Lactate Production In Sertoli Cells Research Articles

Microcystin-LR induces lactate production disruption via altering the m6A modification in Sertoli cells

We have previously reported the toxicity of microcystin-LR (MC-LR) to the male reproductive system, which results in functional changes in mouse testes. In this study, mice were orally exposed to MC-LR at 1, 7.5, 15, or 30 μg/L daily for 180 days. We found an increase in germ cell apoptosis in the seminiferous tubules and low-quality sperm in the epididymis. A decrease in lactate dehydrogenase A (Ldha) expression in testes through high-throughput sequencing was observed. We validated that MC-LR disrupted lactate production in Sertoli cells by suppressing the expression of Ldha. Further studies identified that methyltransferase 3 (Mettl3) catalysed N6-methyladenosine (m6A) methylation of Ldha mRNA. Mettl3 was downregulated in Sertoli cells following exposure to MC-LR, decreasing m6A levels of Ldha. The stability of Ldha mRNA decreased when m6A levels of Ldha were inhibited. In conclusion, these results showed that MC-LR inhibits the expression of Ldha in an m6A-dependent manner, which might result in the apoptosis of spermatogenic cells and a decline in sperm quality. Our work provides a new perspective to understanding MC-LR-induced male infertility.

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.

Ldha-Dependent Metabolic Programs in Sertoli Cells Regulate Spermiogenesis in Mouse Testis.

Sertoli cells play indispensable roles in spermatogenesis by providing the advanced germ cells with structural, nutritional, and regulatory support. Lactate is regarded as an essential Sertoli-cell-derived energy metabolite that nurses various types of spermatogenic cells; however, this assumption has not been tested using genetic approaches. Here, we have reported that the depletion of lactate production in Sertoli cells by conditionally deleting lactate dehydrogenase A (Ldha) greatly affected spermatogenesis. Ldha deletion in Sertoli cells significantly reduced the lactate production and resulted in severe defects in spermatogenesis. Spermatogonia and spermatocytes did not show even mild impairments, but the spermiogenesis of Ldha conditional knockout males was severely disrupted. Further analysis revealed that 2456 metabolites were altered in the sperm of the knockout animals, and specifically, lipid metabolism was dysregulated, including choline, oleic acid, and myristic acid. Surprisingly, choline supplementation completely rescued the spermiogenesis disorder that was caused by the loss of Ldha activities. Collectively, these data have demonstrated that the interruption of Sertoli-cell-derived lactate impacted sperm development through a choline-mediated mechanism. The outcomes of these findings have revealed a novel function of lactate in spermatogenesis and have therapeutic applications in treating human infertility.

TGF-β3 induces lactate production in Sertoli cell through inhibiting the Notch pathway.

In the testis, spermatocytes and spermatids rely on lactate produced by Sertoli cells (SCs) as energy source. Transforming growth factor-beta 3 (TGF-β3) is one of the generally accepted paracrine regulatory factors of SC-created blood-testis barrier (BTB), yet its role in SC glycolysis and lactate production still remains unclear. To investigate the effect of TGF-β3 on glycolysis and lactate production in SCs and determine the role of lethal giant larvae 2 (Lgl2) and Notch signaling activity during this process. Primary cultured rat SCs and TM4 cells were treated with different concentrations of TGF-β3. In some experiments, cells were transfected with siRNA specifically targeting Lgl2 and then treated with TGF-β3 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Lactate concentration, glucose and glutamine (Gln) consumption in the culture medium, activity of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glutaminase (Gls), ATP level, oxygen consumption, extracellular acidification, and mitochondrial respiration complex activity were detected using commercial kits. The protein level of Lgl2, LDH, monocarboxylate transporter 4 (MCT4), and activity of Akt, ERK, p38 MAPK, and Notch pathway were detected by Western blot. The stage-specific expression of Jagged1 was examined by immunohistochemistry (IHC) and qPCR after laser capture microdissection. Spermatogenesis in rat testis injected with recombinant Jagged1 (re-Jagged1) was observed by HE staining, and lactate concentration in testis lysate was measured at a different day point after re-Jagged1 treatment. Significant enhancement of lactate concentration was detected in a culture medium of both primary SCs and TM4 cells treated with TGF-β3 at 3 or 5ng/ml. Besides, other parameters of glycolysis, that is, glucose and Gln consumption, enzyme activity of PFK, LDH, and Gls displayed different levels of increment in primary SCs and TM4 cells after TGF-β3 treatment. Mitochondria respiration of SCs was shown to decrease in response to TGF-β3. Lgl2, MCT4, activity of ERK, and p38 MAPK were up-regulated, whereas Akt and Notch pathway activity were inhibited by TGF-β3. Silencing of Lgl2 in SCs affected lactate production and attenuated the previous effects of TGF-β3 on SC glycolysis except for Gln consumption, Gls activity, and activity of Akt, ERK, and p38. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment in SCs antagonized glycolysis suppression caused by Lgl2-silencing. In vivo analysis revealed a stage-specific expression of Jagged1 in contrary with TGF-β3. Activating Notch signaling by re-Jagged1 resulted in restorable hypospermatogenesis and lowered lactate level in rat testis. TGF-β3 induces lactate production in SC through up-regulating Lgl2, which weakened the Notch signaling activity and intensified glycolysis in SCs. Thus, besides the known function of TGF-β3 as the BTB regulator, TGF-β3-Lgl2-Notch may be considered an important pathway controlling SC glycolysis and spermatogenesis.

Smtnl2 regulates apoptotic germ cell clearance and lactate metabolism in mouse Sertoli cells

Smtnl2 is an epithelial Smoothelin that binds to actin filaments and is crucial for epithelial morphogenesis. We examined the role of Smtnl2 in Sertoli cells, which undergo dynamic cytoskeleton reorganization to phagocytose apoptotic germ cells, a process known as efferocytosis. We observed Smtnl2 expression in primary mouse Sertoli cells and the 15P1 Sertoli cell line. Smtnl2 expression increased in 15P1 cells committing efferocytosis. Smtnl2-deficient Sertoli cells exhibited defective ability to engulf apoptotic germ cells and importantly, the phenomenon occurred in the setting of an unaffected maturation of phagosome. We demonstrated that Smtnl2 regulates the engulfment process through the function of branched actin nucleation protein ARP3, an actin assembly dictator. Intriguingly, a shift in glucose metabolism that restricts lactate production in Sertoli cells was induced upon Smtnl2 depletion, leading to the activation of downstream AMPK and AKT signaling. Using an in vivo RNAi approach, we found that silencing of Smtnl2 in testis triggers an obvious disruption in cytoskeleton architecture and blood-testis barrier integrity across seminiferous epithelium, causing the detachment of massive germ cells from their nest, as evidenced by their exfoliation into the lumen. Overall, our study identifies Smtnl2 as a determinant for Sertoli cells’ functioning in supporting spermatogenesis.

Identification of testicular Foxq1 as a critical modulator of lactate metabolism in mouse Sertoli cells.

Postmeiotic germ cells require the lactate produced by the adjacent Sertoli cells (SCs) as their sole energy fuels. Lactate production in SCs is elaborately regulated by monitoring the transcription of the lactate dehydrogenase A (Ldha) gene. However, the transcription factors that are responsible for the control of Ldha transcription in SCs remain ill defined. Herein, the expression of forkhead box Q1 (FOXQ1), a central modulator of glucose metabolism in liver, was demonstrated in mouse testis throughout postnatal development, with maximum levels in adult specimens. At this age, FOXQ1 was immunolocalized in the nuclei of the functionally mature SCs. Testicular levels of FOXQ1 were overtly modulated by germ cells (GCs)-derived IL-1α, in a dose- and time-dependent manner. To further clarify the biological functions of FOXQ1, we disrupted the mouse Foxq1 gene using a Cas9/RNA-mediated gene targeting strategy. Foxq1-/- males were subfertile and showed oligoasthenozoospermia due to lactate deficiency. Moreover, we provided the molecular evidence that FOXQ1 may regulate lactate production by directly targeting the transactivation of the Ldha gene in SCs. From a functional standpoint, overexpression of the exogenous Ldha ameliorated Foxq1 deficiency-impaired lactate synthesis in the SCsFoxq1-/- cells. Thus, these findings collectively underscore a reproductive facet of this recently characterized transcription factor, which may operate as a novel transcriptional integrator linking energy homeostasis and nursery function in SCs.

Participation of HIFs in the regulation of Sertoli cell lactate production

Hypoxia Inducible Factors (HIFs) are master regulators of glycolytic metabolism. HIFs consist of a constitutive HIFbeta (HIFβ) subunit and a HIFalpha (HIFα) subunit, whose half-life depends on prolyl-hydroxylases activity. Inhibition of prolyl-hydroxylases by hypoxia or transition metals, or augmentation of HIFα subunit levels by hormonal stimuli lead to a higher HIF transcriptional activity. On the other hand, it is well known that lactate produced by Sertoli cells is delivered to and used by germ cells as an energy substrate. The aim of this work was to investigate whether HIFs participate in the regulation of lactate production in rat Sertoli cells and whether they are involved in the FSH mechanism of action. In order to reach a higher HIF transcriptional activity, Sertoli cells were treated with CoCl2. We observed that a higher HIF transcriptional activity leads to an augmentation of: lactate production, glucose uptake and LDH activity. Besides, an increase in Glut1, Pkm2 and Ldha mRNA levels was observed. These findings suggested that HIFs may participate in the modulation of Sertoli cell nutritional function. As FSH regulates lactate production, we evaluated whether HIFs were involved in FSH action. Sertoli cells were stimulated with FSH in the absence or presence of LW6, a drug which promotes HIFα subunit degradation. On the one hand, we observed that FSH increases HIF1α protein, Hif1α and Hif2α mRNA levels and, on the other hand, that LW6 inhibits FSH-stimulated lactate production, glucose uptake, Glut1, Pkm2 and Ldha expression. It is proposed that HIFs are key components of the intricate pathways utilized by FSH to regulate the provision of lactate for germ cells. Considering that FSH is the master endocrine regulator of Sertoli cells, it is not surprising that this hormone may employ several regulatory mechanisms to fulfill the nourishing functions of this cell type.

Novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells.

The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the physiological relevance of this mechanism. As for the regulation of PDC, analysis of pyruvate dehydrogenase kinase (Pdk) expression showed that FSH increases Pdk3 and decreases Pdk4 mRNA levels while bFGF increases the expression of all Pdks. In addition, we showed that bFGF increases phosphorylated PDC levels and that bFGF-stimulated lactate production is partially inhibited in the presence of a PDK inhibitor. Altogether, these results add new information regarding novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells. Considering that lactate is essential for the production of energy in spermatocytes and spermatids, these mechanisms might be relevant in maintaining spermatogenesis and male fertility.

Effect of white tea (Camellia sinensis (L.)) extract in the glycolytic profile of Sertoli cell

Many health benefits have been attributed to tea (Camellia sinensis (L.)), and tea infusions are used as dietary agent and included in food supplements. Herein, we report the effect of a white tea (WTEA) extract in Sertoli cell (SC) metabolism. The SC is responsible for the nutritional support of the developing germ cells. An aqueous WTEA extract was prepared and analyzed by (1)H-NMR. Rat SCs were cultured with or without the WTEA extract. mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by qPCR and western blot. LDH activity was assessed and metabolite production/consumption determined by (1)H-NMR. WTEA-exposed SCs presented decreased protein and mRNA levels of GLUT1 and decreased glucose uptake. However, intracellular LDH activity was increased and SC lactate production was stimulated by the presence of the WTEA extract. Interestingly, alanine production was also found to be stimulated in WTEA extract-exposed SCs. WTEA extract altered the glycolytic profile of cultured SCs, stimulating lactate production. Since lactate is used as metabolic substrate and has an anti-apoptotic effect in the developing germ cells, the supplementation with WTEA extract may be advantageous to improve male reproductive health.

The AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-b-d-ribonucleoside, regulates lactate production in rat Sertoli cells

The aim of the present study was to investigate whether the AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in Sertoli cells and whether its activation by 5-aminoimidazole-4-carboxamide-1-b-d-ribonucleoside (AICAR) results in the regulation of cell metabolism to ensure lactate supply for germ cell development. Sertoli cell cultures from 20-day-old rats were used. Western blot analysis for the alpha-subunit of AMPK showed that high levels of AMPK are present in Sertoli cells. Treatment of the cultures with AICAR resulted in a dose- and time-dependent increase of P-AMPK levels indicating activation of the enzyme. A possible effect of AICAR on Sertoli cell lactate production was then analyzed. A dose- and time-dependent increment in lactate secretion was observed. The participation of AMPK activation in different biochemical processes that may be implicated in the regulation of lactate production was also analyzed. AICAR stimulated glucose uptake in a dose- and time-dependent manner. Additionally, AICAR increased the glucose transporter 1 (GLUT1) and decreased the glucose transporter 3 (GLUT3) mRNA levels. As for the role of AMPK in the regulation of the monocarboxylate transporters 1 and 4 (MCT1 and MCT4), it has been observed that AICAR treatment decreased MCT1 and increased MCT4 mRNA levels. In summary, the results presented herein show that AMPK is present in Sertoli cells and that its activation by AICAR increases lactate production as a result, at least in part, of a) an increase in glucose uptake, b) an increase in GLUT1 expression, and c) a decrease in MCT1 and an increase in MCT4 levels. Altogether, these results suggest an important role of AMPK in modulating the nutritional function of Sertoli cells.

Participation of phosphatidyl inositol 3-kinase/protein kinase B and ERK1/2 pathways in interleukin-1β stimulation of lactate production in Sertoli cells

Interleukin-1beta (IL1beta ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1beta regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1beta showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1beta . PD and W, but not R, decreased IL1beta-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1beta was also analyzed. It was observed that W decreased IL1beta-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1beta , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1beta stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1beta utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1beta utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.

Possible role of arachidonic acid in the regulation of lactate production in rat Sertoli cells.

The aim of the study was to determine whether arachidonic acid (AA) is involved in the regulation of Sertoli cell lactate production and if this fatty acid participates in follicle-stimulating hormone (FSH) regulation of Sertoli cell function. In a first set of experiments the effect of AA and porcine pancreas phospholipase A2 (PLA2) on lactate production, glucose uptake, lactate dehydrogenase (LDH) activity and LDH A mRNA levels in Sertoli cell cultures obtained from 20-day-old rats was evaluated. In a second set of experiments the effect of two PLA2 inhibitors--quinacrine (Q) and AACOCF3--on FSH stimulation of the above-mentioned parameters of Sertoli cell function was investigated. Treatment with PLA2 and AA increased Sertoli cell lactate production. The observed action of exogenously added PLA2 involved its catalytic properties responsible for AA release. PLA2 and AA treatments also stimulated Sertoli cell glucose uptake, LDH activity and LDH A mRNA levels. In order to determine whether AA participates in FSH regulation of Sertoli cell lactate production cells were incubated with FSH in the absence or presence of the PLA2 inhibitors Q and AACOCF3. Both drugs partially inhibited the ability of FSH to stimulate lactate production, glucose uptake and LDH activity. The present investigation suggests that AA is involved in the regulation of lactate production, glucose transport, LDH activity and LDH A mRNA levels. In addition, these results suggest that cytosolic PLA2 and AA may participate in FSH-regulation of Sertoli cell energetic metabolism.

Developmental and hormonal regulation of the monocarboxylate transporter 2 (MCT2) expression in the mouse germ cells.

During spermatogenesis, postmeiotic germ cells utilize lactate produced by Sertoli cells as an energy metabolite. While the hormonal regulation of lactate production in Sertoli cells has been relatively well established, the transport of this energy substrate to the germ cells, particularly via the monocarboxylate transporters (MCTs), as well as the potential endocrine control of such a process remain to be characterized. Here, we report the developmentally and hormonally regulated expression of MCT2 in the testis. At Day 18, MCT2 starts to be expressed in germ cells as detected by Northern blot. The mRNA are translated into protein (40 kDa) in elongating spermatids. Ultrastructural analysis demonstrated that MCT2 protein is localized to the outer face of the cell membrane of spermatid tails. MCT2 mRNA levels are under the control of the endocrine, specifically follicle-stimulating hormone (FSH) and testosterone, and paracrine systems. Indeed, a 35-day-old rat hypophysectomy resulted in an 8-fold increase in testicular MCT2 mRNA levels. Conversely, FSH and LH administration to the hypophysectomized rats reduced MCT2 mRNA levels to the basal levels observed in intact animals. The decrease in MCT2 mRNA levels was confirmed in vitro using isolated seminiferous tubules incubated with FSH or testosterone. FSH or testosterone inhibited in a dose-dependent manner MCT2 mRNA levels with maximal inhibitory doses of 2.2 ng/ml and 55.5 ng/ml for FSH and testosterone, respectively. In addition to the endocrine control, TNFalpha and TGFbeta also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFbeta and TNFalpha, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell-germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.

Regulation of lactate production and glucose transport as well as of glucose transporter 1 and lactate dehydrogenase A mRNA levels by basic fibroblast growth factor in rat Sertoli cells.

By using cultured rat Sertoli cells as a model, both the action of basic fibroblast growth factor (bFGF) on lactate production and the site of this action were studied. bFGF stimulated Sertoli cell lactate production in a dose-dependent manner (basal: 7.3+/-0.5; 0.1 ng/ml bFGF: 7.5+/-0.5; 1 ng/ml bFGF: 7.5+/-0.6; 10 ng/ml bFGF: 10.3+/-1.0; 30 ng/ml bFGF: 15.2+/-1.5; 50 ng/ml bFGF: 15.4+/-1.6 microg/microg DNA). Two major sites for the action of this growth factor were identified. First, bFGF was shown to exert short- and long-term stimulatory effects on glucose transport (basal: 1170+/-102; 30 ng/ml bFGF for 120 min: 1718+/-152 and basal: 718+/-64; 30 ng/ml bFGF for 48 h: 1069+/-69 d.p.m./microg DNA respectively). Short-term bFGF stimulation of glucose transport was not inhibited by the protein synthesis inhibitor cycloheximide. These results indicate that short-term bFGF stimulation of glucose uptake does not involve an increase in the number of glucose transporters. On the other hand, stimulation with bFGF for periods of time longer than 12 h increased glucose transporter 1 (GLUT1) mRNA levels. These increased mRNA levels were probably ultimately responsible for the increments in glucose uptake that are observed in long-term treated cultures. Secondly, bFGF increased lactate dehydrogenase (LDH) activity (basal: 31.0+/-1.4; 30 ng/ml bFGF: 45.7+/- 2.4 mIU/microg DNA). The principal subunit component of those LDH isozymes that favors the transformation of pyruvate to lactate is subunit A. bFGF increased LDH A mRNA levels in a dose- and time-dependent manner. In summary, the results presented herein show that glucose transport, LDH activity and GLUT1 and LDH A mRNA levels are regulated by bFGF to achieve an increase in lactate production. These observed regulatory actions provide unequivocal evidence of the participation of bFGF in Sertoli cell lactate production which may be related to normal germ cell development.

Toxic effects of polychlorinated biphenyls on cultured rat Sertoli cells.

Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental contaminants. In mammals, PCBs affect spermatogenesis and may be associated with Sertoli cell changes. Therefore, our aim was to evaluate in vitro toxic effects of hydroxylated PCB (PCB-22; 2',3',4',5'-tetrachloro-4-biphenylol) and PCB congener (PCB-77; 3,3',4,4'-tetrachlorobiphenyl) on Sertoli cells isolated from 19- to 21-day-old male rats. Sertoli cells incubated for 24 hours in 10(-7) M PCB-22 and 10(-8) M PCB-77, but not in 0.05% ETOH or 10(-7) M 17beta-estradiol (E2) showed morphological changes. Sertoli cells demonstrated progressive damage with higher concentrations of PCB-77 (10(-7) M). After 24 hours, 10(-7) M PCB-22 killed 20% of the Sertoli cells and equimolar PCB-77 killed 45% of the Sertoli cells in culture. At 10(-8) M, PCB-22 did not kill any significant number of Sertoli cells, whereas PCB-77 killed 40% of cells in culture. This result showed differential effects of PCB compounds, with PCB-77 being more cytotoxic than PCB-22 as tested on Sertoli cells. Because PCB-77 produced greater toxic effects, we further tested this congener on Sertoli cell lactate production. After 24 hours, lactate production by Sertoli cells treated with 10(-7) or 10(-8) M PCB-77 was significantly increased. Finally, Sertoli cells exposed to 10(-7) M PCB-77 showed disorganized and less intense F-actin staining. The results demonstrate that PCBs, but not E2, is directly toxic to Sertoli cells in vitro, and suggest this toxic effect is independent of estrogenic action.

Interleukin 1alpha stimulates lactate dehydrogenase A expression and lactate production in cultured porcine sertoli cells.

By using cultured porcine Sertoli cells as a model, the action of interleukin 1alpha (IL-1alpha) on lactate production and the site of this action were studied. IL-1alpha stimulated Sertoli cell lactate production in a time- and dose-dependent manner (with a half-maximal effect [ED50] of 6 pM). Two major sites involved in IL-1alpha action were identified. First, IL-1alpha was shown to increase the uptake of glucose substrate in a time- and dose-dependent manner. The maximal effect, with an ED50 of 10 pM, was observed after 24 h of treatment. Second, IL-1alpha increased the activity of the lactate dehydrogenase (LDH) A4 isoform, which is involved in the conversion of pyruvate into lactate. This increase in LDH A4 activity was detected at 12 h and was maximal, with an ED50 of 9 pM, after 24-h treatment with IL-1alpha. The increase was related to an increase in LDH A4 expression, since IL-1alpha stimulated LDH A mRNA (size: 1.5 kilobases, evidenced through Northern blotting analysis) in a dose- and time-dependent manner. Assuming that IL-1alpha might be produced in the seminiferous tubules by both Sertoli and germ cells, which utilize lactate for their energy metabolism, we suggest that these results together show 1) that the cytokine may represent a signal in the metabolic cooperation existing between Sertoli cells and germ cells, and 2) that a redistribution of LDH isoforms in favor of LDH A4 under IL-1alpha control is a key mechanism(s) in such cooperation used by germ cells to enhance lactate production in Sertoli cells.

Tumor necrosis factor-alpha-stimulated lactate production is linked to lactate dehydrogenase A expression and activity increase in porcine cultured Sertoli cells.

By using, as a model, cultured testicular immature Sertoli cells, the action of tumor necrosis factor-alpha (TNF alpha) and the site of action of the cytokine on lactate production were studied. TNF alpha stimulated in a time- and dose-dependent manner (with an ED50 of 0.1 nM) Sertoli cell lactate production. Two major sites involved in TNF alpha action were identified. Firstly, TNF alpha was shown to increase the uptake of glucose substrate in a time- and dose-dependent manner. The maximal effect was observed after 24 h of treatment, with an ED50 of 0.1 nM. Secondly, TNF alpha increased the activity of lactate dehydrogenase (LDH) A isoform, which is involved in the conversion of pyruvate into lactate. This increase in LDH-A activity was detected at 12 h and was maximal after 24 h of treatment with TNF alpha. The stimulatory effect of the cytokine on the LDH-A isoform was observed with an ED50 of 0.05 nM. Such an increase in LDH-A activity was related to an increase in LDH-A expression, because TNF alpha stimulated LDH-A messenger RNA (size, 1.5 kilobases, determined by Northern blotting analysis). Together, assuming that in the seminiferous tubules, TNF alpha is produced by spermatids that use lactate for their energetic metabolism, we suggest that the cytokine may potentially represent a signal used by germ cells to enhance lactate production in Sertoli cells through, at least, a redistribution of LDH isoforms in favor of LDH-A.

Developmental regulation of Sertoli cell lactate production by hormones and the testicular paracrine factor, PModS

Testicular peritubular cells produce a paracrine factor termed PModS that mediates mesenchymal-epithelial interactions and modulates Sertoli cell functions essential for the process of spermatogenesis. Sertoli cells produce lactate as a preferred energy metabolite for developing spermatogenic cells. The current study was designed to examine the actions of PModS and hormones on Sertoli cell lactate production at various stages of pubertal development. Sertoli cells were isolated from pre-pubertal (10 day), midpubertal (20 day) and late pubertal (35 day) rat testes. Lactate accumulation in the conditioned-medium of cultured Sertoli cells was measured. Basal lactate production increased approximately fivefold during pubertal Sertoli cell development. Therefore, lactate production increases as the Sertoli cell differentiates during pubertal development. The ability of regulatory agents such as FSH or a combination of FSH, insulin, retinol and testosterone (FIRT) to stimulate lactate production decreased during pubertal development as Sertoli cell differentiation increased. Purified PModS stimulated lactate production in Sertoli cell preparations throughout pubertal development. PModS had a greater effect than FSH in stimulating late pubertal Sertoli cell lactate production. PModS in combination with FIRT resulted in an additive stimulation of lactate production suggesting a distinct mechanism of action for PModS. Observations support the proposal that the locally produced paracrine factor PModS mediates mesenchymal-epithelial cell interactions during pubertal development and that these interactions promote Sertoli cell differentiated functions (i.e. lactate production) required for the developing spermatogenic cells.

Effect of mono-(2-ethylhexyl) phthalate on the metabolism of energy-yielding substrates in rat sertoli cell-enriched cultures

The effect of the testicular toxin mono-(2-ethylhexyl) phthalate (MEHP) on the metabolism of energy-yielding substrates in Sertoli cell-enriched cultures has been studied. MEHP stimulated glucose utilization and oxidation. Stimulation of 14CO 2 production was greater with [1- 14C]- than with [6- 14C]glucose. Oxidation of [1- 14C]pyruvate and [U- 14C]acetate to 14CO 2 was reduced by MEHP treatment in the presence but unaffected in the absence of glucose. MEHP increased the incorporation of radioactivity from [1- 14C]- and [6- 14C]glucose but not from [U- 14C]acetate into fatty acids. MEHP markedly increased the production of lactate by Sertoli cells cultured in the presence of 5.5 m m-glucose and 0.5 m m-pyruvate. Decreasing the glucose concentration reduced this stimulatory effect. In glucose-free medium containing 0.5 or 2.5 m m-pyruvate MEHP had no effect on Sertoli cell lactate production. Thus, MEHP appears to stimulate the utilization of glucose by the Sertoli cell but does not appear to have a direct effect on the conversion of pyruvate to lactate, on the conversion of pyruvate to acetyl CoA or a direct effect on the metabolism of acetyl CoA through the Krebs cycle. Increased glucose utilization appears to be a consequence of the increased metabolism of glucose that occurs through the glycolytic and pentose phosphate pathways. These results suggest that in contrast to its effects on the liver, MEHP does not exert a primary effect on the Sertoli cell mitochondrion.

The effect of mono-(2-ethylhexyl) phthalate and other phthalate esters on lactate production by Sertoli cells in vitro

Sertoli cells produce lactate and pyruvate as energy substrates for the developing germ cells in the testis. Since the Sertoli cells are thought to be the initial target for phthalate esters causing testicular atrophy, the effect of some phthalates on lactate and pyruvate production by primary Sertoli cellenriched cultures was studied. Mono-(2-ethylhexyl) phthalate (0.1–200 μM) produced a concentrationdependent stimulation of lactate, but not pyruvate production over a 24 h treatment period and an increase in the ratio of lactate/pyruvate concentration in the culture medium. Di-(2-ethylhexyl) phthalate and 2-ethylhexanol (200 μM) had no such effects. Other phthalate monoesters known to cause testicular atrophy also increased Sertoli cell lactate production and the lactate/pyruvate ratio, whereas monoesters not associated with testicular damage in vivo had no such effects. The results suggest that loss of germ cells in phthalate-induced testicular atrophy is not due to inhibition of energy substrate production by the Sertoli cells and that stimulation of lactate production may be a useful in vitro marker for phthalate esters and related compounds that cause testicular injury.