Time course of hypo-osmotic swellings of human spermatozoa: evidence of ordered transition between swelling subtypes

The hypo-osmotic swelling test (HOST or HOS test) usually takes into consideration the total HOS response value with no emphasis either on the value of the response subtypes or the response evaluation time. This study investigated the time course of HOS responses and analysed their physiological relevance. Raw semen spermatozoa and Percoll washed spermatozoa were used in the experiment. The morphological changes in the sperm tail were monitored by incubating the spermatozoa in the hypo- osmotic solution for 16 different time periods. The HOS reactive spermatozoa and the type of HOS reaction (swelling subtypes) of the samples subjected to different duration of treatment were identified under a phase contrast microscope. Also the fate of individual spermatozoa in a hypo-osmotic environment were monitored for 30 min. In spermatozoa exposed to a hypo-osmotic solution, the motility lasted usually less than 2 min and motility characteristics were uniquely different from that of the spermatozoa under iso-osmotic conditions. The HOS response development was permanent but the motility loss due to hypo-osmotic shock was reversible up to 1 min of incubation. There was an indication of ordered transition among the HOS swelling subtypes apparently initiating with subtype b destined to c, d, e, f and g. Further, the subtypes a and g showed gradual decrease and increase, respectively, while subtype b showed abrupt initial increase and then gradual decrease. Transition from b to g could be direct or via one or more than one subtypes. Ultrastructure based analysis indicated that HOS response subtypes are the apparent reflection of the differences in the cytoskeletal assembly of the sperm tail and thus may be identifying different physiological variants in the sperm population. These results indicate that shorter incubation is essential to document the kinetics of various HOS responses but the conventional HOS test misses these important HOS features because of lengthy incubation. Since the time course of ordered transition of HOS responses will vary more than the total HOS response in semen of different aetiologies, the importance of HOS response subtypes and response evaluation time should be taken into consideration when applying HOS test.      

Comparison of three methods to measure absolute cerebral hemoglobin concentration in neonates by near-infrared spectrophotometry

Three methods by which to determine absolute total cerebral hemoglobin concentration (tHb in micromol/L) by near-infrared spectrophotometry (NIRS) have evolved: (1) tHbo, requiring oxygenation changes and arterial oxygen saturation measurements as a reference using a relative NIRS algorithm, (2) tHbg, using a geometrical multidistance principle and (3) tHbgo, a combination of both. The aim of this study was to compare the three methods quantitatively. Sixteen clinically stable preterm infants with a mean gestational age of 29.6 (range of 25.1-36.4) weeks, birthweight of 1386 (680-2820) g and a postnatal age of 2.5 (0.5-6) days, who needed supplemental oxygen, were enrolled. The mean+/-standard deviation tHbg was 150.2+/-41.8 micromol/L (range of 61.6-228.9 micromol/L), the tHbo was 62.1+/-27.2 micromol/L (26.0-110.8 micromol/L) and the tHbgo was 89.3+/-45.6 micromol/L (26.5-195.9 micromol/L). The correlation coefficient among the three methods were tHbg and tHbgo r=0.736; tHbo and tHbgo r=0.938; tHbg and tHbo r=0.598. A multiple regression with variable selection by Mellow's C(p) showed, that tHbg was correlated to the birthweight, the postnatal age, the heart rate and the pCO2 (r(2)=0.588), tHbo and tHbgo were associated with the hemoglobin concentration in the blood, the mean arterial blood pressure and the pCO2 (r(2)=0.493 and 0.406, respectively). The three methods (tHbg, tHbo, and tHbgo) give systematically different tHb readings and large intersubject variability.     

Blockade der chemischen Thermogenese und Auslösung von Muskelzittern durch Adrenolytica und Ganglienblockade beim neugeborenen Meerschweinchen

Summary As has been previously shown young newborn guinea pigs do not shiver on cold exposure although oxygen consumption is considerably increased, i.e. chemical thermogenesis (non-shivering thermogenesis) is the prevailing mechanism of heat production. In the course of the first weeks of life non-shivering thermogenesis is gradually replaced by shivering thermogenesis. In the present study it has been shown that the cold-induced oxygen uptake can be reduced in guinea pigs, 0 to 2 days of age, from 69 to 38 ml/kg min by Alderlin, an adrenergic -receptor blocking agent. This effect was accompanied by an onset of shivering demonstrating that the shivering mechanism is already developed at the time of birth but is not set in function under normal conditions. Similar results have been obtained using Hexamethonium-Bromide and Dichloroisoproterenol. The elicitation of shivering following the administration of the drugs prevents the oxygen uptake from being reduced to the basal level (ca. 20 ml/kg min). When shivering was already present as in older animals (2 to 3 weeks of age) it remained uninfluenced by Alderlin (as veryfied by electromyography); the agent was thus considered suitable to block selectively chemical thermogenesis. On this basis, it was estimated from further data that the contribution of nonshivering thermogenesis to the total heat production is reduced from 91 to 26% within the first three weeks of life. Moreover, it could be shown that the shivering mechanism is less effective than chemical thermogenesis.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment

The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP- sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.      

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

Neuropathological Diagnostic Criteria for Creutzfeldt-Jakob Disease (CJD) and Other Human Spongiform Encephalopathies (Prion Diseases)

Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human transmissible spongiform encephalopathies (prion diseases) are proposed for the following disease entities: CJD - sporadic, iatrogenic (recognised risk) or familial (same disease in 1st degree relative): spongiform encephalopathy in cerebral and/or cerebellar cortex and/or subcortical grey matter; or encephalopathy with prion protein (PrP) immuno-reactivity (plaque and/or diffuse synaptic and/or patchy/perivacuolar types). Gerstmann-Sträussler-Scheinker disease (GSS) (in family with dominantly inherited progressive ataxia and/or dementia): encephalo(myelo)pathy with multicentric PrP plaques. Familial fatal insomnia (FFI) (in member of a family with PRNP178 mutation): thalamic degeneration, variable spongiform change in cerebrum. Kuru (in the Fore population). Without PrP data, the crucial feature is the spongiform change accompanied by neuronal loss and gliosis. This spongiform change is characterised by diffuse or focally clustered small round or oval vacuoles in the neuropil of the deep cortical layers, cerebellar cortex or subcortical grey matter, which might become confluent. Spongiform change should not be confused with non-specific spon-giosis. This includes status spongiosus (“spongiform state”), comprising irregular cavities in gliotic neuropil following extensive neuronal loss (including also lesions of “burnt-out” CJD), “spongy” changes in brain oedema and metabolic encephalopathies, and artefacts such as superficial cortical, perineuronal, or perivascular vacuolation; focal changes indistinguishable from spongiform change may occur in some cases of Alzheimer's and diffuse Lewy body diseases. Very rare cases might not be diagnosed by these criteria. Then confirmation must be sought by additional techniques such as PrP immunoblotting, preparations for electron microscopic examination of scrapie associated fibrils (SAF), molecular biologic studies, or experimental transmission.