Becerro-Rey Laura, Martín-Cano Francisco E, Silva-Rodríguez Antonio, Ortega-Ferrusola Cristina, da Silva-Álvarez Eva, Ortiz-Placín Cándido, Tapia Jose Antonio, Gil María Cruz, Peña Fernando J
Reproduction. 2025 Jun 5;170(1). doi: 10.1530/REP-24-0436. Print 2025 Jul 1.
Three isoforms of lactate dehydrogenase (LDH) - LDHA (cytoplasmic), LDHB (mitochondrial), and LDHC (flagellar) - have been identified and localized in stallion spermatozoa. Functional inhibition assays indicate that these three isoforms constitute a lactate shuttle of crucial importance for sperm function.
Stallion spermatozoa use different energy sources; while oxidative phosphorylation predominates, glycolysis and beta-oxidation of fatty acids are also present. Glycolysis depends on the availability of NAD+ as an electron acceptor. During glycolysis, NAD+ is reduced to NADH. To ensure glycolysis can continue, NAD+ must be regenerated. This regeneration typically occurs when NADH donates its electrons to the electron transport chain (specifically at Complex I), where it is oxidized back to NAD+. If mitochondria are damaged, the regeneration of NAD+ may be compromised, leading to reduced glycolysis and altering sperm metabolism. However, alternative ways to regenerate NAD+ may be present. We hypothesized that aerobic glycolysis is present in the stallion spermatozoa as a backup mechanism to regenerate NAD+. We incubated spermatozoa in two Tyrode's modified media with either 67 mM glucose and 1 mM pyruvate or 67 mM glucose and 10 mM pyruvate. The addition of 10 mM pyruvate improved sperm motility (P < 0.001). Spermatozoa incubated in 67 mM glucose and 1 mM pyruvate for 3 h at 37°C showed a significant decrease in motility (58.1 ± 1.8% vs 81.2 ± 1.8%, P < 0.0001). In contrast, spermatozoa incubated in 67 mM glucose and 10 mM pyruvate retained motility (77.1 ± 1.4%), viability, and mitochondrial membrane potential. We studied the metabolic proteome and metabolome and identified three different isoforms of the enzyme lactate dehydrogenase (LDH), LDHA (cytosolic), LDHB (mitochondrial, with higher affinity for pyruvate), and LDHC (cytosol, motile cilium). Functional experiments using a specific inhibitor of LDHC demonstrated that this isoform may be essential for sperm function. We concluded that activation of aerobic glycolysis in a high-glucose medium improves sperm survival through the regeneration of NAD+.
已在种马精子中鉴定并定位了乳酸脱氢酶（LDH）的三种同工型——LDHA（细胞质型）、LDHB（线粒体型）和LDHC（鞭毛型）。功能抑制试验表明，这三种同工型构成了对精子功能至关重要的乳酸穿梭系统。
种马精子使用不同的能量来源；虽然氧化磷酸化占主导，但脂肪酸的糖酵解和β-氧化也存在。糖酵解依赖于作为电子受体的NAD⁺的可用性。在糖酵解过程中，NAD⁺被还原为NADH。为确保糖酵解能够继续，NAD⁺必须再生。这种再生通常发生在NADH将其电子传递给电子传递链时（特别是在复合体I处），在那里它被氧化回NAD⁺。如果线粒体受损，NAD⁺的再生可能会受到影响，导致糖酵解减少并改变精子代谢。然而，可能存在再生NAD⁺的替代途径。我们假设种马精子中存在有氧糖酵解作为再生NAD⁺的备用机制。我们将精子在两种改良的Tyrode's培养基中孵育，一种含有67 mM葡萄糖和1 mM丙酮酸，另一种含有67 mM葡萄糖和10 mM丙酮酸。添加10 mM丙酮酸可改善精子活力（P < 0.001）。在37°C下于67 mM葡萄糖和1 mM丙酮酸中孵育3小时的精子活力显著下降（58.1 ± 1.8%对81.2 ± 1.8%，P < 0.0001）。相比之下，在67 mM葡萄糖和10 mM丙酮酸中孵育的精子保持了活力、生存能力和线粒体膜电位。我们研究了代谢蛋白质组和代谢组，并鉴定出乳酸脱氢酶（LDH）的三种不同同工型，LDHA（胞质型）、LDHB（线粒体型，对丙酮酸具有更高亲和力）和LDHC（胞质型，运动纤毛型）。使用LDHC特异性抑制剂的功能实验表明，这种同工型可能对精子功能至关重要。我们得出结论，在高糖培养基中激活有氧糖酵解可通过NAD⁺的再生提高精子存活率。
