Metabolism of Round Spermatids: Kinetic Properties of Pyruvate Kinase

Round spermatids (steps 1-8) were isolated from rat testes and kinetic properties of pyruvate (PK) in their extract were examined. A plot of PK activity against phosphoenolpyruvate (PEP) or ADP concentration appeared sigmoidal. Km values for PEP and ADP were 0.12 and 0.29 mM, respectively. However, fructose 1,6-bisphosphate (FBP) stimulated the enzyme markedly by increasing its affinity for PEP. FBP (0.35 microM) was required for 50% activation of PK, when the PK activity was measured at 25 microM PEP and 0.2 mM ADP. In contrast, ATP (Ki = 6.5 mM) inhibited the PK activity. On the other hand, in the presence of 5 mM glucose, the level of FBP in spermatids increased markedly, while that of ATP declined rapidly. The level of ADP remained constant. When the activity of PK in spermatid extract was measured at intracellular levels of FBP, ADP and ATP, it was maximum. The results suggest that PK becomes probably fully activated when glucose is metabolized in the glycolytic pathway of spermatids. It seems unlikely that PK is the rate-limiting step in glycolysis of spermatids.     

Studies of Metabolism of Round Spermatids: Cytochalasin B Binding to Cell Membrane in Relation to Glucose Transport

Cytochalasin B inhibited uptake of 2-deoxy-D-glucose in rat spermatids (Ki = 6.3 X 10(-7) M). This inhibition was reversible. The [3H]cytochalasin B was found to bind to spermatid plasma membranes at low drug concentrations. Lineweaver-Burk plots of binding data indicated that there was a class of high affinity sites (Kd = 2.9 X 10(-7) M). The results suggest that the high affinity cytochalasin B binding sites of spermatids are membrane-associated substances and are intimately related to the glucose transport system.     

Studies on the Male Antifertility Agent Gossypol Acetic Acid. VI. Effect of Gossypol Acetic Acid on the Fertility of Bonnet Monkey, Macaca radiata

Gossypol acetic acid (4 mg/day/5 days a week) suspended in Tonoferon tonic was given to male bonnet monkeys, Macaca radiata, by oral route for 3 months. Marked reduction in the sperm count/ejaculate and sperm motility were observed after gossypol treatment; both motility and sperm count/ejaculate returned to the normal level 8-10 weeks after termination of gossypol treatment. The citric acid and fructose levels in the semen of gossypol treated monkeys were not different from those of the controls.     

Gossypol inhibits proliferation of endometrioma cells in culture

Aim： To evaluate the anti-proliferative activity and mitochondrial toxicity of gossypol in endometrioma cells maintained in short-term cultures. Methods： （A） Three endometrioma cell lines from patients were treated with 25 or 50 nmol/L gossypol for up to 12 days. The effect of gossypol on the cell growth was recorded. （B） A phosphorescence oxygen analyzer was used to determine the effects of gossypol on mitochondrial oxygen consumption of six endometrioma cell lines from patients. （C） Cellular gossypol accumulations in three endometrioma cell lines from patients were measured by high-pressure liquid chromatography. Results： Proliferation of the endometrioma cells was inhibited by 25 and 50 nmol/L gossypol. Respiration of the endometrioma cells was inhibited by 10 μmol/L gossypol. Cellular gossypol was detected in the endometrioma cell lines that were treated for 24 h with l0 and 0.3 μmol/L gossypol. Conclusion： Gossypol invokes a potent toxicity on cultured endometrioma cells.     

Studies on the Male Antifertility Agent Gossypol Acetic Acid VII. Effect of Motility Stimulated Factors on the Revival of Human Spermatozoal Motility after Gossypol Treatment in vitro

Human spermatozoa were incubated with gossypol acetic acid (100 micrograms/1 X 10(6) spermatozoa/ml) at 37 degrees C for 30 min. The drug treatment inhibited the spermatozoal motility significantly. Washing of the spermatozoa, after gossypol treatment, did not effect their motility. A partial revival in the motility of the spermatozoa was observed when gossypol treated spermatozoa were incubated, after washing, with motility stimulating factors, e.g. theophylline, dibutyryl-cAMP and Kallikrein.     

Effects of neuroprotective cocktails on hippocampal neuron death in an in vitro model of cerebral ischemia

Cocktails of neuroprotectants acting at different parts of the ischemic injury cascade may have advantages over single agents. This study investigated, singly and in combination, the neuroprotective efficacy of an energy substrate (3.5 mM fructose 1,6-bisphosphate, FBP), an antagonist of NMDA receptors (1 and 10 microM MK-801), a free-radical scavenger (100 microM ascorbate), an adenosine A1 receptor agonist (10 microM 2-chloroadenosine), and an inhibitor of neurotransmission (2% isoflurane). These agents were evaluated for their ability to prevent loss and morphologic damage of CA1 neurons in rat hippocampal slices when these agents were administered during 30 minutes in vitro ischemia (combined oxygen/glucose deprivation at 37 degrees C) followed by 5 hours of recovery. Ten microM MK-801, alone or in combination with the other compounds, prevented loss of CA1 neurons and preserved their histologic appearance. Isoflurane, which prevents glutamate receptor-dependent cell death in this model, was also protective. Protection against neuron loss was also found when a subtherapeutic concentration of MK-801 (1 microM) was combined with 2-chloroadenosine (which indirectly causes NMDA receptor suppression), but not FBP or ascorbate. The authors conclude that in this model, the strategy of antagonizing NMDA receptors appears more protective than fructose-1,6-bisphosphate, 2-chloroadenosine or ascorbate.     

Protective effect of fructose-1,6-bisphosphate in the cold storage solution for liver preservation in rat hepatic transplantation

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods. FBP maintains ATP levels and thereby cellular energy metabolism, which is important to the liver during cold preservation. In the present study, we evaluated the effects of FBP on the composition of storage solutions for cold liver preservation. Adult male Wistar rats were randomly divided into three experimental groups. Hepatic perfusion and preservation were performed with UW, UW plus 10 mmol/L FBP (UWM), and FBP 10 mmol/L (FBPS) alone solutions. Biochemical measurements of AST, ALT, and TBARS were performed on samples of the cold storage solution at 0, 12, 18, and 24 hours preservation. FBPS and UW solutions showed similar preservation grades during 18 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade. FBP appears to protect the liver from injury caused by free radicals when the preservation time is less than 18 hours. Therefore, FBP may exert a protective effect for the preservation of livers during cold storage, and could represent an important component of new cold storage solutions.     

Transport of transferrin-bound iron into rat Sertoli cells and spermatids

Transferrin (Tf), a major secretory protein of Sertoli cells, may transport iron to spermatogenic cells. This was assessed by measuring the uptake of Fe from 59Fe-125I-labelled rat Tf by Sertoli cells and round spermatids in vitro. Uptake of Fe from labelled Tf by Sertoli cells after a 72-h pre-incubation period was linear for 20 h (approximately 18 pmol/10(6) cells/20 h), whereas the uptake of Fe from labelled Tf by round spermatids after a 16-h pre-incubation period reached a plateau by 2 h (approximately 5 pmol/10(6) cells/2 h). The corresponding net uptake of Tf by both cell types was less than 0.1 pmol. High speed supernatants prepared from Sertoli cells and spermatids labelled with 59Fe-125I-Tf were fractionated by gel permeation chromatography. Separate peaks of protein-bound 59Fe and 125I-Tf were observed. Protein bound 59Fe could be precipitated with an antiserum to rat ferritin. It is concluded that iron from exogenous Tf is transported into Sertoli cells and round spermatids in vitro, and is complexed to intracellular ferritin. However, the present results do not exclude the possibility that Sertoli cell Tf may serve purposes other than iron transport.     

Short Term Metabolic Effects of the Anti-Fertility Agent, Gossypol, on Various Reproductive Organs of Male Mice

In order to evaluate the short term metabolic effects of gossypol on the testes as well as any possible effects on the secondary sex organs, Balb C mice were injected subcutaneously with various doses of gossypol (0.25-25.0 mg/kg body weight) in corn oil for 10 days. Wet weights of several different secondary sex reproductive organs decreased during gossypol treatment. However, wet weights of the testes during treatment remained equal to or greater than control values. Following 10 days of gossypol treatment, incorporation of [³H]thymidine or [³H]amino acids into trichloroacetic acid precipitable macromolecules was inhibited in the seminal vesicles and ventral prostates normalized to either DNA or wet weight. Treatment with gossypol also had an inhibitory effect on epididymal sperm count at the two highest doses.
These results demonstrate that gossypol will decrease sperm count at high dose levels after treatment of male mice for as short as 10 days. However, its overall effects are not limited to the testes and spermatogenesis but, in addition, it has dramatic inhibitory effects on protein and nuclei acid metabolism in the secondary sex organs.     

The luminal P2Y receptor in the isolated perfused mouse cortical collecting duct

Extracellular nucleotides regulate renal ion transport. With the use of in vitro perfusion and [Ca(2+)](i) imaging, this study investigated whether mouse and rabbit cortical collecting ducts (CCD) respond to luminal nucleotides. In mouse CCD, luminal ATP (EC(50): 10 microM) and UTP (EC(50): 9.7 microM) increased [Ca(2+)](i) with an initial peak and a plateau. To make certain that basolateral P2 receptors were not activated by luminal nucleotides via leak diffusion, luminal trypsin (1 microM), a known agonist for basolateral proteinase-activated receptors, was perfused. Mouse CCD that were responsive to luminal ATP were nonresponsive to luminal trypsin but always showed [Ca(2+)](i) elevations by basolateral trypsin (10 or 100 nM). Luminal alpha,beta- and beta,gamma-methylene ATP, 2-methyl-S-ATP, ADP, UDP, and 2',3'-O-4-benzoylbenzoyl ATP had no effect (100 microM, n = 9). Without external Ca(2+), luminal ATP still stimulated a [Ca(2+)](i) increase. Mouse CCD also responded to basolateral ATP (EC(50): 23 microM) and UTP (EC(50): 23 microM) with smaller [Ca(2+)](i) elevations. Confocal microscopy of perfused CCD showed that luminal ATP (100 microM) rapidly increased [Ca(2+)](i) in nearly all cells (n = 6) and the same cells that responded to luminal ATP responded to basolateral ATP (100 microM). In contrast, rabbit CCD did not respond to luminal ATP/UTP (n = 8) despite ATP's known effect from the basolateral side (EC(50): 34 microM). These data indicate the expression of luminal P2Y receptors (probably P2Y(2)) in principal cells of mouse CCD but not in rabbit CCD.     

Studies on the Male Antifertility Agent - Gossypol Acetic Acid. III. Effect of Gossypol Acetic Acid on Rat Testis

Gossypol acetic acid at the dose of 5 mg/rat/day for 2 and 4 weeks did not cause any significant effect on the body weight, testis, epididymis, seminal vesicle and prostate weight, nor gossypol treatment had any significant effect on the activities of acid phosphatase and succinic dehydrogenase in the testis. Changes in the testis ATPase activity were, however, significant after gossypol treatment. During the course of present investigations no effect of gossypol treatment on 3H thymidine incorporation into DNA of testicular cells was observed, nor there were any changes in the DNA and total protein content of the testis after gossypol treatment. Gossypol treatment did not cause any effect on the plasma Na+ level. However, transient decrease in the plasma K+ level was observed; decrease in K+ level two weeks after gossypol treatment was restored to normal after 4 weeks of gossypol treatment. No changes in the histology of the testis were observed 2 weeks after gossypol treatment but marked inhibition of spermatogenesis was observed 4 weeks after gossypol treatment. Motility of vas deferens spermatozoa was also markedly inhibited 4 weeks after gossypol treatment. In the light of the present observations and those of others, there is a clear demonstration that gossypol acts directly on the spermatozoa and on the testis; at both the sites the drug interferes in the ATPase activity.     

Transperitoneal transport of glucose in vitro

The effect of fluid mixing intensification, damage of mesothelial cells, gentamicin, and icodextrin on the diffusive glucose transport across the peritoneal membrane were evaluated in in vitro studies. A mathematical model of mass transport was used to calculate the diffusive permeability, expressed as a diffusive permeability coefficient (P). In the control conditions, the rate of glucose transfer from the interstitial to the mesothelial side of membrane (I-->M) and in the opposite direction (M-->I) remained constant, and the P value at mean was 2,731 +/- 1,493 x 10-4 (cm x s-1). The change of the stirring rate from 5.5 to 11 ml/min increased P values by about 74% for transport direction I-->M and 58% for M-->I, and the change from 11 to 22 ml/min enhanced P at mean by about 42% for both directions. The damage of the mesothelial layer, using sodium deoxycholate (2.5 mmol/L; 103.6 mg%), increased the glucose transfer from the interstitial to the mesothelial side of the peritoneum by 41% and to the opposite direction by 70%. Addition of icodextrin to the glucose solution increased glucose bidirectional transport at mean by about 14% for I-->M and 24% for M-->I. Furthermore, gentamicin did not change the I-->M transfer, but diminished M-->I transport by about 12%. In conclusion, the reduction of unstirred fluid layers at the mesothelium and the interstitium-fluid interfaces, removal of mesothelium, and addition of icodextrin increased the diffusive glucose transport in vitro; unstirred fluid layers restricted glucose transfer (I-->M) more than the mesothelium; and peritoneal glucose transport, directed from the mesothelial to the interstitial side of the peritoneum, decreased slightly after the addition of gentamicin.     

Dynamics of renal glucose reabsorption in rat

Summary: Glucose filtered load (FL), glomerular filtration rate (GFR) and tubular glucose reabsorption (TG) were simultaneously measured in the right, control kidney and in the left kidney with cortical necrosis (CN kidney) in rats with increasing plasma glucose concentrations (PG). Urinary glucose excretion starts when plasma concentrations (threshold value) averages 2.69 ± 0.20 mg/mL in the control kidney and 2.78 ± 0.16 mg/mL in the CN kidney (non significant; NS). For PG above the threshold value, increases in glucose FL were associated with increases in TG reabsorption. Maximal glucose reabsorption was not reached even when PG levels were 10 mg/mL. Moreover, there is a linear correlationship between TG reabsorbed and glucose FL in this range of PG. Among the two factors determining FL, our results support a linear correlationship between glucose reabsorption and GFR. Finally, no significant differences in tubular glucose reabsorption dynamics appears between control and CN kidney suggesting similar dynamics for glucose reabsorption in cortical and juxtamedullary nephrons.     

5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR), a compound that activates 5'-AMP-activated protein kinase (AMPK), has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study, we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes, in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently, closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation, assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle, in which AMPK stimulation promotes glucose transport to provide ATP as a fuel, AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis, to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may, therefore, give insight into the mechanism of insulin action.     

Inhibition kinetics of NAD-linked enzymes by gossypol acetic acid

Effects of gossypol on human spermatozoal enzymes were investigated. This compound was found to be a potent inhibitor of the NAD-linked enzymes, glucelaldehyde-3-phosphate dehydrogenase (GA3PDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). MDH was inhibited by gossypol when the reaction was carried out in malate-oxalacetate (direct) or oxalacetate-malate (reverse) directions. The I50 of gossypol for the direct reaction was 2.9 microM, whereas that for the reverse reaction was 1.2 microM. Reciprocal plots due to Lineweaver-Burk showed that MDH is inhibited in a noncompetitive manner with respect to both reactions. LDH was also inhibited by this compound when pyruvate or alpha-ketobutyrate was used as a substrate. Gossypol was a noncompetitive inhibitor for LDH. The I50 of gossypol for LDH were 9.8 microM and 11.3 microM, when using pyruvate and alpha-ketobutyrate, respectively as substrates. The I50 of gossypol for GA3PDH and ICDH were 110 microM and 2.7 microM, respectively.     

棉酚对睾丸支持细胞间隙连接蛋白表达的影响

目的:探讨药物棉酚对睾丸Sertoli细胞间隙连接蛋白Cx43表达的影响。方法:培养TM4睾丸Sertoli细胞,用1.25、2.5、5、10μmol/L的棉酚分别染毒细胞6、12、24、48 h。CCk-8试剂盒检测细胞毒性,应用细胞免疫荧光化学、RT-PCR检测Cx43在正常TM4细胞和在不同浓度及不同染毒时间的TM4细胞中的表达情况。结果:半定量RT-PCR及免疫荧光结果显示,正常TM4细胞中有较多的Cx43表达;棉酚染毒24 h后,Cx43 mRNA水平开始随时间增加而逐渐下降(P<0.05),且随剂量增加Cx43蛋白表达强度逐渐减弱(P<0.05)。结论:棉酚可抑制TM4细胞表达Cx43,可能是其抗生育的机制之一。     

Gossypol with methyltestosterone and ethinylestradiol male does not affect rat spermatogonial stem cell differentiation

The purpose of this study was to investigate whether administration of the regimen of gossypol at 12 mg/kg/day combined with methyltestosterone at 20 mg/kg/day and ethinylestradiol at 100 microg/kg/day for a long term of twenty-four weeks could affect the existence and differentiation of rat spermatogonial stem cell. This was assessed by conducting TdT-mediated dUTP nick end-labeling detection, spermatogonial stem cell transplantation and fertility recovery evaluation. Our results showed that spontaneous apoptosis was observed in normal rats' testes from the control group with an apoptotic index (AI) average of 10.24+/-1.52. In the regimen-treated group, the predominant apoptotic cells were spermatocytes and spermatids in the seminiferous tubules. Spermatogonia were not apoptotic (AI averaged 113.42+/-13.24). Two to three months after transplantation of spermatogonial stem cells isolated from regimen-treated rats into recipient nude mice, elongated rat spermatids were identified in the seminiferous tubules of recipient nude mice. Six weeks after withdrawal of the administration, fertility of the regimen-treated rats was recovered compared with that of the control group. The number of litters produced by females mated with regimen-treated males averaged 9.88+/-0.166 matched 10.30+/-0.171 of control group and the litters of the first generation appeared to be normal. These results indicated that the administration of this regimen did not affect the existence and differentiation potential of spermatogonial stem cells of the regimen-treated rats.     

Effect of dynorphin on insulin and somatostatin secretion, calcium uptake, and c-AMP levels in isolated rat islets of Langerhans

Dynorphin-[1-13], at concentrations of 5.8 X 10(-12) to 5.8 X 10(-9) M, stimulated insulin secretion from isolated islets of Langerhans of the rat, in medium containing 6 mM glucose. Higher concentrations of dynorphin had no significant effect on secretion. Dynorphin (5.8 X 10(-9) M) was unable to initiate insulin release from islets in the presence of 2 mM glucose, or to increase insulin secretion further in the presence of 20 mM glucose or 6 and 12 mM glyceraldehyde. Dynorphin-induced insulin secretion from islets was blocked by verapamil (5 microM) or by chlorpropamide (72 microM), but not by a mu opiate receptor antagonist, naloxone (0.11 microM), or by ICI 154129, a specific antagonist for the delta receptor (0.25 microM). Dynorphin had no effect on islet somatostatin secretion, under conditions in which insulin secretion was greatly stimulated. Glucose (20 mM) and glyceraldehyde (6 and 12 mM) significantly increased both insulin and somatostatin secretion. Dynorphin (5.8 X 10(-9) M) increased 45Ca2+ uptake into islets, and also increased intracellular islet c-AMP levels. These changes persisted when higher concentrations of dynorphin were used. These results suggest that (1) dynorphin is a very potent stimulus for insulin secretion; (2) dynorphin does not affect somatostatin secretion in static incubations of islets, in the same way as does glucose and glyceraldehyde; (3) dynorphin's effects may involve increased calcium ion movement and can be blocked by verapamil; (4) dynorphin can also increase islet c-AMP, and could thereby modulate the responsiveness of other secretagogues; (5) the actions of dynorphin on insulin secretion are not mediated by delta or mu opiate receptors in islets.     

Effect of fructose-1,6-bisphosphate in the cold storage solution after 12 and 36 hours of rat liver preservation

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods because it maintains ATP levels during cold preservation. In the present study, we evaluated the effects of addition of FBP to storage solutions for cold liver preservation during 12 or 36 hours. Adult male Wistar rats were randomly divided into three experimental groups. The hepatic perfusion and preservation were performed with these solutions: UW; UW plus 10 mmol/L FBP; and FBP 10 mmol/L (FBPS) alone. The biochemical measurements of AST and ALT were performed on samples of the cold storage solution after 12- or 36-hour preservation. UW and FBPS solutions showed similar preservation grades at 12 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade during 12 or 36 hours. UW solution was better than FBPS after 36 hours preservation. UW solution continues to offer a superior performance for liver preservation during long times; however, FBPS may be an alternative for short cold preservation times.     

The effect of chloroquine on the binding, intracellular distribution, and action of insulin on isolated mouse pancreatic acini

The effects of chloroquine on the binding, intracellular distribution, and action of insulin were studied in isolated pancreatic acini prepared from diabetic mice. Chloroquine had three effects on these cells. First, chloroquine altered cellular morphology by inducing an increase in the number and size of autophagic vacuoles and vesicles in the Golgi-lysosomal region. Second, chloroquine, in a dose-dependent fashion, increased the amount of 125I-insulin associated with acini. A detectable effect of chloroquine was seen at 10 microM, and a maximal effect was seen at 30-100 microM where cell-associated insulin was more than doubled. Employing electron microscope autoradiographs, this accumulation of hormone was observed in the Golgi-lysosome area of the pancreatic acinar cell. Third, chloroquine had selective effects on the action of insulin. Preincubation with chloroquine had no effect on basal [3H]2-deoxy-D-glucose uptake, but in a dose-dependent fashion it decreased the stimulatory effect of insulin on this function; at 100 microM chloroquine, the effect of insulin was abolished. In contrast, chloroquine had negligible effects on the stimulation of 2-deoxy-D-glucose uptake into acini by cholecystokinin. Chloroquine in dose-dependent fashion partially inhibited basal [3H]leucine incorporation into acinar cell protein, but in contrast to its effects on 2-deoxy-D-glucose uptake, the drug had no effects on the stimulation of this function by insulin.+2