Sertoli cells (SCs) are somatic cells located in seminiferous tubules that play a crucial role in spermatogenesis. The shape of the SC is generally high columnar or conical, and the nucleus is irregularly shaped or triangular, located at the base, with an obvious nucleolus. Round clumps of chromatin, known as para-nucleolar corpuscles, are observed beside or on either side of the nucleolus, and their morphological features can be used to distinguish SCs by light microscopy [1]. The SC cytoplasm contains abundant endoplasmic reticulum and mitochondria and an obvious Golgi apparatus with lipid droplets and glycogen granules. Methods for isolating SCs from the testis have been summarized for a long time, and SCs are cultured in DMEM supplemented with fetal bovine serum or other additives [2]. The purity of SC is mostly determined by detecting its specific markers, such as vimentin, GATA-4, and cytokeratin 18 [3]. SCs provide nutritional and structural support to spermatogenic cells [4] and secrete lactic acid [5] and cytokines [6], which are important for spermatogenesis, sperm movement, and seminiferous fluid pH. Adjacent SCs can form the blood-testis barrier, which provides an immunoprotected environment for protecting germ cells and a stable microenvironment for germ cell differentiation [7]. Each SC provides support for a limited number of spermatogenic cells, and the number of SCs determines sperm production ability [8]. SC proliferative viability is a vital determinant of cell number. Boar SC proliferation exhibits two phases: from birth to one month of age and from 3 to 4 months of age until puberty [9]. The proliferation of SCs can be regulated by hormones and growth factors, and estrogens play an important role in this process [10]. Our previous study indicated that estrogen has both positive and negative effects on SC proliferation, and low doses of 17β-estradiol (0.0001–0.1 μM) were found to promote immature boar SC proliferation, while a higher dose (1 μM) decreased SC proliferative viability [11].

Adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and regulator of cellular energy metabolism, can affect the proliferative activity of SCs [12]. Specific deletion of AMPK in mouse SCs was found to increase the proliferation rate of SCs and abnormal spermatozoa [13]. AMPK activation leads to the inhibition of rat SC proliferation by inhibiting the mammalian target of rapamycin and stimulating cyclin-dependent kinase inhibitor expression [14]. Compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidin), as a selective and ATP-competitive AMPK inhibitor, has been reported to reduce cisplatin-induced renal tubular cell apoptosis in a dose-dependent manner [15]. The adenosine analog 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a widely used AMPK activator, inhibits tumor cell proliferation and induces cell cycle arrest and apoptosis [16]. Our previous study showed that high concentrations of 17β-estradiol inhibited cell viability and the cell cycle of immature boar SCs by activating AMPK [17]. Apart from specific drugs that can change AMPK activity, microRNAs (miRNAs), which target mRNA through base pairing and lead to mRNA destabilization and translation repression [18], can also regulate AMPK expression and its pathway [19,20]. However, it is still unclear how AMPK regulates the proliferative viability of immature boar SCs.

Mammalian cell proliferation is regulated by cell cycle proteins (Cyclin) and Cyclin-dependent kinases (CDKs). Increased Cyclin D1 expression was found in follicle-stimulating hormone-induced immature SC proliferation [21]; decreased Cyclin D2 expression and increased p21 and p53 protein levels were observed in cultured SCs with inactivated insulin-like growth factor 1 receptor [22]; up-regulated CDK4 was involved in thyroid hormone-regulated SC proliferation [23]. Inactivation of CDK4/Cyclin D3 fails to phosphorylate retinoblastoma protein (Rb), resulting in a blockade of the G1 phase at the beginning of the cell cycle, thus inhibiting cell proliferative viability [24]. Studies in knockout animals for Cyclin-dependent kinase inhibitors (CDKIs) p21 and p27 have demonstrated that they are important cell cycle inhibitors of SC proliferation, and that loss of CDKIs leads to a large increase in the adult SC population, as well as an increase in daily sperm production [25]. Phosphorylated AMPK induces p38 activation [26] and p21 activation [27]. P21 can bind to proliferating cell nuclear antigen (PCNA) to block DNA synthesis [28] and inhibit the activity of the CDK4/Cyclin D3 complex [29]. Our previous study found that AMPK activation increased p53 and p27 expression but inhibited Skp 2 expression in immature boar SCs [17]. However, whether AMPK is involved in regulating immature boar SC proliferation by affecting the CDK4/Cyclin D3 pathway remains unclear.

Mitochondrial function affects cell proliferative viability. Most of the ATP required for cell growth, proliferation, and metabolism is produced by the mitochondria [30]. ATP production was increased in immature SCs during the proliferative period [31]. Oxidative phosphorylation is the main ATP-producing reaction that occurs in the mitochondria [32], where ATP production is coupled to electron transport in the respiratory chain [33]. The activities of ATP synthase, NADH-Q oxidoreductase, and cytochrome c oxidase, which are involved in electron transfer in the respiratory chain, affect ATP synthesis in the mitochondria [34]. Studies have shown that the activities of mitochondrial respiratory chain enzymes are significantly decreased in drug-induced cell cycle inhibition in SCs [31,35]. AMPK can sense the energy status of cells through changes in the AMP/ATP or ADP/ATP ratio, and is activated when the ATP level in cells is low to promote ATP production, thus maintaining the energy stability of cells and regulating cell proliferation [36,37]. Our previous study showed that ATP concentration decreased in AICAR-treated immature boar SCs [17]. Moreover, AMPK phosphorylation and ATP5A (ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit) expression exhibited opposite changes in metformin-treated immature chicken SCs [31]. However, whether AMPK has a direct regulatory role in mitochondria in the regulation of SC proliferation is unclear.

In this study, we investigated the effects of AMPK on immature boar SC proliferative viability, ultrastructure, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and proliferation-related protein expression by altering AMPK activity. Exploring the mechanism by which AMPK regulates the proliferative activity of immature SCs is conducive to controlling the number of SCs and spermatogenesis and helps to provide ideas for preventing and treating male sterility.