Synergistic reduction in albuminuria in type 2 diabetic mice by esaxerenone (CS-3150), a novel nonsteroidal selective mineralocorticoid receptor blocker, combined with an angiotensin II receptor blocker.

Esaxerenone is a novel selective mineralocorticoid receptor (MR) blocker that was recently approved in Japan to treat hypertension. In phase II and III studies, esaxerenone plus a renin-angiotensin system inhibitor markedly reduced the urinary albumin-to-creatinine ratio (UACR) in hypertensive patients with diabetic nephropathy. To evaluate a direct renoprotective effect by MR blockade independent of an antihypertensive effect in the context of diabetic nephropathy, esaxerenone (3 mg/kg), olmesartan (an angiotensin II receptor blocker; 1 mg/kg), or both were orally administered to KK-Ay mice, a type 2 diabetes model, once daily for 56 days. Urinary albumin (Ualb), UACR, and markers, such as podocalyxin, monocyte chemoattractant protein-1 (MCP-1), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), were measured, along with systolic blood pressure (SBP), fasting blood glucose, and serum K+ levels. Prior to the initiation of drug administration, KK-Ay mice showed higher blood glucose, insulin, Ualb excretion, and UACR levels than C57BL/6 J mice, a nondiabetic control, indicating the development of diabetic renal injury. Combined treatment with esaxerenone and olmesartan significantly reduced the change in UACR from baseline compared with the change associated with vehicle at week 8 (-1.750 vs. 0.339 g/gCre; P < 0.002) and significantly inhibited the change in Ualb from baseline compared with the change associated with vehicle at week 8 (P < 0.002). The combination treatment also reduced urinary excretion of podocalyxin and MCP-1, but did not influence 8-OHdG excretion, SBP, blood glucose, or serum K+ levels. Overall, esaxerenone plus olmesartan treatment ameliorated diabetic nephropathy in KK-Ay mice without affecting SBP, suggesting that the renoprotective effects of esaxerenone could be exerted independently of its antihypertensive effect.

CS-3150, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, Shows Preventive and Therapeutic Effects On Renal Injury in Deoxycorticosterone Acetate/Salt-Induced Hypertensive Rats.

The present study was designed to assess both preventive and therapeutic effects of (S)-1-(2-Hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide (CS-3150), a novel nonsteroidal mineralocorticoid receptor antagonist, on renal injury in deoxycorticosterone acetate (DOCA)/salt-induced hypertensive rats (DOCA rats). From 7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt diet. In experiment 1, CS-3150 (0.3-3 mg/kg) was orally administered once a day for 4 weeks coincident with DOCA administration. In experiment 2, after establishment of renal injury by 4 weeks of DOCA/salt loading, CS-3150 (3 mg/kg) was orally administered once a day for 4 weeks with or without continuous DOCA administration. In experiment 1, DOCA/salt loading significantly increased systolic blood pressure (SBP), which was prevented by CS-3150 in a dose-dependent manner. Development of renal injury (proteinuria, renal hypertrophy, and histopathological changes in glomeruli and tubule) was also suppressed by CS-3150 with inhibition of mRNA expression of fibrosis, inflammation, and oxidative stress markers. In experiment 2, under continuous DOCA treatment, CS-3150 clearly ameliorated existing renal injury without lowering SBP, indicating that CS-3150 regressed renal injury independent of its antihypertensive action. Moreover, CS-3150 treatment in combination with withdrawal of DOCA showed further therapeutic effect on renal injury accompanied by reduction in SBP. These results demonstrate that CS-3150 not only prevents but also ameliorates hypertension and renal injury in DOCA rats. Therefore, CS-3150 could be a promising agent for the treatment of hypertension and renal disorders, and may have potential to promote regression of renal injury.

CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist, prevents hypertension and cardiorenal injury in Dahl salt-sensitive hypertensive rats.

The present study was designed to evaluate the antihypertensive and cardiorenal protective effects of CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist, in Dahl salt-sensitive hypertensive rats (DS rats), and to compare the effects with spironolactone and eplerenone. DS rats were fed a control diet (0.3% NaCl) or high salt diet (8% NaCl) from 7 weeks of age. CS-3150 (0.25-2mg/kg), spironolactone (10-100mg/kg) or eplerenone (10-100mg/kg) were orally administered once a day to DS rats fed a high salt diet for 7 weeks. The high salt diet significantly increased systolic blood pressure, which was prevented by treatment with CS-3150 in a dose-dependent manner with no hyperkalemia (>5.5mEq/L). The antihypertensive effect of CS-3150 (0.5mg/kg) was equivalent to that of spironolactone (100mg/kg) and eplerenone (100mg/kg). CS-3150 also suppressed proteinuria and renal hypertrophy induced by the high salt diet. Histopathological examination of kidneys showed that CS-3150 markedly ameliorated glomerulosclerosis, tubular injury and tubulointerstitial fibrosis. In addition, CS-3150 inhibited left ventricular hypertrophy and elevation of plasma brain natriuretic peptide level. In contrast, the cardiorenal protective effects of spironolactone or eplerenone were partial, and the dose-dependency was not clear, especially in eplerenone-treated rats. These results indicate that chronic treatment with CS-3150 exerts antihypertensive and cardiorenal protective effects in a DS hypertensive rat model, and its potency is much superior to that of spironolactone or eplerenone. Thus, CS-3150 could be a promising agent for the treatment of hypertension and cardiorenal disorders.

Pharmacological profile of CS-3150, a novel, highly potent and selective non-steroidal mineralocorticoid receptor antagonist.

The present study was designed to characterize the pharmacological profile of CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist. In the radioligand-binding assay, CS-3150 inhibited (3)H-aldosterone binding to mineralocorticoid receptor with an IC50 value of 9.4nM, and its potency was superior to that of spironolactone and eplerenone, whose IC50s were 36 and 713nM, respectively. CS-3150 also showed at least 1000-fold higher selectivity for mineralocorticoid receptor over other steroid hormone receptors, glucocorticoid receptor, androgen receptor and progesterone receptor. In the reporter gene assay, CS-3150 inhibited aldosterone-induced transcriptional activation of human mineralocorticoid receptor with an IC50 value of 3.7nM, and its potency was superior to that of spironolactone and eplerenone, whose IC50s were 66 and 970nM, respectively. CS-3150 had no agonistic effect on mineralocorticoid receptor and did not show any antagonistic or agonistic effect on glucocorticoid receptor, androgen receptor and progesterone receptor even at the high concentration of 5µM. In adrenalectomized rats, single oral administration of CS-3150 suppressed aldosterone-induced decrease in urinary Na(+)/K(+) ratio, an index of in vivo mineralocorticoid receptor activation, and this suppressive effect was more potent and longer-lasting than that of spironolactone and eplerenone. Chronic treatment with CS-3150 inhibited blood pressure elevation induced by deoxycorticosterone acetate (DOCA)/salt-loading to rats, and this antihypertensive effect was more potent than that of spironolactone and eplerenone. These findings indicate that CS-3150 is a selective and highly potent mineralocorticoid receptor antagonist with long-lasting oral activity. This agent could be useful for the treatment of hypertension, cardiovascular and renal disorders.

Effect of fibroblast growth factor and enamel matrix derivative treatment on root resorption after delayed replantation.

BACKGROUND: Periodontal ligament (PDL) healing and long term prognosis of replanted avulsed teeth should rely on several factors including length of extra-oral dry time and type of the storage medium. The status of periodontal ligament is critical for the healing of replanted teeth. Different substances have been used for root surface treatment to promote formation of PDL and increase the survival of avulsed teeth submitted to replantation. AIM: The purpose of this study was to assess the effect of recombinant basic fibroblast growth factor 2 (bFGF) and enamel matrix derivative (EMD) on root resorption after delayed replantation. DESIGN: 18 freshly extracted single-rooted incisor and premolar teeth were extracted from the beagle dogs and immersed in whole bovine milk for 45 and 60 min (n = 3 each). Following storage period, sockets washed and teeth were treated with bFGF and EMD and replanted into the sockets. After 8 weeks, dogs were sacrificed, specimens processed to 4-µm thick serial sections for histopathologic examination and morphometric assessments. Thus, the proportions of the roots that exhibited signs of surface resorption, inflammatory resorption, and replacement resorption, that is, ankylosis and normal PDL were noted. RESULTS: The percentage of root resorption was in the following order: EMD>milk>bFGF for 45 min and milk>EMD>bFGF for 60 min. For all groups, teeth stored 60 min showed significantly higher incidence of PDL resorption than those stored for 45 min (P < 0.01). The highest incidence of replacement resorption was observed in teeth treated with EMD for 60 min. After 8 weeks, the least resorption was found in bFGF-treated group (P < 0.01). CONCLUSIONS: The findings of this study suggest that use of bFGF favored the formation of new periodontal ligament; prevent ankylosis and resorption process following delayed replantation of teeth while EMD shows replacement resorption, which may turn to ankylosis.

Effect of plasma-irradiated silk fibroin in bone regeneration.

We have recently identified plasma-irradiated silk fibroin (P-AF) as a key regulator of bone matrix properties and composition. Bone matrix properties were tested in 48 femur critical size defects (3.25 mm in diameter) with the expression of osteoblast specific genes at 1 and 2 weeks after surgery. The scaffolds were characterized by various states of techniques; the scanning electronic microcopy revealed the large sized pores in the aqueous-based silk fibroin (A-F) scaffold and showed no alteration into the architecture by the addition of plasma irradiation. The contact angle measurements confirmed the introduction of plasma helped to change the hydrophobic nature into hydrophilic. The histological analyses confirmed the presence of silk fibroin in scaffolds and newly formed bone around the scaffolds. Immunohistochemical examination revealed the increased expression pattern in a set of osteoblast specific genes (TGF-ß, TGF-ß type III receptor, Runx2, type I collagen and osteocalcin). These data were the first to show that the properties of bone matrix are regulated, specifically through Runx2 pathway in P-AF group. Thus, an employment of P-AF increases several compositional properties of bone, including increased bone matrix, mineral concentration, cortical thickness, and trabecular bone volume.

Silk fibroin-based scaffolds for bone regeneration.

Porous scaffolds were prepared using regenerated Bombyx mori silk fibroin dissolved in water or hexafluoroisopropanol (HFIP). The effects of these two preparations on the formation and growth of new bone on implantation into the rabbit femoral epicondyle was examined. The aqueous-based fibroin (A-F) scaffold exhibited significantly greater osteoconductivity as judged by bone volume, bone mineral content, and bone mineral density at the implant site than the HFIP-based fibroin (HFIP-F) scaffold. Micro-CT analyses showed that the morphology of the newly formed bone differed significantly in the two types of silk fibroin scaffold. After 4 weeks of implantation, new trabecular bone was seen inside the pores of the A-F scaffold implant while the HFIP-F scaffold only contained necrotic cells. No trabecular bone was seen within the pores of the latter scaffolds, although the pores of these did contain giant cells and granulation tissue.

Spironolactone, but not eplerenone, impairs glucose tolerance in a rat model of metabolic syndrome.

Although some clinical studies have suggested that spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, appears to increase the blood glucose levels, experimental studies have not supported this notion. Here, we investigated the effect of SPL on blood glucose levels in SHR/NDmcr-cp(cp/cp) (ND) rats, an animal model of metabolic syndrome, in comparison with that of eplerenone (EPL), another MR antagonist. At the same dose of 100 mg/kg, SPL and EPL increased the urinary sodium-to-potassium ratio to a comparable extent, indicating that both agents have similar renal MR antagonistic efficacy in ND rats. Interestingly, SPL but not EPL significantly increased the level of blood glucose. The oral glucose tolerance test revealed that treatment with SPL led to glucose intolerance. The levels of serum insulin and adiponectin, regulators of the blood glucose level, were virtually unaffected by treatment with SPL. On the other hand, SPL induced a marked increase in the blood level of aldosterone, known to be a risk factor for insulin resistance. These results demonstrate that in comparison with EPL, SPL characteristically impairs glucose tolerance in an animal model of metabolic syndrome, in association with a higher blood level of aldosterone.

Activation of renal angiotensin type 1 receptor contributes to the pathogenesis of progressive renal injury in a rat model of chronic cardiorenal syndrome.

Although chronic cardiac dysfunction is known to progressively exacerbate renal injury, a condition known as type 2 cardiorenal syndrome (CRS), the mechanism responsible is largely unknown. The present study was undertaken to clarify the mechanism of renal injury in rats with both unilateral nephrectomy (NX) and surgically induced myocardial infarction (MI), corresponding to a model of type 2 CRS. Compared with a control group, rats with both MI and NX (MI+NX) exhibited progressive proteinuria during the experimental period (34 wk after MI surgery), whereas proteinuria was not observed in rats with MI alone and was moderate in rats with NX alone. The proteinuria in rats with MI+NX was associated with renal lesions such as glomerulosclerosis and infiltration of mononuclear cells and upregulation of the renal proinflammatory and -fibrotic cytokine and angiotensin II type 1a receptor (AT1aR) genes. In contrast, plasma renin activity was lowered in rats with MI+NX. Immunohistochemistry revealed that the increased AT1R protein was present mainly in renal interstitial mononuclear cells. Olmesartan medoxomil, an AT1R blocker, markedly reduced the proteinuria and infiltration of mononuclear cells, whereas spironolactone, a mineralocorticoid receptor blocker, did not. The present findings demonstrate the pathogenetic role of renal interstitial AT1R signaling in a model of type 2 CRS, providing evidence that AT1R blockade can be a useful therapeutic option for this syndrome.

Growth hormone inhibition of glucose uptake in adipocytes occurs without affecting GLUT4 translocation through an insulin receptor substrate-2-phosphatidylinositol 3-kinase-dependent pathway.

Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4.

Novel 3,3a,5,9b-tetrahydro-2H-furo[3,2-c][2] benzopyran derivatives: synthesis of chiral glycol benzyl ether herbicides.

Novel tricyclic 3,3a,5,9b-tetrahydro-2H-furo[3,2-c][2]benzopyran (TFB) derivatives were synthesized, and their herbicidal activities were elucidated. They were synthesized from D-glucose as a natural chiral source. The formation of the TFB skeleton was achieved by a Friedel-Crafts type intramolecular cyclization of methyl 5-deoxy-2,3-O-dibenzyl-5-C-methyl-D-xylofranosides. The intramolecular cyclization was dependent upon the electronic effects of the substituents at the C-2 benzyloxy group of methyl xylofranosides. Some TFBs exhibited a remarkable herbicidal activity to annual paddy weeds, such as Echinochloa sp, without injury to the rice.

Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells: (tubulin/spermatocytes/spermatids/rat).

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[35 S]methionine ([35 S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [35 S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.

Metabolism of Round Spermatids: A Possible Regulation of Hexose Transport: (hexose transport/ATP/rat spermatids).

Round spermatids (steps 1-8) were isolated from rat testes and glucose transport into the cells was examined. The exposure of spermatids to glucose resulted in an extremely low level of ATP. In contrast, the level of ATP remained constant in the presence of pyruvate. Transport of a glucose analogue, 2-deoxy-D-[3 H]glucose ([3 H]dGlc) into spermatids was correlated with intracellular levels of ATP and was much greater in cells with higher rather than lower levels of ATP. [3 H]dGlc transport into spermatids with low levels of ATP was partially reversed when the cells were incubated with pyruvate. Inhibition of [3 H]dGlc transport was exerted on Vmax and not on Km. Moreover, glucose acted as a competitive inhibitor of [3 H]dGlc uptake (Km increased; Vmax unaltered). These results suggest that glucose transport into spermatids is active in vitro and probably regulated by the intracellular level of ATP.

Metabolism of Round Spermatids: Pyruvate cannot Maintain the ATP Level: (ATP synthesis/α-ketoacid/rat spermatids).

Round spermatids (steps 1-8) were isolated from rat testes and the effect of pyruvate on their intracellular ATP level was examined. Results showed that although the spermatids consumed a considerable amounts of pyruvate, this substrate alone did not maintain their ATP level. However, their ATP level was maintained in the presence of both pyruvate and α-ketovalerate or α-ketobutyrate. Maintenance of the ATP level by these substrates was associated with electron trasnport and oxidative phosphorylation. α-Ketoacid inhibited pyruvate reduction to lactate in the lactate dehydrogenase (LDH) reaction, but increased pyruvate oxidation to CO2 . The NADH level in spermatids was too low to be detectable, but the NAD level remained unchanged in the presence of pyruvate and α-ketovalerate. These results suggest that pyruvate by itself is not an adequate energy-yielding substrate for spermatids and that a high NADH/NAD ratio may be essential for maintenance of their ATP level.

Stimulation of Spermatid Phosphofructokinase by Fructose 2,6-Bisphosphate from Rat Testes: (fructose 2, 6-bisphosphate/phosphofructokinase/spermatids/rats).

Effect of fructose 2, 6-bisphosphate on 6-phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) in spermatid extract from rat testes was studied. Fructose 2, 6-bisphosphate stimulated the enzyme greatly by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Fructose 2, 6-bisphosphate (0.8 µM) was required for 50% activation of 6-phosphofructokinase (PFK). In addition, fructose 2, 6-bisphosphate, AMP and fructose 6-phosphate acted cooperatively to stimulate the activity of PFK. This stimulation may play an important role in the regulation of glycolysis in spermatids of rat testes.

Synergistic Inhibition of Fructose 1,6-Bisphosphatase by Fructose 2,6-Bisphosphate and Adenosine Monophosphate in Round Spermatids of Rats: (fructose 1, 6-bisphosphatase/AMP/fructose 2, 6-bisphosphate/spermatids/rats).

The effects of adenosine monophosphate (AMP) and fructose 2, 6-bisphosphate (fruc-2, 6-P2 ) on the key-enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (fruc-P2 ase; D-fructose 1, 6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in spermatid extract from rat testes were studied. The fruc-P2 ase activity in the spermatids of rats was suppressed by AMP and fruc-2, 6-P2 . The inhibition of fruc-2, 6-P2 was much stronger at low than at high substrate concentrations, and enhanced synergistically with AMP. The substrate saturation curve was changed by fruc-2, 6-P2 hyperbolic to sigmoidal. Furthermore, the concentration of AMP that decreased the activity to 50% was much lower in the presence than in the absence of fruc-2, 6-P2 . These results indicate the possibility that gluconeogenesis in spermatids of rats is controlled by AMP and fruc-2, 6-P2 .

Influence of Temperature on Glucose Metabolism of Round Spermatids of Rats: (temperature/glucose metabolism/spermatids/rat testis).

Glucose utilization by spermatids was found to be 17.37±0.37 nmoles/hr/106 cells at 34°C and 28.94±1.12nmoles/hr/106 cells at 40°C. A good parallelism was observed between the increased rate of glucose utilization and lactate production at 40°C. There was no significant change in the levels of glycolytic intermediates in the cells, except for marked accumulations of fructose-1, 6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in the presence of glucose (1 mM). Glucose oxidation in the citrate cycle by spermatids was higher at 40°C than at 34°C, but was never greater than 2% of the overall rate of glucose utilization. In addition, glucose did not prevent decrease of ATP at either 34 or 40°C. The effects of temperature on the activities of 11 glycolytic enzymes were examined. The activities of aldolase and phosphoglyceromutase were similar between 30 and 34°C, but increased markedly at 40°C. The higher temperature increased the Vmax values, without affecting the Kms. The activities of other glycolytic enzymes were similar at the different temperatures. These findings indicate that the increased overall rate of glucose utilization in glycolysis at higher temperature is due to increased Vmax values of aldolase and phosphoglyceromutase.

Changes in Activities of Glucose Metabolising Enzymes in Germ Cells during Spermatogenesis in Rats: (glucose-6-phosphate dehydrogenase/germ cells/spermatogenesis/rat).

The rate of 14 CO2 , liberation from [14 C-1]glucose was identical to that from [14 C-6]glucose in spermatids, but more than the latter in spermatogonia. Rotenone (1 µM) completely inhibited 14 CO2 release from [14 C-1]glucose in spermatids, but decreased it only 30% in spermatogonia. The activity of glucose-6-phosphate dehydrogenase, but not 6-phosphogluconate dehydrogenase, was markedly lower in spermatocytes and spermatids than in spermatogonia. The activities of the glycolytic enzymes, glucosephosphate isomerase, fructose diphosphatase, glyceraldehyde-3-phosphate dehydrogenase and enolase, differed only slightly in spermatids and spermatogonia. It is concluded that the low glucose-6-phosphate dehydrogenase activity may contribute to the low activity of the pentose cycle in spermatocytes and spermatids.

Influence of Temperature on Lactate Utilization by Round Spermatids from Rat Testes: (Temperature/lactate/spermatids/rat/oxidative phosphorylation).

Rotenone-sensitive 14 CO2 formation from [14 C]lactate and oxygen consumption by round spermatids were found to be greater at elevated temperatures than at 34°C. More than 96% of the total radioactivity of the metabolized [14 C]lactate was recovered in the released CO2 and the acid soluble fraction of the cells. There was practically no incorporation of [14 C]latctate into the lipid, nucleic acid, and protein fractions. Intracellular level of ATP in spermatids was enhanced in the presence of lactate (20 mM) at 34°C (scrotal temperature), whereas it was decrease at 37°C (body temperature). However, this was reversible when the cells were transferred from the elevated temperature to 34°C. It was also found that oxygen consumption and CO2 production were increased at 34°C by 2, 4-dinitrophenol (DNP), but decreased by oligomycin. On the other hand, oligomycin and DNP had no effect on oxygen consumption and 14 CO2 formation at the elevated temperature. These findings provide evidence that lactate utilization by spermatids is coupled with oxidative phosphorylation at scrotal temperature, but becomes uncoupled at elevated temperature, although more lactate is consumed.