; Zou et al., 2019; Hardy et al., 2005).

Mol Reprod Dev. 2021;1–11. wileyonlinelibrary.com/journal/mrd © 2021 Wiley Periodicals LLC | 1

Several studies have suggested that glucocorticoids induce the apoptosis of spermatogenic cells in mice (Orazizadeh et al., 2010; Orazizadeh et al., 2009) and rats (Yazawa et al., 2000; Mogilner et al., 2006). During mammalian spermatogenesis, more than 75% of developing spermatogenic cells undergo apoptosis before maturation (Johnson et al., 1983). Apoptotic spermatogenic cells can provide energy for Sertoli cells (Xiong et al., 2009). The rapid and efficient degradation of apoptotic germ cells by Sertoli cells has been suggested to be crucial for appropriate germ cell development and differentiation. If the ability of Sertoli cells to phagocytose is impaired, then the number of apoptotic germ cells will increase, and they cannot be eliminated to turn into energy in time (Boussouar & Benahmed, 2004; Peng et al., 2012) leading to noninfectious inflammatory responses in the testis (Zhang et al., 2013). Moreover, developing germ cells have specific me- tabolic requirements, preferentially using lactate as a substrate for ATP production. Glucose uptake in the testis takes place predominantly in Sertoli cells, which metabolize glucose to lactate, which is then transported to postmeiotic germ cells and provides the primary metabolic fuel. Not only is it a metabolic substrate, but lactate has also been reported to inhibit germ cell apoptosis in human testis (Erkkila et al., 2002).

Taken together, the functions of Sertoli cells, including the phagocytosis of apoptotic germ cells and production of lactate, play vital roles in spermatogenesis.Glucocorticoids act via glucocorticoid receptors and miner- alocorticoid receptors, which are ligand‐inducible transcription factors belonging to the nuclear receptor superfamily. Detectable glucocorticoid receptor expression has been described in the Sertoli cells of mice (Hazra et al., 2014), rats (Levy et al., 1980), and cows (Jenkins & Ellison, 1986). Glucocorticoid‐induced upregulation of androgen‐binding protein (Lim et al., 1996) and stanniocalcin 1 (Li & Wong, 2008), which are associated with a number of endocrine and andrology diseases, have been observed in cultured Sertoli cell.

In addition, Sertoli cell‐specific glucocorticoid receptor knockout mice provide in vivo evidence of the need for functional glucocorticoid receptor in Sertoli cells to maintain normal testicular Sertoli/germ cell numbers and circulating gona- dotropin levels, and downregulated Leydig cell steroidogenic transcripts (Hazra et al., 2014). However, whether glucocorticoids affect Sertoli cell functions and how they impair spermatogenesis is unknown.In the present study, we sought to gain insight into the me- chanisms by which glucocorticoids impair spermatogenesis. As mentioned above, the phagocytosis of apoptotic germ cells by Sertoli cells and lactate production in Sertoli cells are essential for sper- matogenesis. Thus, we used mice chronically treated with oral cor- ticosterone and TM4 cells (an immortalized mouse Sertoli cell line) as in vivo and in vitro models, respectively, to address whether pha- gocytosis and lactate metabolism in Sertoli cells were altered byglucocorticoids. The results will help to delineate the potential me- chanisms involved in the stress‐induced impairment of male re- productive health.

2 | RESULTS

2.1 | Effect of corticosterone treatment on serum hormone and sperm quality

To investigate the effect of corticosterone on sperm quality,
we established an experimental hypercortisolism male mouse model (fed a corticosterone drink ([100 μg/ml]) and a control model (fed untreated water). After corticosterone treatment for
4 weeks, the serum corticosterone was higher in the corticosterone‐treated mice compared with control (Figure 1a; p = 0.0144).
The serum testosterone levels were significantly reduced in corticosterone‐treated mice than in the control mice (Figure 1b; p = 0.0111). However, the serum luteinizing hormone (LH) (Figure 1c; p = 0.6046) and follicle‐stimulating hormone (FSH) (Figure 1d; p = 0.3101) levels were not significantly betweengroups.

The testis weight showed a slight decrease in the corticosterone‐ treated mice (Figure 1e; p = 0.0515). Of particular interest to this study, the sperm count was significantly decreased in the corticosterone‐ treated mice (Figure 1f; p = 0.0086). Moreover, the rate of ter- atozoospermia was significantly increased in the corticosterone‐treated mice (Figure 1g; p < 0.0001).

2.2 | Effect of corticosterone on germ cell apoptosis, meiosis, and inflammation in testis

Testis morphology was observed by hematoxylin and eosin staining. The results showed that there were more multi- nucleated giant cells in the seminiferous tubules in the corti- costerone group, implying that the spermatocytes did notundergo complete meiosis (Figure 2a‐c). Moreover, down- regulation of the meiotic marker Synaptonemal complex protein
3 (Scp3) suggested that corticosterone inhibited meiosis (Figure2d,e; p = 0.0343). The terminal deoxynucleotidyl trans- ferase dUTP nick‐end labeling (TUNEL) assay on testicular sec- tions revealed more apoptotic germ cells (number of positive cells per tubule) in corticosterone‐treated mice than in control mice (Figure2f‐i; p < 0.0001). Based on the results of
immunohistochemistry targeting F4/80, dramatically increased numbers of macrophages were observed in the testicular inter- stitial spaces after corticosterone treatment (Figure2j,k), which was indicative of a local inflammatory condition. Moreover, the expression of major inflammatory genes in the testis was de- termined using quantitative reverse‐transcription polymerase
chain reaction (qRT‐PCR). The expression levels of TNF‐α, IL‐1β, and CD163 were upregulated in the corticosterone‐treated mice. By contrast, the MCP‐1 messenger RNA (mRNA) level was not significantly upregulated and the CD68 mRNA level was down- regulated in corticosterone treatment (Figure 2l).

Effects of corticosterone on blood corticosterone, testosterone, LH and FSH concentration, testis weight and sperm quality. Male mice were administered water supplemented with corticosterone for 4 weeks. (a) Blood corticosterone, (b) testosterone, (c) LH, and (d) FSH concentration were measured by ELISA (corticosterone, LH and FSH) or radioimmunoassay (testosterone) after corticosterone treatment for 4 weeks. Values are expressed as mean ± SEM (n = 8 in the control [CON] group, n = 8 in the corticosterone [CORT] group). (e) Testis weight after corticosterone treatment for 4 weeks. (f) Sperm from the cauda epididymis were counted in a blood count plate under a light microscope. (g) Statistical analysis of the teratozoospermia rate. Values are expressed as mean ± SEM (n = 16 in the control [CON] group, n = 10 in the corticosterone [CORT] group). ELISA, enzyme‐linked immunosorbent assay; FSH, follicle‐stimulating hormone; LH, luteinizing hormone

2.3 | Effect of corticosterone on lactate metabolism in Sertoli cells in vivo and in vitro

Lactate produced by Sertoli cells not only provides nutrients for spermatogenic cells, but it also increases the ability of sperma-
togenic cells to resist apoptosis (Figure 3a). The lactate content in the testis was significantly lower in the corticosterone‐treated mice than in the control mice (Figure 3b; p = 0.0343). Subse- quently, we used qRT‐PCR to detect the expression levels of genes participating in lactate secretion. Compared to the control group, the mRNA expression levels of PFKFB1, PDP1, and PDP2 were increased in the testis of the mice treated with corticos- terone (Figure 3c). To verify the effect of corticosterone on lactate production in Sertoli cells, we further treated TM4 Sertoli cells with corticosterone. Lactate in the medium of the TM4 cells decreased after corticosterone treatment in a dose–response
manner (Figure 3d). In addition, the mRNA levels of PDP1 and PDP2 were significantly upregulated in the TM4 cells, while the mRNA levels of GLUT1, GLUT3, LDHB, and MCT4 were down- regulated (Figure 3e).

2.4 | Effect of corticosterone on apoptotic germ cells clearance by Sertoli cells in vitro

To investigate whether corticosterone affected the ability of Sertoli cells to engulf apoptotic germ cells, in vitro phagocytosis assays were
performed with TM4 cells. The apoptotic germ cells were labeled with 4′,6‐diamidino‐2‐phenylindole (DAPI), and engulfment of apoptotic germ cells by the TM4 cells was observed as phagocytic cups. Fluorescence microscopy showed that the TM4 cells had sig- nificantly compromised ability to engulf DAPI‐labeled apoptotic germ cells, as determined by reduced percentages of TM4 cells taking up the apoptotic germ cells as well as reduced numbers of apoptotic germ cells engulfed per TM4 cell (Figure 4a, p = 0.0381). Mitochondrial metabolic activity is critical for continued clear- ance of apoptotic cells in Sertoli cells (Park et al., 2011; Wang et al., 2017). We found that the mitochondrial membrane potential (ΔΨm) of the TM4 cells was decreased after corticosterone treat- ment (Figure 4b, p = 0.0031). Pyruvate dehydrogenase kinase‐4
(PDK4) is an important mitochondrial matrix enzyme in cellular metabolism shift, and it participates in mitochondrial activity

Effects of corticosterone on morphology of testis, germ cell apoptosis, meiosis, and inflammatory markers in the testis. Male mice were administered water supplemented with corticosterone for 4 weeks. Representative hematoxylin and eosin staining images of the testis of the CON (a) and CORT‐treated (b–c) mice. The arrow shows a multinucleated giant cell. Scale bar = 50 µm. (d–e) Western blots and histogram showing the protein levels of meiosis‐specific protein Scp3 in the testis. Values are expressed as mean ± SEM (n = 6). (f) Histogram of apoptotic germ cells in testis sections showing the average number of apoptotic germ cells per seminiferous tubule. Representative TUNEL staining images of the testis of the CON (G) and CORT‐treated (H–I) mice in the control (g) and corticosterone‐treated (h–i) mice. Arrows indicate apoptotic germ cells. Scale bar = 50 µm. Values are expressed as mean ± SEM (n = 3). (j–k) Immunohistochemistry determination of macrophage‐specific marker antigen F4/80 in the testis of the CON (j) and CORT‐treated (k) mice. Arrows indicate F4/80 positive cells. Scale bar = 50 µm. (l) Effect of corticosterone on inflammatory cytokine expression in the testis. Values are expressed as mean ± SEM (n = 6). *p < 0.05; **p < 0.01. CON, control; CORT, corticosterone; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling

Content of lactate and expression of lactate metabolism‐related genes in testicular tissue and TM4 cells. (a) Schematic representation of the lactate metabolism pathways in Sertoli cells. (b) Lactate content in the testicular tissue attained by measuring the centrifuged supernatant of testicular tissue homogenate. (c) Effects of corticosterone on the mRNA levels of lactate metabolism‐related genes in the testis. (d) Lactate content in the medium of TM4 cells exposed to corticosterone. (e) Effects of corticosterone on the mRNA levels of lactate metabolism‐related genes in TM4 cells. Values are expressed as mean ± SEM (n = 6). Different letters indicate significant differences at p < 0.05 in D. *p < 0.05; **p < 0.01 in E. CON, control; CORT, corticosterone; mRNA, messenger RNA regulation. Here, the expression of PDK4 was upregulated in the testis (Figure 4c, p = 0.0167) and TM4 cells (Figure 4d, p = 0.0002) after corticosterone treatment. We also used the PDK4 inhibitor sodium dichloroacetate to investigate whether corticosterone induced the downregulation of ΔΨm and phagocytosis capacity in the
TM4 cells via PDK4. The results showed that sodium dichloroacetate blocked the corticosterone‐induced downregulation of ΔΨm and phagocytosis capacity in the TM4 cells (Figure 4e,f).

3 | DISCUSSION

Stress is a widespread condition, and stressors are increasingly present in modern society (Leaver, 2016; Gabrielsen & Tanrikut, 2016; Skoracka et al., 2020). The effect of stress on male fertility has received considerable attention, but less is known about the related signaling pathway. In the present study, by using in vivo and in vitro models, we showed that corticosterone resulted in lowered sperm counts and poor sperm quality. Moreover, dysfunc- tional phagocytosis capacity and lactate metabolism in Sertoli cells
was found to participate in the corticosterone‐induced impairment of
spermatogenesis.

Spermatogenesis is an elaborate process of germ cell prolifera- tion and differentiation leading to the production and release of
sperm from the testis. Sertoli cells provide morphological support via cell‐cell interactions and also biochemical components via secreting lactate (Crisostomo et al., 2018; Yin et al., 2017). The testis are oxygen‐deprived organs, and germ cells mainly depend on glycolytic metabolism to obtain energy, metabolizing glucose to lactate, which is then transported to postmeiotic germ cells and provides the pri- mary metabolic fuel (Boussouar & Benahmed, 2004). Here, we found that corticosterone treatment induced decrease of lactate content in testis and TM4 cells. The results of previous in vivo and in vitro studies on the influence of glucocorticoids on lactate levels are contradictory (Orazizadeh et al., 2009; Kilger et al., 2003). The ad- dition of dexamethasone, a glucocorticoid receptor agonist, to the incubation medium of adipose tissue explant cultures has been shown to decrease lactate production, but the results differ de- pending on the species. In ovine adipose tissue, for example, the addition of dexamethasone alone was found to decrease lactate production during the first 5 days of incubation, whereas no sig- nificant effect was found in bovine samples during the same period (Riera et al., 2009). In another study, the relationship between lac- tate secretion and glucocorticoids was proved by multivariate re- gression, and the effect of dexamethasone on lactate production was found to rely on its hyperglycemic effect (Ottens et al., 2015).
Sertoli cells produce lactate mainly through the following bio- chemical reactions. First, the glucose is transported to Sertoli cells

Effects of corticosterone on mitochondrial activity and the phagocytosis of apoptotic germ cells in Sertoli cells. (a) Histogram showing the percentage of TM4 cells engulfing apoptotic germ cells in CON and CORT‐treated group, as derived from the immunofluorescence analysis. (b) Histogram showing the mitochondrial membrane potential (ΔΨm) in the TM4 cells by JC‐1 staining and flow cytometric analysis. (c–d) Effects of CORT on the mRNA levels of Pdk4 in the testis (C) and TM4 cells (d). (e) Effects of CORT and Pdk4 inhibitor (sodium dichloroacetate [SD]) on ΔΨm in the TM4 cells. (f) Effects of CORT and SD on the TM4 cells engulfing apoptotic germ cells. Values are expressed as mean ± SEM (n = 6). Values with different superscripts are significantly different from each other (p < 0.05). BLK, without SD; CON, control; CORT, corticosterone; mRNA, messenger RNA via glucose transporters. Second, the ingoing glucose is converted to 6‐phospho‐glucose by the action of hexokinase. Third, the excess pyruvate is converted to lactate under the action of lactate dehydrogenase. Finally, the lactate is catalyzed by monocarboxylate transporters to cross the plasma membranes of the Sertoli cells for spermatogenic cells (Alves et al.,). In the present study, we found that the expression levels of GLUT1 and GLUT3 were downregulated the corticosterone‐treated TM4 cells. GLUT1 and GLUT3 are ex-
pressed at the plasma membrane and are believed to be the main executors transporting glucose from the extracellular milieu (Riera
et al., 2009).

It has been reported that dysregulation of the mmu‐
miR‐320‐3p/GLUT3 cascade and consequent lactate deficiency may be a key molecular event contributing to germ cell loss via Sertoli cell
dysfunction (Zhang et al., 2018). In addition, the expression levels of PDP1 and PDP2 were upregulated by corticosterone in the testis and TM4 cells in this study. The mitochondrial pyruvate dehydrogenase complex is crucial for glucose homeostasis in mammalian cells, as it plays a crucial role in the fate of pyruvate, converting pyruvate to acetyl coenzyme A. The upregulation of PDP1 and PDP2 leads to hyperphosphorylation and the inhibition of pyruvate dehydrogenase complex activity and the ratio of pyruvate inversion to acetyl
coenzyme A and the level of lactate catalyzed by lactate dehy- drogenase (Maj et al., 2006; Bedoyan et al., 2019; Sugden & Holness, 2006). Moreover, in this study, the expression levels of LDHB and MCT4, which participate in pyruvate conversion to lactate and transport across the plasma membrane, were downregulated by corticosterone. These data suggest that corticosterone induces tes- ticular metabolic disorders and results in impaired spermatogenesis and decreased sperm quality.

Sertoli cells engulf and degrade residual bodies and apoptotic spermatogenic cells, which are obligatory processes for healthy spermatogenic cells to proceed through spermatogenesis (Ren et al., 2010; Zou et al., 2019). If the ability of Sertoli cells to pha- gocytose is impaired, then the number of apoptotic germ cells will be increased, and they cannot be eliminated to turn into energy in time (Boussouar & Benahmed, 2004; Peng et al., 2012), leading to non- infectious inflammatory responses in the testis (Zhang et al., 2013). Here, our results suggested that corticosterone induced the down-
regulation of mitochondria‐mediated apoptotic germ cell phagocy-
tosis in Sertoli cells, and it also induced inflammation in the testis. Olivares‐Morales et al. demonstrated that glucocorticoids impair
phagocytosis and induce anti‐inflammatory polarization after

adherent‐invasive Escherichia coli infection in macrophages (Olivares‐Morales et al., 2018).
We and others have reported that the mitochondrial membrane potential is a key determinant of apoptotic cell clearance in Sertoli cells (Park et al., 2011; Gong et al., 2018). In the present study, corticosterone resulted in decreased mitochondrial membrane po- tential in Sertoli cells. PDKs are mitochondrial enzymes that control the conversion of pyruvate to acetyl coenzyme A via inhibitory phosphorylation of the pyruvate dehydrogenase complex within the glucose metabolism pathway (Mogilner et al., 2006; Choiniere et al., 2018). PDK4 participates in mitochondrial activity regulation (Xu et al., 2020), and in this study, it was upregulated in the testis and the TM4 cells after corticosterone treatment. Glucocorticoids reg- ulate PDK4 gene expression through complex hormone response units and glucocorticoid receptors, and the transcription factor forkhead box protein O1 participates in this process (Connaughton et al., 2010). We used a PDK4 inhibitor, sodium dichloroacetate, to investigate whether corticosterone induced the downregulation of mitochondrial membrane potential in TM4 cells via PDK4. The re-
sults showed that sodium dichloroacetate blocked the corticosterone‐induced downregulation of mitochondrial membrane potential and phagocytosis capacity in the TM4 cells.

Taken together, our data suggest that the molecular mechanism of corticosterone‐induced spermatogenesis impairment involves 2 parts. First, corticosterone led to disordered lactate metabolism in Sertoli cells. Second, the phagocytic activity of corticosterone‐ treated Sertoli cells was downregulated, and this was accompanied
by decreased mitochondrial activity via upregulated PDK4. These results provide new insights regarding the underlying mechanisms of stress‐induced damage to male reproductive health.

4 | MATERIALS AND METHODS

4.1 | Reagents

Corticosterone (C0388) was purchased from Shanghai Development CO., Ltd. Chemical Industry. The TUNEL kits (C1091) were pur- chased from Beyotime Institute of Biotechnology. The JC‐1 (5,5′,6,6′‐
tetrachloro‐1,1′,3,3′tetraethylbenzymid‐azolyl carbocyanine iodide) mitochondrial membrane potential (ΔΨm) kits were purchased from Nanjing KeyGen Development Co., Ltd. Lactate determination kits
(MB‐W‐B206) were purchased from Nanjing MALL‐BIO. Sodium di- chloroacetate (HY‐Y0445A) was purchased from MedChemExpress (MCE) Chemical Corporation.

4.2 | Animal treatment

Adult (8 weeks) male C57BL/6 mice were bought from the Animal Research Center of Yangzhou University. Mice were maintained under a light: dark cycle of 12: 12 h, at a temperature of 23°C and humidity of 50%‐70%; they had free access to food (chow diet) and
water. In accordance with a previous reports, corticosterone (Aladdin) was dissolved in 100% ethanol, and then diluted into the drinking water of corticosterone‐treated mice to a final con-
centration of 1% vol/vol ethanol and 100 μg/ml corticosterone
(Fenton et al., 2019; Karatsoreos et al., 2010). Bottles were re- placed three times a week. The corticosterone solution was made fresh and stored at 4°C before use. Fluid intake for both groups was determined by weighing the drinking bottles, before replacing the bottles.

4.3 | Hormone and biochemical measurements

Serum corticosterone levels were quantified using an enzyme‐linked immunosorbent assay (ELISA) according to the manufacturer's in- structions by Mouse CORT (Corticosterone) ELISA Kit (E‐EL‐0161c, Elabscience Biotechnology Co. Ltd). Serum Testosterone were mea-
sured by [125I] Testosterone Radioimmunoassay Kit (Beijing North Institute of Biotechnology). Serum LH and FSH were measured by ELISA Kits (Beijing North Institute of Biotechnology). The experi-
mental procedures were as described in the manufacturer's in- structions. The detection limit, intra‐ and inter‐assay coefficients of variation are presented in Table 1.

4.4 | Sperm count and abnormalities measurement

The cauda epididymis was minced in 1 mL of phosphate buffered saline (pH 7.4) and incubated for 20 min at 37°C to release the sperm. The sperm count was quantified in a Hemocytometer under a light microscope (Nikon). A drop of spermatozoa filtrate was pressed and dried naturally at room temperature, followed by methanol
fixation and drying, and then stained with Eosin Y (2%) for 1–2h
(Ding et al., 2020). The sperm was washed by water and dried, ex- amined by high‐power microscope, and the abnormal sperm were
observed, including hooked, banana‐shaped, fat head and small head,
amorphous, etc. A total of 200 sperm were examined for each animal directly under microscope to detect the morphological abnormalities in sperm.

The detection limits, intra‐, and inter‐assay coefficients of different kits used in the study

Item Detection limit Intra‐assay coefficient (%) Inter‐assay coefficient (%)
Corticosterone 1.93 ng/ml 10 15
Testosterone 0.02 ng/ml 10 15
LH 0.5 mIU/ml 15 20
FSH 0.5 mIU/ml 15 20
Abbreviations: FSH, follicle‐stimulating hormone; LH, luteinizing hormone.

4.5 | Histological studies

For histological studies, animals were dissected, and their testis were removed and fixed in Bouin's stationary liquid for 24 h, dehydrated in graded ethanol, embedded in paraffin and sectioned (4 µm in thick- nesses). The sections were stained with hematoxylin/eosin (H&E). The morphological changes were examined under a microscope (BX63F OLYMPUS Micro Image System, OLYMPUS).

4.6 | Protein extraction and Western blot analysis

Testis samples were homogenized in RIPA buffer (50 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na‐deoxycholate, 1 mM PMSF, 1 mM sodium orthovanadate with Roche EDTA‐free complete
mini protease inhibitor cocktail, no. 11836170001). The protein concentration was measured with the BCA Protein Assay Kit (Pierce) according to a previous publication (He et al., 2016). Fourty micro- grams of protein extract were used for electrophoresis on a 15% or
10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gel.
The mouse Anti‐Scp3 antibody (1:1000, ab97672; Abcam) was used as primary antibody. Protein loading controls for each experiment using rabbit anti‐α‐tubulin antibody (1:1000, bs1699; Bioworld). All the operations were carried out according to the recommended
protocols provided by the manufacturers.

4.7 | Terminal deoxynucleotidyl transferase dUTP nick‐end labeling assay

The TUNEL assay was performed using Colorimetric TUNEL Apop- tosis Assay Kit according to the manufacturer's protocol. The solu- tion without Tdt enzyme was applied as a negative control. Positive
staining cells were counted by using an Image‐Pro Plus 6.0 system
from nine randomly selected microscopic fields for each section. The cells displaying brown staining in the nucleus were counted as po-
sitive apoptotic cells. The TUNEL‐positive nuclei in two slices per
animal (n=3 mice per group) was observed. The numbers of TUNEL‐ positive cells of six separate fields were chosen at random in every
slice were counted under light microscopy. The percentage of apoptotic, TUNEL‐positive cells was expressed as the average num- ber of apoptotic cells within 20 seminiferous tubes.

4.8 | Immunohistochemical staining

We carried out immunohistochemical analyses on Bouin's stationary liquid‐fixed and paraffin‐embedded testicular section. The sections were incubated with 1X phosphate‐buffered saline (PBS) contain-
ing 3% H2O2 for 15 min to block endogenous peroxidase activity. Fetal bovine serum (10% [vol/vol]) was used to block nonspecific staining for 1 h at room temperature, the sections were incubated with primary antibodies F4/80 (1:400; Cell Signaling Technology) overnight at 4°C. After rinsing with PBS, the sec- tions were incubated with biotinylated appropriated secondary antibodies at room temperature for 30 min. Negative controls
were incubated with the pre‐immune sera instead of the primary
antibodies.

4.9 | RNA extraction, reverse transcription, and real time quantification PCR

Total RNA was extracted from testis and TM4 cells using TRIzol Reagent (15596‐026; Invitrogen) and then treated with DNase I (RNase Free, D2215; Takara) to eliminate possible contamination of genomic DNA according to the manufacturer's instructions. Concentration of the extracted RNA was measured using Nano Drop 1000 Spectrophotometer (ND‐1000; Thermo Fisher Scien- tific). Ratios of absorption (260/280 nm) were between 1.9 and

2.1. RNA integrity was confirmed by denaturing agarose electro-
phoresis. Two micrograms of total RNA were reverse‐transcribed in a final volume of 25 μl with M‐MLV reverse transcriptase (Promega) and random hexamer primers (SunShine) following the manufacturer's instructions. Reverse transcription was performed
in a Thermal Cycler PTC0200 (Bio‐Rad). Two microliters of diluted complementary DNA (1:20) were used for real‐time PCR. All pri- mers (Table 2) were synthesized by Generay Biotech Co., Ltd. Real‐time quantitative PCR was performed with a Mx3000P real‐ time PCR detection system (Stratagene). The peptidylprolyl Iso-merase A was chosen as reference genes in quantitative mRNA profiling assays. The amplification specificity of each gene was checked by melting curve analysis.

4.10 | Cell culture and treatment

The TM4 cells line used in this study was established by Mather in 1980 from primary cultures of SC isolated from 11 to 13 days old BALB/c mice Mather. They were seeded (1 × 105 cells/dish) and cultured at 37°C in Dulbecco's modification of Eagle's medium (DMEM/F12) (Gibco BRL) containing 10% fetal bovine serum, Peni- cillin/Streptomycin (100 mU/ml) in a saturated atmosphere of 5% CO2. Corticosterone treatment begins before the cells grow to about 50%.

4.11 | Measurement of lactate concentrations in testis and conditioned culture media

The testicular extracts were prepared for measurement of in-
tratesticular lactate concentrations using lactate Assay Kit, according to the manufacturer's instruction (Nanjing MALL‐BIO). Briefly, 10% (wt/vol) testicular homogenates were prepared in pre‐cold extracting
solution at 4°C for 12,000 g, 20 min, then supernatant was taken to detect lactic acid content. The conditioned cell culture media that were collected 24 h after corticosterone treatment, along with the above‐mentioned tissue supernatants, were subjected to measure-
ment of lactate concentrations.

4.12 | Phagocytosis assay

The procedure of phagocytosis of apoptotic cells by Sertoli cells was
performed based on previous protocol with modifications (Shiratsuchi et al., 1997). The testicular cells of three‐week‐old C57BL/6J mice were primary cultured at 32.5°C. Sertoli cells ad-
hered to the culture containers and spermatogenic cells attached lightly to the Sertoli cells. Spermatogenic cells were recovered by
gentle pipetting from testicular cells cocultured in collagen‐coated
multi‐well plates for two days. The non‐adherent germ cell fraction was collected, with 40%–50% of cells being apoptotic, which was
confirmed by annexin V staining. Apoptotic germ cells were then labeled with DAPI for 5 min at room temperature in the dark, fol- lowed by washing and incubation with TM4 Sertoli cells. After 3 h of coincubation, engulfed apoptotic germ cells were observed under the microscope or were subjected to flow cytometry analysis.

4.13 | Measurement of the mitochondrial membrane potential (ΔΨm)

TM4 cells were measured using a JC‐1 fluorescent probe. Cells with corticosterone treatment at 1 × 106 cells/ml were incubated at 37°C
for 24 h before analysis by flow cytometry analysis according to our
previous research (Guo et al., 2017). Briefly, treated cells were centrifuged at 800 g for 10 min and then stained with 2.5 μg/ml JC‐1
for 15 min at 37°C. Then, cells were washed with ice‐cold PBS three
times, samples were analyzed via flow cytometry, and 10,000 events were acquired on the flow cytometer. JC‐1 emissions from excitation
at the 488 nm were collected at 525 nm (JC‐1 green) and 585 nm
(JC‐1 red). Gates, including the final gate for dye excluding cells were subjectively set based on the flow cytometry images; however,
within each experiment, the same gate settings were used to de- termine dye exclusion cohort percentile changes that resulted from experimental maneuvers.

4.14 | Statistical analysis

All data were tested for normality and variance homogeneity before statistical analysis. Independent‐samples t test was performed to assess the differences between groups. Two‐way analysis of variance
was performed to assess the main effects of corticosterone and SD, as well as their interactions on ΔΨm and phagocytic activity using the GLM, followed by least significant difference post hoc analysis to evaluate differences between specific groups. All analyses were
performed using SPSS 20.0 software. Data are expressed as mean ± SEM. Two tailed p values less than 0.05 were considered statistically significant.

ACKNOWLEDGMENTS
The study is funded by National Natural Science Foundation of China (grant number: 31872436; 32072807), Natural Science
Foundation of Jiangsu Province (grant number: BK20181323) and Priority Academic Program Development of Jiangsu Higher Educa- tion Institutions.

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
Bin He conceived and designed the experiments. Li Ren, Yanwen Zhang, Yining Xin, Guo Chen, Xiaoxiao Sun, and Yingqi Chen per- formed the experiments. Li Ren and Bin He analyzed the data and wrote the paper. All authors critically revised and drafted the manuscript.

ETHICS STATEMENT
The Institutional Animal Care and Use Committee (IACUC) of Nanjing Agricultural University approved all animal procedures. The
“Guidelines on Ethical Treatment of Experimental Animals” (2006)
No. 398 set by the Ministry of Science and Technology, China and
the Regulation regarding the Management and Treatment of Ex- perimental Animals” (2008) No. 45 set by the Jiangsu Provincial People's Government, was strictly followed during the slaughter and sampling procedures.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/mrd.23515

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

REFERENCES
Bedoyan, J. K., Hecht, L., Zhang, S., Tarrant, S., Bergin, A., Demirbas, D., Yang, E., Shin, H. K., Grahame, G. J., DeBrosse, S. D., Hoppel, C. L., Kerr, D. S., & Berry, G. T. (2019). A novel null mutation in the pyruvate dehydrogenase phosphatase catalytic subunit gene (PDP1)
causing pyruvate dehydrogenase complex deficiency. JIMD Reports, 48, 26–35.
Boussouar, F., & Benahmed, M. (2004). Lactate and energy metabolism in
male germ cells. Trends in Endocrinology and Metabolism, 15, 345–350.
Choiniere, J., Lin, M. J., Wang, L., & Wu, J. (2018). Deficiency of pyruvate
dehydrogenase kinase 4 sensitizes mouse liver to diethylnitrosamine and arsenic toxicity through Sodium dichloroacetate inducing apoptosis. Liver Research, 2, 100–107.
Connaughton, S., Chowdhury, F., Attia, R. R., Song, S., Zhang, Y.,
Elam, M. B., Cook, G. A., & Park, E. A. (2010). Regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) gene expression by glucocorticoids and insulin. Molecular and Cellular Endocrinology, 315, 159–167.
Crisostomo, L., Alves, M. G., Gorga, A., Sousa, M., Riera, M. F.,
Galardo, M. N., Meroni, S. B., & Oliveira, P. F. (2018). Molecular mechanisms and signaling pathways involved in the nutritional support of spermatogenesis by Sertoli cells. Methods in Molecular Biology, 1748, 129–155.
Ding, N., Zhang, X., Zhang, X. D., Jing, J., Liu, S. S., Mu, Y. P., Peng, L. L.,
Yan, Y. J., Xiao, G. M., Bi, X. Y., Chen, H., Li, F. H., Yao, B., &
Zhao, A. Z. (2020). Impairment of spermatogenesis and sperm motility by the high‐fat diet‐induced dysbiosis of gut microbes. Gut, 69, 1608–1619.
Erkkila, K., Aito, H., Aalto, K., Pentikainen, V., & Dunkel, L. (2002). Lactate inhibits germ cell apoptosis in the human testis. Molecular Human Reproduction, 8, 109–117.
El Hajj, N., Schneider, E., Lehnen, H., & Haaf, T. (2014). Epigenetics and
life‐long consequences of an adverse nutritional and diabetic intrauterine environment. Reproduction, 148, R111–R120.
Fenton, C. G., Webster, J. M., Martin, C. S., Fareed, S., Wehmeyer, C.,
Mackie, H., Jones, R., Seabright, A. P., Lewis, J. W., Lai, Y. C.,
Goodyear, C. S., Jones, S. W., Cooper, M. S., Lavery, G. G., Langen, R., Raza, K., & Hardy, R. S. (2019). Therapeutic glucocorticoids prevent bone loss but drive muscle wasting when administered in chronic polyarthritis. Arthritis Research & Therapy, 21, 182.
Gabrielsen, J. S., & Tanrikut, C. (2016). Chronic exposures and male fertility: the impacts of environment, diet, and drug use on spermatogenesis. Andrology, 4, 648–661.
Gong, Y., Zhang, Z., Chang, Z., Zhou, H., Zhao, R., & He, B. (2018).
Inactivation of glycogen synthase kinase‐3alpha is required for mitochondria‐mediated apoptotic germ cell phagocytosis in Sertoli cells. Aging (Albany NY), 10, 3104–3116.
Guerrero‐Bosagna, C., & Skinner, M. K. (2014). Environmental epigenetics
and effects on male fertility. Genetic Damage in Human Spermatozoa, 791, 67–81.
Guo, H. D., Gong, Y. B., He, B., & Zhao, R. Q. (2017). Relationships
between mitochondrial DNA content, mitochondrial activity, and boar sperm motility. Theriogenology, 87, 276–283.
Hardy, M. P., Gao, H. B., Dong, Q., Ge, R., Wang, Q., Chai, W. R., Feng, X., &
Sottas, C. (2005). Stress hormone and male reproductive function.
Cell and Tissue Research, 322, 147–153.
Hazra, R., Upton, D., Jimenez, M., Desai, R., Handelsman, D. J., & Allan, C. M. (2014). In vivo actions of the Sertoli cell glucocorticoid receptor. Endocrinology, 155, 1120–1130.
He, B., Zhang, N., & Zhao, R. (2016). Dexamethasone downregulates
SLC7A5 expression and promotes cell cycle arrest, autophagy, and apoptosis in BeWo Cells. Journal of Cellular Physiology, 231, 233–242.
Jenkins, N., & Ellison, J. (1986). Corticosteroids suppress plasminogen
activation in the bovine Sertoli cell. The Journal of Endocrinology, 108, R1–R3.
Johnson, L., Petty, C. S., & Neaves, W. B. (1983). Further quantification of
human spermatogenesis: germ cell loss during postprophase of meiosis and its relationship to daily sperm production. Biology of Reproduction, 29, 207–215.
Karatsoreos, I. N., Bhagat, S. M., Bowles, N. P., Weil, Z. M.,
Pfaff, D. W., & McEwen, B. S. (2010). Endocrine and physiological changes in response to chronic corticosterone: A potential model of the metabolic syndrome in mouse. Endocrinology, 151, 2117–2127.
Kilger, E., Weis, F., Briegel, J., Frey, L., Goetz, A. E., Reuter, D., Nagy, A.,
Schuetz, A., Lamm, P., Knoll, A., & Peter, K. (2003). Stress doses of hydrocortisone reduce severe systemic inflammatory response syndrome and improve early outcome in a risk group of patients after cardiac surgery. Critical Care Medicine, 31, 1068–1074.
Lane, M., Robker, R. L., & Robertson, S. A. (2014). Parenting from before
conception. Science, 345, 756–760.
Leaver, R. B. (2016). Male infertility: An overview of causes and treatment options. British Journal of Nursing, 25, S35–S40.
Levy, F. O.Ree A. H.Eikvar L.Govindan M. V.Jahnsen T., & Hansson V.
(1989). Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells. Endocrinology, 124, 430–436.

Li, L., & Wong, C. K. (2008). Effects of dexamethasone and dibutyryl cAMP on stanniocalcin‐1 mRNA expression in rat primary Sertoli and Leydig cells. Molecular and Cellular Endocrinology, 283, 96–103.
Lim, K., Yoon, S. J., Lee, M. S., Byun, S. H., Kweon, G. R., Kwak, S. T., & Hwang, B. D. (1996). Glucocorticoid regulation of androgen binding protein expression in primary Sertoli cell cultures from rats. Biochemical and Biophysical Research Communications, 218, 490–494.
Maj, M. C., Cameron, J. M., & Robinson, B. H. (2006). Pyruvate dehydrogenase
phosphatase deficiency: Orphan disease or an under‐diagnosed condition? Molecular and Cellular Endocrinology, 249, 1–9.
Mather, J. P. (1980). Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biology of Reproduction, 23, 243–252.
Mogilner, J. G., Elenberg, Y., Lurie, M., Shiloni, E., Coran, A. G., &
Sukhotnik, I. (2006). Effect of dexamethasone on germ cell apoptosis in the contralateral testis after testicular ischemia‐reperfusion injury in the rat. Fertility and Sterility, 85(Suppl 1), 1111–1117.
Nordkap, L., Jensen, T. K., Hansen, A. M., Lassen, T. H., Bang, A. K., Joensen, U. N., Blomberg Jensen, M., Skakkebaek, N. E., & Jorgensen, N. (2016). Psychological stress and testicular function: A cross‐sectional
study of 1,215 Danish men. Fertility and Sterility, 105, 174–187.
Olivares‐Morales, M. J., De La Fuente, M. K., Dubois‐Camacho, K., Parada, D., Diaz‐Jimenez, D., Torres‐Riquelme, A., Xu, X., Chamorro‐ Veloso, N., Naves, R., Gonzalez, M. J., Quera, R., Figueroa, C.,
Cidlowski, J. A., Vidal, R. M., & Hermoso, M. A. (2018). Glucocorticoids impair phagocytosis and inflammatory response against Crohn’s disease‐associated adherent‐invasive Escherichia coli. Frontiers in Immunology, 9, 1026.
Orazizadeh, M., Hashemitabar, M., & Khorsandi, L. (2009). Protective effect of minocycline on dexamethasone induced testicular germ cell apoptosis in mice. European Review for Medical and Pharmacological Sciences, 13, 1–5.
Orazizadeh, M., Khorsandi, L. S., & Hashemitabar, M. (2010). Toxic effects
of dexamethasone on mouse testicular germ cells. Andrologia, 42, 247–253.
Ottens, T. H., Nijsten, M. W., Hofland, J., Dieleman, J. M., Hoekstra, M.,
van Dijk, D., & van der Maaten, J. M. (2015). Effect of high‐dose dexamethasone on perioperative lactate levels and glucose control: a randomized controlled trial. Critical Care, 19, 41.
Park, D., Han, C. Z., Elliott, M. R., Kinchen, J. M., Trampont, P. C., Das, S., Collins, S., Lysiak, J. J., Hoehn, K. L., & Ravichandran, K. S. (2011). Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein. Nature, 477, 220–224.
Peng, H., Shi, J., Zhang, Y., Zhang, H., Liao, S., Li, W., Lei, L., Han, C.,
Ning, L., Cao, Y., Zhou, Q., Chen, Q., & Duan, E. (2012). A novel class of tRNA‐derived small RNAs extremely enriched in mature mouse sperm. Cell Research, 22, 1609–1612.
Ren, L., Li, X., Weng, Q., Trisomboon, H., Yamamoto, T., Pan, L., Watanabe, G., & Taya, K. (2010). Effects of acute restraint stress on sperm motility and secretion of pituitary, adrenocortical and
gonadal hormones in adult male rats. Journal of Veterinary Medical Science, 72, 1501–1506.
Riera, M. F., Galardo, M. N., Pellizzari, E. H., Meroni, S. B., &
Cigorraga, S. B. (2009). Molecular mechanisms involved in Sertoli cell adaptation to glucose deprivation. American Journal of Physiology, Endocrinology and Metabolism, 297, E907–E914.
Shiratsuchi, A., Umeda, M., Ohba, Y., & Nakanishi, Y. (1997). Recognition
of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoli cells of the rat. Journal of Biological Chemistry, 272, 2354–2358.
Skoracka, K., Eder, P., Lykowska‐Szuber, L., Dobrowolska, A., & Krela‐ Kazmierczak, I. (2020). Diet and nutritional factors in male infertility‐underestimated factors. Journal of Clinical Medicine, 9.
Soubry, A. (2015). Epigenetic inheritance and evolution: A paternal
perspective on dietary influences. Progress in Biophysics & Molecular Biology, 118, 79–85.
Soubry, A., Hoyo, C., Jirtle, R. L., & Murphy, S. K. (2014). A paternal
environmental legacy: Evidence for epigenetic inheritance through the male germ line. BioEssays, 36, 359–371.
Stuppia, L., Franzago, M., Ballerini, P., Gatta, V., & Antonucci, I. (2015).
Epigenetics and male reproduction: The consequences of paternal lifestyle on fertility, embryo development, and children lifetime health. Clinical Epigenetics, 7, 120.
Sugden, M. C., & Holness, M. J. (2006). Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate
dehydrogenase kinases. Archives of Physiology and Biochemistry, 112, 139–149.
Wang, Y., Subramanian, M., Yurdagul, A., Jr., Barbosa‐Lorenzi, V. C.,
Cai, B., de Juan‐Sanz, J., Ryan, T. A., Nomura, M., Maxfield, F. R., &
Tabas, I. (2017). Mitochondrial fission promotes the continued clearance of apoptotic cells by macrophages. Cell, 171, 331–345.
Xiong, W., Wang, H., Wu, H., Chen, Y., & Han, D. (2009). Apoptotic
spermatogenic cells can be energy sources for Sertoli cells.
Reproduction, 137, 469–479.
Xu, C., Kasimumali, A., Guo, X., Lu, R., Xie, K., Zhu, M., Qian, Y., Chen, X.,
Pang, H., Wang, Q., Fan, Z., Dai, H., Mou, S., Ni, Z., & Gu, L. (2020).
Reduction of mitochondria and up regulation of pyruvate dehydrogenase kinase 4 of skeletal muscle in patients with chronic kidney disease. Nephrology (Carlton), 25, 230–238.
Yazawa, H., Sasagawa, I., & Nakada, T. (2000). Apoptosis of testicular
germ cells induced by exogenous glucocorticoid in rats. Human Reproduction, 15, 1917–1920.
Yin, J., Ni, B., Tian, Z. Q., Yang, F., Liao, W. G., & Gao, Y. Q. (2017).
Regulatory effects of autophagy on spermatogenesis. Biology of Reproduction, 96, 525–530.
Zhang, L. L., Ma, J., Yang, B., Zhao, J., Yan, B. Y., Zhang, Y. Q., & Li, W.
(2018). Interference with lactate metabolism by mmu‐miR‐320‐3p via negatively regulating GLUT3 signaling in mouse Sertoli cells. Cell
Death & Disease, 9, 964.
Zhang, X. Y., Wang, T., Deng, T. T., Xiong, W. P., Lui, P., Li, N., Chen, Y. M., & Han, D. S. (2013). Damaged spermatogenic cells induce inflammatory gene expression in mouse Sertoli cells through the activation of Toll‐like receptors 2 and 4. Molecular and Cellular
Endocrinology, 365, 162–173.
Zou, P., Wang, X., Yang, W., Liu, C., Chen, Q., Yang, H., Zhou, N., Zeng, Y.,
Chen, H., Zhang, G., Liu, J., Cao, J., Ao, L., & Sun, L. (2019).
Mechanisms of stres‐induced spermatogenesis impairment in male rats following unpredictable chronic mild stress (uCMS). International Journal of Molecular Sciences, 20, 4470.