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While glycolysis and oxidative phosphorylation (OXPHOS) are established ATP sources, they cannot fully explain motility maintenance under glucose\u2010deprived conditions. This study examined whether gluconeogenesis functions as a third metabolic pathway supporting sperm energy production.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Sperm from mice, humans, cattle, and horses were comparatively analyzed under glucose\u2010containing and glucose\u2010free conditions with inhibitors of OXPHOS (FCCP) and fructose\u20101,6\u2010bisphosphatase 1 (FBPase1). Motility and ATP levels were measured, and FBPase1 expression was assessed by Western blotting and immunofluorescence.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Mouse sperm maintained motility without glucose, and inhibition of FBPase1 significantly reduced ATP and rapid progressive motility. FBPase1 was expressed in the midpiece and principal piece, indicating active gluconeogenesis. Similar patterns were observed in human and bovine sperm, suggesting a conserved mechanism among mammals.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>Gluconeogenesis serves as a third metabolic platform that complements glycolysis and OXPHOS, enabling efficient substrate use and sustaining sperm motility. This discovery provides new insight into sperm metabolic flexibility and may contribute to treatments for male infertility and improved livestock reproduction.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/rmb2.70017","type":"journal-article","created":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T07:36:10Z","timestamp":1768635370000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["The Three Pillars of\n                    <scp>ATP<\/scp>\n                    Production in Mammalian Sperm: Integrating Gluconeogenesis Into the Metabolic Framework"],"prefix":"10.1002","volume":"25","author":[{"given":"Kosei","family":"Oishi","sequence":"first","affiliation":[{"name":"Graduate School of Engineering Science Yokohama National University  Yokohama Kanagawa 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