Calcium-binding proteins from the outer acrosomal membrane of ram spermatozoa: potential candidates for involvement in the acrosome reaction.

After an intracellular calcium influx, fusion of the sperm plasma membrane and outer acrosomal membrane (the acrosome reaction) precedes mammalian fertilization in vivo. This study describes the isolation of outer acrosomal membrane from ram spermatozoa and the subsequent characterization of calcium-binding proteins. Pooled ejaculates were diluted, cooled slowly and washed. Incubation with Hyamine 1622 (benzethenium chloride) and subsequent slow centrifugation gently dislodged and concentrated acrosomal membranes, the fragments of which were isolated on a two-step discontinuous sucrose gradient. The acrosomal membrane material stained with Giemsa, whereas spermatozoa from the gradient pellet stained intensely only in the equatorial segment. The acrosomal fraction showed a limited number of polypeptides by SDS-PAGE. Incubation with 45Ca2+ revealed two radioactive bands at 34 and 39 kDa. Extraction in the presence of EGTA implied that these proteins are not peripheral proteins associated with the membrane only in the presence of calcium ions, but are integral membrane proteins. Polyclonal antisera raised to the two bands showed specific binding to the anterior acrosomal region and demonstrated the intracellular location of the proteins. Sequence data of protein A revealed 83% homology with calnexin homologue precursor and 70% homology with annexin XI. Protein B showed 68% homology with protein SP-10 precursor and 64-72% homology with various annexins. However, crossreactivity with a range of commercial annexin antibodies and a specific antibody to a synthetic motif encompassing the annexin calcium-binding site was not demonstrable. It is concluded that the isolated proteins are unlikely to be annexins, but are possibly novel calcium-binding proteins.

Single water channels of aquaporin-1 do not obey the Kedem-Katchalsky equations.

The Kedem-Katchalsky (KK) equations are often used to obtain information about the osmotic properties and conductance of channels to water. Using human red cell membranes, in which the osmotic flow is dominated by Aquaporin-1, we show here that compared to NaCl the reflexion coefficient of the channel for methylurea, when corrected for solute volume exchange and for the water permeability of the lipid membrane, is 0.54. The channels are impermeable to these two solutes which would seem to rule out flow interaction and require a reflexion coefficient close to 1.0 for both. Thus, two solutes can give very different osmotic flow rates through a semi-permeable pore, a result at variance with both classical theory and the KK formulation. The use of KK equations to analyze osmotic volume changes, which results in a single hybrid reflexion coefficient for each solute, may explain the discrepancy in the literature between such results and those where the equations have not been employed. Osmotic reflexion coefficients substantially different from 1.0 cannot be ascribed to the participation of other 'hidden' parallel aqueous channels consistently with known properties of the membrane. Furthermore, we show that this difference cannot be due to second-order effects, such as a solute-specific interaction with water in only part of the channel, because the osmosis is linear with driving force down to zero solute concentration, a finding which also rules out the involvement of unstirred-layer effects. Reflexion coefficients smaller than 1.0 do not necessitate water-solute flow interaction in permeable aqueous channels; rather, the osmotic behaviour of impermeable molecular-sized pores can be explained by differences in the fundamental nature of water flow in regions either accessible or inaccessible to solute, created by a varying cross-section of the channel.

Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore.

Published values for sperm membrane water permeability (L(p)) obtained using a time-to-lysis methodology have produced anomalous results when used to model optimal cooling rates for cryopreservation of spermatozoa. As the lysis method is dependent on potentially questionable assumptions, we describe an alternative method for measuring sperm L(p). Spermatozoa were exposed to hypo- and hyperosmotic conditions using a stopped-flow apparatus and the time course of resulting volume changes was measured using concentration-dependent self-quenching of the entrapped fluorophore, carboxyfluorescein (CF). L(p) was measured for boar, rabbit, and ram spermatozoa using a range of osmotic stresses (+/-50-100 mOsm). Values for exosmotic and endosmotic flow showed no evidence of rectification. Mean L(p) values were 0.84 microm/min/atm (boar), 0.28 microm/min/atm (rabbit), and 2.79 microm/min/atm (ram). These values are lower than the lysis method estimates, with the ram value reduced by approximately two-thirds using the current methodology. The value for boar spermatozoa showed good agreement with published values obtained using an electronic cell-sizing technique. Substitution of the revised values for L(p) into the model for optimal cooling rates brings the calculated optimal rate closer to the lower empirically observed value but does not fully account for the previously reported discrepancies.

Cryopreservation of semen from domestic livestock.

Fifty years after the first successful cryopreservation of spermatozoa, the technique is an integral part of the cattle breeding industry but has failed to establish itself commercially in the production of other breeds of domestic livestock. New assessment techniques have shown that the ejaculate consists of a heterogeneous population of cells, which achieve their full fertility potential at different rates within the female tract and thus maximize the chances of a fertile spermatozoon successfully combining with an egg. It is becoming apparent that the freeze-thaw process results in a more homogeneous cell population, which may be functionally compromised. One aspect of sperm function that has been demonstrated to be affected by cryopreservation is the process of capacitation. Chlortetracycline staining has shown that frozen-thawed spermatozoa undergo an accelerated 'capacitation-like' process which has implications for their interaction with the female tract, ability to establish sperm reservoirs in vivo and hence for their life expectancy after insemination. In addition to heterogeneity within the ejaculate, there is increasing evidence for variation between individuals in the success of sperm freezing. Post-thaw sperm survival may be consistently poor for certain individual animals even though pre-freeze parameters appear normal. The mechanisms that may underlie such differences in cryosensitivity remain unclear. A greater role for the use of frozen semen in livestock production can come only from an improvement in the preservation of the functional competence of the cryopreserved spermatozoon after insemination into the female tract.

The potential transmission of infectious agents by semen packaging during storage for artificial insemination.

Plastic straws, of a type widely used for semen cryopreservation, sealed using three different methods, (PVA powder, plastic spheres and plasticine modelling clay) were tested for leakage of low molecular weight dye (methylene blue), bacteria (Escherichia coli) and virus (Newcastle disease virus). Leakage was found to be dependent on the method used to fill the straws. Straws filled using a traditional 'dip and wipe' method and sealed with PVA powder demonstrated a significant degree of methylene blue leakage (0.0269% of the total straw contents) probably associated with contamination of the powder sealing plug. Straws filled using an aseptic filling technique showed no detectable leakage of any agent with any of the sealing methods. This study highlights the need to establish good-practice guidelines for the packaging of semen collected for freezing and future AI from non-domestic livestock where disease-free status cannot be guaranteed and unsophisticated technology is used.

Simultaneous detection of the acrosomal status and viability of incubated ram spermatozoa using fluorescent markers.

Incubation of diluted ram spermatozoa at 39 degrees C results in a high percentage of acrosome reactions, but previously we have not been able to demonstrate the viability of these cells. Detection of the viability and stages of acrosomal exocytosis, either spontaneous or induced, was carried out using fluorescent probes. Propidium iodide (PI) was used to determine cell viability and, simultaneously, FITC-Pisum sativum lectin (FITC-PSA) was used to assess acrosomal status by staining glycoproteins in the acrosome of permeabilised spermatozoa. Diluted ram semen was incubated for 6 hours at 39 degrees C. At 2 hourly intervals, samples were taken and examined for evidence of a spontaneous acrosome reaction. In addition, calcium ionophore A23187 was used to induce the acrosome reaction and samples were examined at 10 minute intervals. PI was added and then washed out by filtration. Smears were made and air-dried, permeabilised with absolute ethanol and then stained with FITC-PSA. The slides were later viewed under the fluorescence microscope with a peak excitation wavelength of 488 nm. With this combination of two fluorescent probes using a single excitation wavelength, both the cell viability and the acrosomal status could be simultaneously and easily visualized. Results showed four categories of staining: PI-ve/PSA + ve (Live and acrosome-intact), PI + ve/PSA + ve (dead and acrosome-intact), PI - ve/PSA - ve (live and acrosome-reacted) and PI + ve/PSA - ve (dead and acrosome-degenerated). About 75% spermatozoa that were acrosome-reacted were still viable after 4 h incubation in the absence of ionophore, and approximately 90% spermatozoa were acrosome-reacted and still viable after 30 min incubation in the presence of ionophore.

Surface area and volume measurements for ram and human spermatozoa.

Surface area and volume measurements were made for ram and human spermatozoa. Measurements were made using different techniques in an attempt to confirm estimates arrived at by independent methodologies. Sperm head projected surface areas were calculated from published formulae using linear micrometry measurements or were obtained directly by image analysis. Total surface areas were calculated as twice the projected head area plus the flagella area, estimated from published dimensions. Spermatozoon surface area was also measured from electron micrographs using a stereological method. Micrometric methods gave values of 135 microns2 for ram spermatozoa and 106 microns2 for human spermatozoa. Stereology methods gave values of 142 microns2 for ram spermatozoa and 106.5 microns2 for human spermatozoa, offering good agreement between the two methods. Sperm volumes were estimated by stereology and by radiolabel volume exclusion methods. From stereology, ram sperm volume was 13.3 microns3 and human sperm volume was 22.2 microns3. From the volume exclusion method, ram sperm volume was estimated as 25 microns3. Attempts to measure total volume and water volume simultaneously using and adaptation of this method were unsuccessful. Separate estimation of water volume for ram spermatozoa gave a value or 12.8 microns3, suggesting a 50% water space. Results from this study are compared with existing published values.

The presence of water channel proteins in ram and human sperm membranes.

Ram and human spermatozoa have a high coefficient of osmotic water permeability (Pf) with a low activation energy (Ea), suggesting the presence of water channels within their plasma membranes. Sperm membranes were examined for the presence of two known water channel proteins, CHIP28 and glucose transporters belonging to the GLUT family of proteins. The water permeability of ram spermatozoa was not inhibited by mercuric chloride to which the CHIP28 channel is sensitive. The CHIP28 protein was not located in western blots of ram sperm membrane preparations that used an anti-CHIP28 antibody. The water permeability of ram and human spermatozoa was inhibited in the presence of phloretin, an inhibitor of glucose transport. Rabbit spermatozoa, which have a low Pf and a high Ea value, suggesting a non-porous membrane, were unaffected by phloretin. These results indicate that the erythrocyte and proximal tubule water channel, CHIP28, is not present in sperm membranes but that sperm membrane glucose transporters may have a secondary water channel function.

Determination of water permeability coefficient and its activation energy for rabbit spermatozoa.

Critical tonicity (tonicity at which 50% of cells swell beyond their maximum volume-to-surface area ratio and lyse) of rabbit spermatozoa was measured as 45.6 mOsm at 22 degrees C. To determine the temperature effect on critical tonicity, cells were equilibrated to 15, 25, and 35 degrees C and critical tonicity was measured as 52.9, 42.2, and 32.4 mOsm, respectively. The time taken for rabbit sperm to achieve lysis at these temperatures was measured by exposing cells for increasing times to distilled water. From these results values for the permeability coefficient of rabbit spermatozoa to water (Lp) and its activation energy (Ea) were calculated. At 25 degrees C, Lp was 0.63 micron/min/atm and was clearly temperature dependent; Ea was 17.8 kcal/mol. Rabbit spermatozoa appear to have a low Lp and high Ea, the opposite of the situation seen with spermatozoa from all other species examined to date which have high Lp and low Ea. Nevertheless, the values obtained permit modeling of cooling rates for cell survival during cryopreservation in keeping with cooling rates commonly employed.

Health services delivery to students with special health care needs in Texas public schools.

A statewide survey of 2,875 Texas public school nurses was conducted to determine the characteristics, needs, and involvement of nurses in the health and education management of students with special health care needs (SSHCN). The 1,574 survey respondents (response rate = 55%) were primarily registered nurses (84%) with a mean of 8.6 years (SD = 7.1) of experience in the school setting. Respondents served 1.5 school campuses on average; the mean nurse-to-student ratio per campus was 1:728 (SD = 518). Respondents identified 106,650 SSHCN (6% of total enrollment). Asthma (47%), attention deficit disorder (26%), and seizure disorders (8%) were the most prevalent conditions encountered among SSHCN. Medication administration (54%), diapering (12%), and inhalation respiratory treatments (11%) were the most common of 48,569 health procedures delivered daily to SSHCN by nurses, clerical staff, assistants, and teachers. Parents were identified as the primary source of both child-specific health (70%) and training (68%) information in the school setting. Although nurses, of all school personnel, are likely best able to speak to the impact of a child's health impairment and needed school services, only 32% of respondents reported routine participation in special education eligibility evaluations and only 18% reported routine attendance at special education meetings for SSHCN. Moreover, 84% and 92%, respectively, reported discomfort at participating in special education eligibility evaluations and attending special education meetings.

Calculated optimal cooling rates for ram and human sperm cryopreservation fail to conform with empirical observations.

The permeability coefficient to water (Lp) and its associated activation energy (Ea) were measured for ram (8.47 microns/min/atm at 25 degrees C, 1.06 kcal/mol) and human (2.89 microns/min/atm at 30 degrees C, 1.93 kcal/mol) spermatozoa. By use of these figures, predictive water loss curves were calculated, from published equations, for different cooling rates from 100 degrees C/min to 100,000 degrees C/min. The calculated curves show that ram spermatozoa cooled at even the fastest rate would be in osmotic equilibrium by -20 degrees C, and human spermatozoa cooled at rates up to 10,000 degrees C/min would be in equilibrium by -15 degrees C. If the nucleation temperature for spermatozoa is taken to be between -20 degrees C and -30 degrees C, then ram and human spermatozoa cooled at these rates would apparently not exhibit any intracellular freezing. There is a significant discrepancy between these calculated optimal cooling rates and the published empirically derived optimal rates of 50 degrees C/min for ram and 10 degrees C/min for human. The failure of ram and human spermatozoa to conform with the established and previously successful model for prediction of optimal cooling rates suggests that damage sustained at high cooling rates may be unrelated to intracellular ice formation.

Osmotic effects on ram and human sperm membranes in relation to thawing injury.

The effects of hyposmotic and hyperosmotic stresses on ram and human spermatozoa were examined. Human spermatozoa exhibited a precipitate decline in survival at osmolalities below 90 mOsm caused by cells swelling beyond their maximum volume-to-surface area ratio and lysing. Ram spermatozoa exhibited a progressive decline in cell survival at relatively small hyposmotic stresses before exceeding their maximum volume-to-surface area ratio; this prelytic cell loss could be prevented by decreasing the osmolality in a series of 25-mOsm steps. Repeated hyposmotic stress experiments indicated that cells sensitive to prelytic damage constitute a discrete subpopulation within the ram ejaculate. Spermatozoa of both species were apparently resistant to hyperosmotic stresses; human spermatozoa maintained membrane integrity when subject to stresses up to 2.5 Osm and ram spermatozoa up to 1 Osm. However, ram spermatozoa suffered an almost complete and irreversible loss of motility above 600 mOsm. Spermatozoa of both species exposed to hyperosmotic stress and returned to isosmotic conditions exhibited significant cell damage, although ram spermatozoa were the more vulnerable. These observations are related to cryopreservation of spermatozoa.

The culture of human epididymal epithelium and in vitro maturation of epididymal spermatozoa.

OBJECTIVE: To promote human sperm maturation in vitro. DESIGN: Spermatozoa from the proximal epididymis were coincubated with epididymal epithelial fragments. SETTING: Hospital and research institute. PATIENTS, PARTICIPANTS: Tissue samples were obtained from men undergoing epididymovasostomy procedures or vasectomy. INTERVENTIONS: Fragments of epididymal epithelium formed everted epithelial spheres that in the presence of androgen maintained cell integrity. Coincubation for up to 48 hours of caput epididymal spermatozoa with 3-day-old epithelial cultures from the cauda epididymis was undertaken. MAIN OUTCOME MEASURES: Morphology of epididymal epithelium was assessed by light and electron microscopy. Pulse labeling of tissue in vitro with 35S-methionine was performed with analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique and fluorography. Spermatozoa were assessed for progressive motility and their capacity to bind to salt-stored human zona pellucidae. RESULTS: Epididymal fragments formed everted epithelial spheres that maintained cell integrity and functional morphology for 5 to 7 days. Specific proteins were synthesized in culture, in particular, proteins of 20, 22, 40, and 66 kd. Coincubation of caput epididymal spermatozoa with cultures from the cauda epididymis induced a significant increase in progressive sperm motility and sperm binding to salt-stored human zona pellucidae compared with control cultures of epithelium incubated in the absence of androgens or overgrown with fibroblasts. CONCLUSIONS: Aspects of human sperm maturation processes can be mimicked in vitro using coculture techniques with epididymal epithelium. This method may be valuable for improving the fertilizing capacity of human spermatozoa retrieved from the proximal region of the excurrent ducts.

Prenatal diagnosis of cystic fibrosis: microvillar enzymes and DNA analysis compared.

We have established reference ranges for three microvillar intestinal enzymes--alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.1), and leucine aminopeptidase (EC 3.4.1.1)--measured in amniotic fluid in a reference population of 1875 women presenting for routine amniocentesis. These data were derived for use in prenatal diagnostic studies in a population at risk (1:4) for cystic fibrosis. False-positive or indeterminate results were noted for fewer than 3.5% of all low-risk cases for each enzyme evaluated. Total alkaline phosphatase and its isoenzymes and leucine amino-peptidase and gamma-glutamyltransferase were measured in amniotic fluid sampled between the 15th and 19th weeks of gestation. Restriction fragment length polymorphism analysis of DNA was also performed when possible. In 52 cases examined for cystic fibrosis thus far, 46 were diagnosed on the basis of DNA analysis and (or) by sweat testing; for the other six cases, only abnormal enzyme results were obtained before termination of pregnancy. Predictions based on microvillar enzyme results were falsely negative in three cases. In only one case was there a discrepancy between enzyme results and DNA analysis. Diagnostic accuracy was highest during the 17th and 18th week of gestation. Preliminary results suggest the false-negative rate of this diagnostic strategy may be greater than or equal to 10%.

Fucosylated glycoconjugates appear on mouse embryos during blastocyst formation.

Mouse embryos were analysed for expression of surface glycoconjugates using a panel of fluorescein-labelled lectins. Morulae and blastocysts but not early cleavage stages stained brightly with fucose binding lectins. By contrast, cleavage stages stained brightly with all the other lectins tested. These findings provide evidence for substantial reorganisation of the cell surface during blastocyst formation and are consistent with a role for fucosylated glycoconjugates in compaction.

Prenatal diagnosis of cystic fibrosis using linked DNA markers and microvillar intestinal enzyme analysis.

Prenatal diagnosis was performed for 47 pregnancies with 1 in 4 risk of cystic fibrosis, including 7 cases analyzed with linked DNA markers, 16 cases analyzed by microvillar intestinal enzyme testing, and 24 cases where both methods of testing were attempted. DNA was obtained by chorionic villus sampling in 10 cases and by amniocentesis in 21 cases, and diagnosis was based on analysis with the tightly linked DNA markers D7S8 and met. DNA analysis using these probes was fully informative in 74.4% of 90 couples with 1 in 4 risk. In 18 cases where both DNA results and microvillar intestinal enzyme data were diagnostic, there was agreement regarding the predicted status of the fetus. No adequate diagnosis was achieved in two cases where both diagnostic tests were attempted. Outcome is known for 24 pregnancies including 10 where DNA analysis was diagnostic, and no errors in diagnosis were detected. Prenatal diagnosis of cystic fibrosis using DNA markers is highly informative and accurate, but microvillar intestinal enzyme analysis remains a valuable part of a complete diagnostic program.