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

Backgrounds: 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.

Objectives: 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.

Materials and methods: 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.

Results: 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 5 ng/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.

Conclusion: 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.