Aberrant fetal growth and development after in vitro culture of sheep zygotes.

The effects of in vitro culture systems for sheep zygotes on subsequent fetal growth and development to day 61 and day 125 of gestation were studied. Zygotes recovered from superovulated Scottish Blackface ewes approximately 36 h after intrauterine insemination using semen from a single Suffolk sire were cultured for 5 days in (a) a granulosa cell co-culture system (co-culture); (b) synthetic oviductal fluid medium without serum (SOF-); and (c) synthetic oviductal fluid medium supplemented with human serum (SOF+). Control embryos were recovered from superovulated donor ewes at day 6 after oestrus. Embryos were transferred at day 6 to synchronous Scottish Blackface recipient ewes. In total, 146 gravid uteri were recovered, comprising 97 at day 61 (20 co-culture, 27 SOF-, 25 SOF+ and 25 control) and 49 at day 125 (13 co-culture, 8 SOF-, 6 SOF+ and 22 control) of gestation. Fetuses derived from co-cultured embryos were 14% heavier (P < 0.01) by day 61 of gestation than those derived from control embryos. Growth coefficients derived from the linear allometric equation logey = logea + b logex (where y = organ mass; x = fetal mass) were significantly greater (P < 0.05) for liver, heart, kidneys and plantaris muscle in fetuses derived from co-cultured embryos, and for liver in fetuses derived from SOF+ embryos than those for control fetuses. Fetuses derived from co-cultured embryos were 34% heavier (P < 0.001) and fetuses derived from SOF+ embryos were 18% heavier (P < 0.01) by day 125 of gestation than those derived from control embryos. Growth coefficients for liver and heart for fetuses derived from co-culture and SOF+ embryos were also significantly greater (P < 0.05) at this stage of gestation than those for control group fetuses. In contrast, allometric coefficients for these organs in fetuses derived from embryos cultured in SOF without serum supplementation were not different from those for controls. Excessive volumes of amniotic fluid (polyhydramnios) were observed in 23% of conceptuses derived from co-cultured embryos. In vitro embryo culture can significantly influence fetal growth and this study provides quantitative evidence of major shifts in the patterns of organ and tissue development.

Temporary exposure of ovine embryos to an advanced uterine environment does not affect fetal weight but alters fetal muscle development.

Embryo transfer techniques may result in fetuses that are heavier at birth and that have been described as highly muscled. The aim of this study was to investigate myogenesis in lambs derived from embryo transfer. Embryos were transferred at Day 3 (estrus = Day 0) to a 3 days-advanced uterine environment, maintained there for 3 days, recovered, and then returned to a synchronous (Day 6) uterus; these fetuses comprised the asynchronous group. Control animals were created by synchronous embryo recovery and single transfer at Day 3. Asynchronous transfer did not affect fetal weight or curved crown-rump length between 46 and 135 days of gestation. No differences were detected between groups at Days 110-135 with respect to muscle mass or protein, RNA, and DNA content. However, total muscle fiber number was significantly increased in plantaris muscles from the asynchronous groups at Day 110 and Day 125, suggestive of prolonged hyperplasia. In addition, the levels of Myf 5 protein and the secondary-to-primary fiber ratio were altered in plantaris muscle from the asynchronous group. The growth data are in contrast to previously reported findings. The results show that fetal myogenesis can be altered by very early events in embryogenesis and suggest that any inferences made solely on the basis of fetal or muscle weight may be fallacious.

Short-term culture of ovine embryos modifies fetal myogenesis.

Certain reproductive techniques culture embryos in vitro; however, little is known about the impact of culture on fetal growth. Coculture of day 1 ovine zygotes on a bovine granulosa cell layer to blastocysts followed by transfer to synchronous recipients increased fetal weight by 11 and 40% at days 61 and 125, respectively, compared with the transfer of in vivo-produced blastocysts. Plantaris muscle weights were increased by 40% in cultured fetuses at day 125. Examination of myogenesis in plantaris muscle showed that primary fiber number was unchanged at day 61 by culture but that primary fiber area was increased significantly by 15 and 25% at days 61 and 125, respectively; secondary fiber area was increased by 40% at day 125 by culture, and the ratio of secondary to primary fiber numbers was 18-20% greater in the cultured groups compared with the controls at days 61 and 125. The results show that coculture of preimplantation embryos may alter myogenic programming. These changes may contribute to the abnormally large muscles observed in oversize fetuses.

Fetal growth and development following temporary exposure of day 3 ovine embryos to an advanced uterine environment.

The effect of exposing Day 3 ovine embryos to an advanced uterine environment for a period of 3 days on subsequent fetal growth and development between Day 35 and Day 135 of gestation was studied. Day 3 embryos were recovered from superovulated donor ewes and transferred to synchronous final or asynchronous temporary recipients for 3 days. Embryos were recovered from these temporary recipients and transferred to Day 6 final recipients. Gravid uteri were recovered, weighed and dissected on Days 35, 45, 60, 90, 110, 125 and 135 of gestation. Fetal weight and length data were analysed by fitting non-linear Gompertz models of the form log(e) y = a - be(-ct), where y is fetal size and t is time from conception. Various terms including treatment, gestational age, embryo stage at transfer and fetal sex were fitted to this model. Fetal development was assessed by relating organ weight to fetal bodyweight using the linear allometric equation log(e) y = log(e) a + b log(e) x, where y is organ weight and x is fetal weight. Temporary exposure of Day 3 embryos to an advanced uterine environment did not increase the rate of embryo development and had no effect on fetal growth and development between Days 35 and 135 of gestation in this study. A single Gompertz model (log(e) y = 10.134 - 17.047e(-0.1733t)) explained 99.8% of the variation in fetal weight. Of terms fitted to this model only gestational age and fetal sex influenced fetal weight, with male fetuses being 5% heavier (P<0.05) than female fetuses. Fetal development was also unaffected by experimental treatment in this study. Allometric coefficients established for various fetal components agreed well with those from previously published studies.

Effects of diabetes on reactivity of sciatic vasa nervorum in rats.

The aim was to investigate the effects of 2 months of streptozotocin-induced diabetes mellitus in rats on the responses of sciatic vasa nervorum to vasoactive drugs. Changes in perineural blood flow were monitored by laser-Doppler flowmetry during drug superfusion in vivo. Laser-Doppler flux was reduced by 53.3% after 2 months of diabetes. A 38-fold increase in norepinephrine sensitivity was found in diabetic compared to nondiabetic rats. Co-superfusion of norepinephrine and a high dose (100 microM) of the nitric oxide synthase inhibitor, NG-nitro-L-arginine, resulted in 116-fold and 3.6-fold increases in norepinephrine sensitivity in nondiabetic and diabetic rats, respectively, such that dose-response curves for changes in vascular conductance were superimposed. This suggests that the increased norepinephrine sensitivity in diabetes was caused by defective endothelial nitric oxide production or action. After norepinephrine preconstriction, acetylcholine caused dose-dependent increases in vascular conductance, sensitivity being 8.1-fold greater in nondiabetic than diabetic rats. In contrast, endothelium-independent responses to the nitrovasodilator, glyceryl trinitrate, were relatively unaffected by diabetes. Thus, diabetes causes a deficit in nitric oxide mediated endothelium-dependent relaxation of vasa nervorum, resulting in increased vasoconstrictor sensitivity which is likely to impair perfusion and contribute to the pathogenesis of neuropathy.

Nerve function and regeneration in diabetic rats: effects of ZD-7155, an AT1 receptor antagonist.

Effects of the angiotensin II AT1 receptor antagonist ZD-7155 on nerve function, blood flow, capillarization, oxygenation, and regenerative capacity after injury were studied in streptozocin-diabetic rats. Deficits in saphenous sensory and sciatic motor conduction velocity measured after 1 or 2 mo of diabetes in anesthetized rats were prevented and corrected by ZD-7155. Sciatic resistance to hypoxic conduction failure, which was increased by 71% by 2 mo of diabetes, was attenuated by 39% with ZD-7155. Endoneurial capillary density, which was unaffected by diabetes, was increased by 34% with 2 mo of ZD-7155 treatment. Sciatic nutritive endoneurial blood flow, which was reduced by 45% by 2 mo of diabetes, remained in the nondiabetic range with ZD-7155. Mean endoneurial O2 tension was reduced 38% by diabetes, which was attenuated by ZD-7155. Punctate freeze damage of sciatic nerve caused complete fiber degeneration. Fourteen days postlesion, there was a 26% deficit in myelinated fiber regeneration distance after 2 mo of diabetes, which was prevented by ZD-7155 treatment from diabetes induction. Thus alterations in the renin-angiotensin system contribute to the neurovascular etiology of nerve dysfunction in experimental diabetes.

Aldose reductase inhibition, nerve perfusion, oxygenation and function in streptozotocin-diabetic rats: dose-response considerations and independence from a myo-inositol mechanism.

We examined the effects of aldose reductase inhibition on nerve biochemistry and function, blood flow and endoneurial oxygenation in experimental diabetes mellitus. After 1 month untreated diabetes in rats, treatment with the novel sulphonylnitromethane aldose reductase inhibitor, ZENECA ZD5522, prevented a progressive increase in sciatic nerve resistance to hypoxic conduction failure (p < 0.05). Motor conduction velocity deficits after 4 months untreated diabetes were rapidly returned to normal within 12 days (p < 0.0001) by ZD5522 treatment. Following 2-months untreated diabetes, examination of 1 month ZD5522 treatment dose-response relationships for correction of nerve sorbitol and fructose accumulations and reduction in myo-inositol concentration, sciatic motor and saphenous sensory conduction velocity and sciatic blood flow by laser-Doppler flowmetry revealed poor agreement between nerve function and biochemical indices. In addition, polyol accumulation differed between sciatic and saphenous nerves, the latter showing ten-fold lower sorbitol concentrations. Laser-Doppler blood flow was 60% decreased by untreated diabetes (p < 0.001) and there was a strong correlation between ZD5522-mediated increases in blood flow and conduction velocity (p < 0.0001). Measurement of nutritive endoneurial blood flow by microelectrode polarography and hydrogen clearance showed 44% and 45% deficits for 1 and 2 months untreated diabetes (p < 0.001) that were prevented by ponalrestat and ZD5522 treatments, respectively. In contrast, 2 months myo-inositol treatment from diabetes induction did not prevent reduction in blood flow or sciatic motor conduction velocity. A 37% reduction in endoneurial oxygen tension after 2 months diabetes (p < 0.001) was completely prevented by ZD5522 treatment (p < 0.001). The data show that a very high degree of polyol pathway blockade is necessary to correct nerve functional deficits and that aldose reductase inhibitors have a neurovascular action that does not depend on restoration of nerve myo-inositol.

Anti-oxidant and pro-oxidant effects on nerve conduction velocity, endoneurial blood flow and oxygen tension in non-diabetic and streptozotocin-diabetic rats.

Increased oxygen free radical activity, coupled with reduced protection against oxidative stress, could play a role in the aetiology of neurovascular abnormalities in experimental diabetes mellitus. To test this hypothesis, non-diabetic and streptozotocin-diabetic rats were treated with the anti-oxidant probucol or the pro-oxidant primaquine. One-month diabetes caused 21.4% and 13.6% reduction in sciatic motor and saphenous sensory conduction velocity (p < 0.001). These deficits were prevented by probucol treatment (p < 0.001). After 1-month untreated diabetes, conduction velocity deficits were reversed by a further month of probucol treatment (p < 0.001). For non-diabetic rats, primaquine treatment caused a 12.9% reduction in motor conduction velocity (p < 0.001), which was prevented by probucol treatment (p < 0.001). Primaquine treatment did not affect diabetic rats. Sciatic nerve nutritive endoneurial blood flow, measured using microelectrode polarography and hydrogen clearance, was 48.0% reduced by 2-month diabetes (p < 0.001). This was completely prevented by probucol treatment (p < 0.001). Primaquine treatment did not affect blood flow in diabetic rats. However, in non-diabetic rats it caused a 30.0% reduction (p < 0.01) which was prevented by probucol treatment (p < 0.05). Sciatic endoneurial oxygen tensions were also measured by microelectrode polarography. Mean tension was 38.8% reduced by diabetes (p < 0.001). This was prevented by probucol treatment. Non-diabetic rats given primaquine treatment showed a 21.7% reduction in endoneurial oxygen tension (p < 0.01). The data suggest that vascular-mediated nerve dysfunction in diabetes depends on oxidative stress, and that similar effects in non-diabetic rats may be produced by pro-oxidant treatment. This provides evidence for the potentially important role of oxygen free radical activity in diabetic neuropathy.

Angiotensin II receptor blockade improves nerve function, modulates nerve blood flow and stimulates endoneurial angiogenesis in streptozotocin-diabetic rats and nerve function.

We examined the effect of the angiotensin II receptor blocker, ZD 8731, on nerve function, capillary density, and blood flow in streptozotocin-diabetic rats. Deficits in sciatic motor and saphenous sensory nerve conduction velocity of 21% and 15%, respectively, were observed after 1 month of diabetes mellitus (p < 0.001). These were completely ameliorated by a further month of ZD 8731 treatment (p < 0.001). Treatment of non-diabetic rats for 1 month with ZD 8731 had no effect on motor or sensory conduction velocity. Sciatic nerve capillary density was not significantly affected by 1- or 2-month untreated diabetes, however, there was a 15% increase in density with ZD 8731 treatment (p < 0.001). Treatment of non-diabetic rats for 1 month had no effect on capillary density. Diabetes prolonged the time taken for 80% conduction failure by 19% (p < 0.05) and 49% (p < 0.001) for 1 and 2 months of diabetes, respectively, when sciatic nerve was exposed to hypoxia in vitro. ZD 8731 treatment during the second month of diabetes limited the prolongation to 22%, not significantly different from 1 month of untreated diabetes but less than for the 2-month diabetic group (p < 0.001). Concentrations of sciatic nerve polyol pathway metabolites were elevated six-fold and myo-inositol was reduced 40% by diabetes; ZD 8731 treatment was without effect. Acute experiments examined the effect of ZD 8731 on sciatic nerve blood flow using laser-Doppler flowmetry. In non-diabetic rats, blood flow changes followed the dose-dependent reductions in systemic arterial pressure and there were no significant variations in sciatic vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological manipulation of vascular endothelium function in non-diabetic and streptozotocin-diabetic rats: effects on nerve conduction, hypoxic resistance and endoneurial capillarization.

We examined the potential for some of the abnormalities of vascular endothelium found in diabetes mellitus to cause neuropathic changes. Non-diabetic rats were treated for 2 months with the cyclo-oxygenase inhibitor flurbiprofen (5 mg.kg-1.day-1) to reduce prostacyclin production, the nitric oxide synthase inhibitor NG-nitro-L-arginine (5 or 25 mg.kg-1.day-1), or combined treatment. There were dose-dependent reductions in sciatic motor and saphenous sensory conduction velocity. The two inhibitors acted synergistically, thus, the 5-6% motor conduction deficits (p < 0.01) produced by either flurbiprofen or NG-nitro-L-arginine (5 mg.kg-1.day-1) increased to 17% (p < 0.001) for combined treatment. With NG-nitro-L-arginine (25 mg.kg-1.day-1) and flurbiprofen, motor and sensory conduction velocity were reduced by 23% (p < 0.001) and 12% (p < 0.001), respectively, matching the deficits following 2-month streptozotocin diabetes. NG-nitro-L-arginine (25 mg.kg-1.day-1) and flurbiprofen together produced a 13% prolongation of the time taken for 80% hypoxic conduction failure in vitro (p < 0.05) and a 10% reduction in sciatic capillary density. A second investigation tested an alternative hypothesis that overproduction of nitric oxide was responsible for vascular-related complications in diabetes, the prediction being that NG-nitro-L-arginine (5 mg.kg-1.day-1) would prevent nerve dysfunction. However, rather than prophylaxis during 2-month streptozotocin diabetes, treatment exacerbated nerve abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-oxidant treatment prevents the development of peripheral nerve dysfunction in streptozotocin-diabetic rats.

We tested the notion that oxidative stress makes an important contribution to the aetiology of diabetic neuropathy. The effect of treatment with a 1% dietary supplement of the anti-oxidant butylated hydroxytoluene was studied during 2 months of streptozotocin-induced diabetes mellitus. In final experiments, sciatic motor and saphenous sensory conduction velocities were measured in vivo, and resistance to hypoxic conduction failure for sciatic trunk was examined in vitro. There were 20% and 12% decreases in motor and sensory conduction velocity, respectively after 2 months of diabetes (p < 0.001). There were completely prevented by butylated hydroxytoluene treatment (p < 0.001). Resistance to hypoxic conduction failure, shown by the time taken for sciatic compound action potential amplitude to decline by 80%, was 55% increased by diabetes, and this was limited to 31% (p < 0.01) by treatment. There were no significant effects of treatment on the 9-10 fold elevation of sciatic nerve sorbitol and fructose levels with diabetes, or on the non-significant 22% reduction in myoinositol content. Butylated hydroxytoluene treatment also did not affect sciatic nerve capillary density. We conclude that oxidative stress makes an important contribution to the aetiology of early experimental diabetic neuropathy. Amelioration of oxidative stress could potentially be a final common mechanism whereby a number of diverse treatments exert a beneficial effect on diabetic nerve function.

Nerve function in experimental diabetes in rats: effects of electrical stimulation.

The effects of unilateral electrical stimulation of the peroneal sciatic nerve branch were studied in streptozocin-diabetic rats of 12-wk duration. Stimulation was carried out over 7 days (10 Hz, 8 h/day) with chronically implanted electrodes. Compared with controls, there was a 25% conduction velocity (CV) deficit for the peroneal nerve supplying tibialis anterior muscle in the unstimulated leg, which was corrected by stimulation. For tibial fibers supplying soleus muscle, a similar diabetic CV deficit (20%) was normalized by stimulation, although soleus axons were not directly activated. In saphenous nerve, which has a functionally separate vascular supply, peroneal stimulation was ineffective. In anesthetized diabetic rats, stimulation caused an 18% reduction in tibialis anterior CV after 4 h. However, serial measurements showed progressive normalization of CV over 4 days of stimulation. On termination, CV returned to diabetic levels over 36-60 h. Sciatic nerve showed a 70% increase in resistance to hypoxic conduction failure with diabetes, which was halved by chronic stimulation. Acute experiments demonstrated that peroneal stimulation increased sciatic vascular conductance by 60%. We conclude that stimulation causes activity-related improvements in diabetic nerve blood flow and metabolism.