Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte.

In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles.

Nitrendipine prolongs rat parturition: no changes occur in progesterone withdrawal.

The mechanism of tocolytic action of the calcium channel-blocking agent nitrendipine [3-ethyl-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) 3,5-pyridinedicarboxylate; BAYER E 5009] had been examined. Pregnant rats (duration of pregnancy, 22 days) were treated with nitrendipine (12.5 mg/kg, sc) on days 18-21 of gestation (group I) or on days 20-21 of gestation (group II) and with vehicle only (control group). Serum levels of progesterone from the three experimental groups were determined on days 20 and 22, and the course of labor was monitored on days 22-23 in the individually caged dams. The duration of labor, delivery times, and other labor parameters were significantly delayed in the treated groups vs. the control group, while there was no difference in serum progesterone levels among the three experimental groups on day 20 or 22 of gestation 136.0 +/- 5.6 (mean +/- SEM; (n = 6) and 43.8 +/- 7.2 (n = 17) ng progesterone/ml serum, respectively. We can conclude that progesterone withdrawal, the primary event in the initiation of rat labor, is not altered by nitrendipine treatment. The delay of labor is apparently related to a decrease in the contractile state of myometrium due to the inhibition of calcium influx by nitrendipine.

Urinary 3-methylhistidine/creatinine ratio as a clinical tool: correlation between 3-methylhistidine excretion and metabolic and clinical states in healthy and stressed premature infants.

We have investigated the role of the urinary 3-methylhistidine (3MH) excretion, a measure of protein catabolism, in the evaluation of the metabolic state of premature infants. Two-hundred and twenty-two 24 hr urine collections and 3MH/Cr ratio determinations (expressed as mumoles of 3MH per mg creatinine) were carried out in 36 infants (average gestational age 32.7 +/- 0.7 wk, weight 1640 +/- 120 grams) and the relationship between the 3MH/Cr ratios and the metabolic and clinical state has been investigated. Five or more 3MH/Cr measurements were carried out on each of 19 infants and serial determinations on four of those babies are presented. The urinary 3MH/Cr ratio of healthy infants with adequate caloric intake and normal growth curve was .148 +/- .039 (S.D.) mumol/mg, about 35% higher than the 3MH/Cr ratio in healthy adults. As long as the premature infants were healthy the degree of prematurity had no effect on the 3MH/Cr ratio. The relationship between 3MH/Cr ratio and nitrogen balance was highly significant (p less than .001). 3MH/Cr ratio also correlates very well with the metabolic status of the infants: in the group with normal 3MH/Cr ratios less than or equal to .175 (.148 + 1 S.D., n = 90) there were four clinically stressed infants (4.4% false negative rate) while in the group with elevated 3MH/Cr ratios greater than .225 (.148 + 2 S.D.; n = 79) there were only three clinically well infants (3.8% false positive rate). In comparing the clinical status and 3MH/Cr ratios, we found that in the group of infants who could not be clearly defined as clinically well or stressed (n = 108) the 3MH/Cr ratio was more useful than clinical judgment in the prediction of metabolic status. It can be concluded that 3MH/Cr ratio is a potentially useful clinical tool which describes with high accuracy the clinical and metabolic status of premature infants. This conclusion is further supported by the data of serial 3MH/Cr determinations.

Urinary 3-methylhistidine excretion in juvenile-onset diabetics: evidence of increased protein catabolism in the absence of ketoacidosis.

Urinary 3-methylhistidine excretion (an indicator of protein catabolism) was measured in ten diabetic patients and in age and weight matched control subjects. The diabetic group, while receiving their usual insulin dose, excreted 42% more 3-methylhistidine than the control group (2.7 versus 1.9 mumole/kg body weight/24 hr). When the insulin dose of the diabetic subject was reduced by 15% or 25%, the concentrations of blood and urinary glucose were significantly increased by the rate of urinary 3-methylhistidine excretion was not increased further. These findings demonstrate augmented protein catabolism in diabetics even in the absence of ketoacidosis. It appears that blood and urine glucose levels are more sensitive to changes in insulin availability than protein catabolism.

Sperm creatine phosphokinase M-isoform ratios and fertilizing potential of men: a blinded study of 84 couples treated with in vitro fertilization.

OBJECTIVE: To examine the value of sperm creatine phosphokinase M-isoform (CK-MM) measurements toward predicting fertilizing potential of men. DESIGN: In 84 in vitro fertilization (IVF) couples without knowing the semen parameters, reproductive history or the outcome of the IVF cycles, we determined the sperm CK-MM ratios (the proportion of sperm CK-MM versus CK-MM+CK-BB). Husbands with less than 10% or greater than or equal to 10% CK-MM ratios were classified as "low likelihood for fertilization" (CKMM-Infertile, n = 22) or "high likelihood for fertilization" (CKMM-Fertile, n = 62), respectively. RESULTS: Both the CKMM-Infertile and CKMM-Fertile groups (CK-MM ratios: 4.9% +/- 0.6% versus 31.1% +/- 1.8%) were in the normospermic range (31.5 +/- 6.9 versus 78.4 +/- 5.9 x 10(6) sperm/mL and 45.6% +/- 5.0% versus 54.0% +/- 2.0% motility). The fertilization rates (6.2 versus 4.9 oocytes inseminated) were 14.2% versus 53.4%, and 72.7% versus 25.8% of the couples failed to achieve any oocyte fertilization. All 14 pregnancies (16.7% rate) occurred in the CKMM-Fertile group. The pregnancy rate in the 62 CKMM-Fertile couples was 22.6%, and considering only the 46 CKMM-Fertile women in whom oocyte fertilization occurred, it was 30.4%. Among the 22 CKMM-Infertile men, 9 were normospermic and 9 of the 62 CKMM-Fertile men were oligospermic. Within the CKMM-Fertile group, 12 and 2 of the 14 pregnancies occurred by the 53 normospermic and 9 oligospermic men (22.6% versus 22.2% rate). CONCLUSIONS: Sperm CK-MM ratios, a measure of normal sperm development, predict fertilizing potential independently from sperm concentrations. Sperm CK-MM ratios also detect unexplained male infertility (infertile men with normospermic semen), a diagnosis that until now could not be substantiated.

Hyaluronic acid (Sperm Select) improves retention of sperm motility and velocity in normospermic and oligospermic specimens.

The effects of Sperm Select (Pharmacia AB, Uppsala, Sweden), a hyaluronic acid medium, on the motility and membrane integrity properties of sperm were studied. In 15 normospermic specimens after overnight incubation, the motility parameters in the control versus the Sperm Select group were as follows (mean +/- SEM): motility, 18.8% +/- 2.8% versus 27.4% +/- 2.9%; velocity, 21.5 +/- 2.4 versus 27.2 +/- 2.2 microns/s; linearity, 3.8 +/- 0.3 versus 4.4 +/- 0.2; lateral head displacement, 1.5 +/- 0.2 versus 1.9 +/- 0.1 microns; and tail beat/cross frequency, 8.8 +/- 1.3 versus 10.8 +/- 1.4 Hz. The density of motile sperm was 10.8 +/- 2.3 versus 18.5 +/- 2.5 X 10(6) sperm/mL. Finally, the velocity coefficient, the multiple of the sperm motility and linear velocity, was 4.6 +/- 1.1 versus 8.1 +/- 1.4. However, we found no Sperm Select related differences when testing sperm membrane integrity with hypoosmotic swelling and supravital staining. Thus, Sperm Select improves the retention of sperm motility (most prominently velocity) apparently due to a direct action of hyaluronic acid on sperm metabolism or contractility rather than to preservation of sperm membrane integrity. In 20 oligospermic specimens, Sperm Select caused similar improvements in sperm motility, and the duration of motility could be predicted from the degree of enhancement in sperm velocity after short-term Sperm Select exposure. A modified Sperm Select protocol is described that further increases motile sperm yield without a centrifugation step.

Creatine kinase immunocytochemistry of human sperm-hemizona complexes: selective binding of sperm with mature creatine kinase-staining pattern.

OBJECTIVE: To examine the clinical significance of the increased sperm cytoplasmic content that is due to a fault of spermatogenesis, we have further studied the relationship between increased sperm creatine kinase (CK) concentrations and diminished fertilizing potential in men. In the present work, we used CK immunocytochemistry of human sperm-hemizona (HZ) complexes to examine whether the distribution of mature (clear heads), intermediate (sperm heads with light stippling), and immature (heads with heavy stippling or with solid CK staining) spermatozoa bound to the HZ would follow the incidence of these sperm in the samples tested, or if there is a preferential binding by the mature sperm. DESIGN: Two pairs of HZ were exposed to washed semen and to their swim-up sperm fractions. The sperm and sperm-HZ complexes were treated with a CK antibody followed by horseradish peroxidase immunostaining, and the sperm were evaluated for maturity. SETTING: Men presenting for fertility evaluation were studied in two university-based andrology laboratories. RESULTS: The binding of the HZ was selective for mature sperm as indicated by the incidence of intermediate and immature sperm in washed semen versus bound to the HZ (intermediate: 20.0% versus 1.4%; immature: 7.6% versus 0.5% [mean +/- SEM]) or in swim-up sperm fractions versus the HZ (intermediate: 18.7% versus 3.4%; immature: 2.5% versus 0.2%). The binding was almost exclusive to normal sperm (96.4% to 98.1%) whether the HZ were exposed to washed semen or swim-up fractions in spite of the five to ten times higher incidence of intermediate and immature sperm. CONCLUSIONS: Mature sperm selectively bind to the zona. We suggest that spermatozoa with immature CK-staining patterns are deficient in the site(s) of oocyte recognition and binding.

A follicular fluid chondroitin sulfate proteoglycan improves the retention of motility and velocity of human spermatozoa.

OBJECTIVE: To examine the effects of two proteoglycans of different structure, isolated from human follicular fluid (FF), on the motility of human spermatozoa. DESIGN: Normozoospermic semen samples and their swim-up sperm fractions were incubated in the presence of 0.4 mg/mL of a larger chondroitin sulfate proteoglycan (CS-PG) for 0, 3, 7, and 16 hours. The effects of a smaller heparan-CS-PG and the chondroitin sulfate side chains of the larger proteoglycan were also investigated in the same conditions. Sperm motility parameters were analyzed using a computer-aided sperm analysis system (CASA; Cryo Research Inc., New York, NY) RESULTS: The larger CS-PG caused an immediate increase in sperm linearity. After 3 and 7 hours, the retention of sperm motility, velocity, linearity, and amplitude of lateral head displacement have increased by an average of 13% compared with the control samples. After a 16-hour incubation, the retention of the motility properties was improved by approximately 40% (range, 27% to 50%) in the samples containing proteoglycan. The effects of the isolated glycosaminoglycan side chains were much lower than those of the intact proteoglycan. The heparan-CS-PG did not affect sperm motility. CONCLUSION: A CS-PG from FF increases retention of motility and velocity of human sperm. These physiological effects may enhance the fertilizing efficiency of spermatozoa in the female reproductive tract.

Enhancement of human sperm motility and velocity in vitro: effects of calcium and creatine phosphate.

Because of their roles in motility regulation and energy transport, calcium and creatine phosphate were examined for their effects on sperm motility and velocity in specimens of normal donors. Semen or migrated sperm fractions were incubated with of 1 mmol of calcium, 5 mmol magnesium, and 10 mmol of creatine phosphate (n = 28) or in the presence of 4 mumol of Verapamil, calcium, and creatine phosphate (n = 10). The samples were subjected to multiple exposure photography (four picture frames of two different drops) at 0, 1, 4, or 5 and at 10 hours and sperm motility and velocity were analyzed. In both calcium and calcium-creatine phosphate conditions, sperm motility and velocity were significantly increased, compared with control values (P = between less than 0.001 and 0.05). Sperm motility declined following Verapamil exposure, but the motility values remained at the level of the control in the presence of additional calcium or creatine phosphate. The effects of calcium and creatine phosphate take place rapidly; within 1 minute all improvements in sperm velocity and motility are fully achieved. There is no loading effect of calcium, and when the sperm is transferred into media without the additional calcium, the velocity decreased to that of the initial control value. Magnesium alone had no effect on motility or velocity. These experiments indicate that calcium or creatine phosphate can support sperm motility and velocity at a significantly increased level. Thus the addition of calcium or creatine phosphate to the insemination media may enhance the fertilizing capacity of sperm during in vitro fertilization or gamete intrafallopian transfer procedures.

Peritoneal fluid from women with moderate or severe endometriosis inhibits sperm motility: the role of seminal fluid components.

OBJECTIVE: To examine the mechanism of sperm motility inhibition by peritoneal fluid (PF) from women with endometriosis. DESIGN: Prospective, randomized study. SETTING: University-based andrology laboratory. PATIENTS: Women with and without endometriosis. INTERVENTIONS: Fresh semen or Percoll-purified sperm fractions were combined with PF from women with endometriosis (n = 20), from fertile women without endometriosis (n = 10), or with physiological saline. MAIN OUTCOME MEASURE: Sperm motility parameters were determined with computer assisted semen analysis. Data were evaluated by the analysis of variance and the Student's t-test. RESULTS: Peritoneal fluid from women with minimal or mild endometriosis did not inhibit sperm motility in semen. Peritoneal fluid from women with moderate or severe endometriosis caused approximately 40%, 50%, and 80% declines in sperm motility and in percent progressive motile sperm after 4,7, and 24 hours, respectively. Sperm velocity was inhibited by approximately 30% and 60% after 7 and 24 hours, respectively. However, in the Percoll-purified sperm fractions the same PF did not inhibit sperm motility within the 4- to 7-hour time frame, and only a 17% to 42% inhibition occurred after the overnight incubation. Sperm velocity was not affected. CONCLUSION: Cellular components of seminal fluid appear to mediate the inhibitory action of PF. Assuming that the leukocyte components of semen and PF are common, the cell-mediated inhibition of sperm motility is a likely contributor to endometriosis related infertility.

Correlation between the rate of lipid peroxidation and cellular maturity as measured by creatine kinase activity in human spermatozoa.

We have demonstrated previously that creatine kinase (CK) activity is a measure of cellular maturity and fertilizing potential in human spermatozoa. In the present work we have examined whether there is a relationship between sperm CK activity and the rate of lipid peroxidation (LP) as measured by malondialdehyde (MDA) formation. Both MDA production and CK activity were higher in oligospermic than in normospermic specimens (P < 0.001, N = 41 and 101, respectively), and there was a close correlation (R = 0.43, P < 0.001) between these two biochemical parameters. As demonstrated previously with the CK measurements, there was a heterogeneity among the groups: About 40% of the oligospermic men had MDA and CK activity values similar to that of the normospermic group, and 12% of the normospermic men had MDA and CK activity values similar to that of the oligospermic group. We have also examined in three experimental paradigms the question of sperm-to-sperm propagation of increased LP and the possible increase in LP following centrifugation as used in sperm preparation for assisted reproduction: The MDA differences among Percoll sperm fractions originating within the same specimens, the lack of change in MDA production after co-centrifugation and co-incubation of samples with high and low sperm LP rates, and the repeated centrifugation of the same specimens without an increase in MDA production all indicated the lack of sperm-to-sperm propagation of LP or increase in LP due to mechanical stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm creatine kinase activity in fertile and infertile oligospermic men.

The authors examined the value of sperm creatine-kinase (CK) activity parameters to predict sperm fertilizing potential of oligospermic men. Two patient groups from our intrauterine insemination program were studied: fertile oligospermic (32 men/46 specimens) and infertile oligospermic (19 men/82 specimens). In the initial specimens, the CK activities were (mean + SEM IU CK/10(8) sperm): 0.53 +/- 0.09 and 1.17 +/- 0.19 (P less than 0.001). The corresponding values in the swim-up fractions were 0.32 +/- 0.06 and 0.67 +/- 0.08 (P less than 0.001). In a subset of samples by fertile (N = 33) and infertile (N = 66) oligospermic men who had close to identical sperm concentrations (11.9 +/- 0.9 vs. 11.9 +/- 0.5 million sperm/ml) and motility values (23.7 +/- 1.7 vs. 23.0 +/- 1.3%), the CK activities were significantly lower in the fertile group in both the initial (P = 0.02) and in the swim-up (P = 0.002) samples. A logistic regression analysis of all 160 study samples (including 21 normal men/32 samples) further demonstrated that CK activities were predictive of fertilizing potential, whereas sperm concentrations of the samples provided no additional contribution. Sperm CK and similar biochemical markers will facilitate selection of men for various approaches in assisted reproduction.

Adenosine triphosphate (ATP) concentrations and ATP/adenosine diphosphate ratios in human sperm of normospermic, oligospermic, and asthenospermic specimens and in their swim-up fractions: lack of correlation between ATP parameters and sperm creatine kinase concentrations.

The authors had previously found an inverse correlation between per sperm creatine phosphokinase activity and sperm concentrations in men. Because creatine phosphokinase is a key enzyme in sperm energy transport, the possible relationship of sperm creatine phosphokinase activity, sperm adenosine triphosphate (ATP) concentrations, sperm ATP/ADP (adenosine diphosphate) ratios, and computer-aided semen analysis sperm motility parameters were then studied. The ATP concentrations and ATP/ADP ratios, measured by high-pressure liquid chromatography in washed sperm, were similar in normospermic and oligospermic specimens (ATP: 123.1 +/- 21.6 vs. 90.0 +/- 24.5 pmol/10(6) sperm; ATP/ADP: 2.8 +/- 0.4 vs. 2.1 +/- 0.4, N = 32 and 17, mean +/- SEM), and in samples with normal and less than 40% sperm motility (ATP: 96.8 +/- 27.2 vs. 122.2 +/- 19.6 pmol/10(6) sperm; ATP/ADP: 2.4 +/- 0.5 vs. 2.8 +/- 0.4, n = 26 and 23). In the swim-up sperm fractions, which showed improved motility, the ATP concentrations, but not the ATP/ADP ratios, were lower than in the initial semen samples (ATP: 152.9 +/- 28.4 vs. 90.3 +/- 10.6 pmol/10(6) sperm, P less than 0.05; ATP/ADP: 3.3 +/- 0.5 vs. 3.9 +/- 0.7, N = 18 pairs of samples). This is consistent with our previous finding of a lower cytoplasmic content in sperm in swim-up fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm function and choice of preparation media: comparison of Percoll and Accudenz discontinuous density gradients.

We compared the sperm populations prepared by Accudenz (35-65%) and Percoll (40-80%) density gradients in 21 normospermic specimens (concentration, 53.6 +/- 3.8 x 10(6) sperm/ml; motility, 44.5 +/- 3.5%). Accudenz facilitated a higher recovery of sperm and motile sperm (68.4 +/- 6.6% vs. 49.3 +/- 4.9%, P < 0.001, and 87.8 +/- 4.1% vs. 77.8 +/- 3.7%, P < 0.01, respectively). Sperm motility was lower in the Accudenz compared to the Percoll pellets; thus the values of total motile sperm recovered were not different (17.1 +/- 2.4 vs. 15 + /- 1 2.2 x 10(6) sperm/ml). The long term retention of sperm motility was substantially improved in Accudenz (at 24 hours, 34.9 +/- 2.8% vs. 26.3 +/- 1.5%; 60% vs. 40% of the initial motility, P < 0.001), and the Accudenz vs. Percoll samples also exhibited a higher retention of total motile sperm (at 24 hours, 9.8 +/-.2 vs. 6.1 = 0.5 x 10(6) motile sperm/ml, P < 0.05). The sperm motility index, a multiple of velocity and motility in the sample that reflects the efficiency of the sperm population in sperm-oocyte interaction, was 75% higher in the Accudenz samples at 24 hours (3.6 +/- 0.4 vs. 2.1 +/- 0.2, mu m/second, P < 0.01). Sperm cellular maturity by the creatine phosphokinase (CK) activity and CK-M to CK-B isoform ratio parameters (in the original samples 0.14 +/- 0.02 lU CK/100 x 10(6) sperm and 57.9 +/- 3.7%, respectively) were improved in both the Accudenz and Percoll pellets (P < 0.001), with no difference between the two sperm fractions. Sperm activation status monitored by chlortetracycline fluorescence indicated that after 4 hours of incubation the incidence of fully acrosome-reacted spermatozoa in the Accudenz versus Percoll pellets was 6.2 +/- 0.3% versus 13.1 +/- 1.0% (P < 0.001), a 100% increase in Percoll. We can conclude that Accudenz yields a higher concentration of motile spermatozoa, with improved retention of motility, velocity, and acrosomal integrity and without an increase of sperm with diminished cellular maturity. Thus, in sperm preparation for intrauterine insemination, in which the timing of ovulation and insemination frequently do not correspond, Accudenz-prepared sperm, with a better retention of motility/velocity and acrosomal integrity and with a consequential higher resistance to activation by the female reproductive tract, are expected to be more effective.

In vitro sperm-binding assay to distinguish differences in populations of human sperm or damage to sperm resulting from cryopreservation.

Annually, >1.3 million men are members of couples seeking help because of infertility. Semen from many of these men contains reasonable numbers of motile and normal sperm, but for a subset of individuals, many sperm are deficient in ability to bind to the zona pellucida during in vitro fertilization. Diagnosis of this defect has been hampered by lack of a low-cost test. Molecular similarity exists between the perivitelline membrane of a hen's egg and the mammalian zona pellucida. These facts and some preliminary data led to evaluation of binding of human sperm during incubation for 60 minutes at 37 degrees C to an extract of chicken perivitelline membrane coated in microwell assay plates. The sperm-binding assay had inter- and intraassay plate variations of 21 and 12%, respectively, using washed fresh sperm. All seminal samples were normal, except a few that had 36 to 50% motile sperm with a low rate of sperm movement (if there is a low rate of movement, World Health Organization [WHO] criterion for normalcy is >50% motile). Nevertheless, this sperm-binding assay detected differences among individuals in percentage of sperm bound. Based on data for two to four ejaculates from each of eight occasional sperm donors, the coefficient of variation for ejaculates within donor averaged 31%, and means for the donors differed (P < 0.02). Percentage of sperm bound ranged from <1 to 38% for fresh semen from 57 men and from <1 to 13% for frozen-thawed semen from 34 men. Percentage of motile sperm accounted for <30% of the variation in percentage of sperm bound. In a direct comparison based on 17 ejaculates, aliquots evaluated fresh averaged 13% sperm bound, versus 2% for frozen-thawed aliquots. We concluded that the egg membrane substrate used in these microwell assay plates might serve as the basis for a diagnostic assay. However, it remains to be established whether samples of human semen with a low percentage of sperm binding indeed have relatively low fertilizing potential.

Increased in vitro binding of fresh and frozen-thawed human sperm exposed to a synthetic peptide.

Prosaposin is a well-characterized, approximately 68-kDa protein found in many tissues and as a normal component of human semen. A fragment of prosaposin apparently is involved in primary sperm-egg binding. We hypothesized that binding of sperm from some men to egg investments would be increased by in vitro exposure of their sperm to a synthetic fragment of human prosaposin (FertPlus peptide). Hence, we evaluated samples of washed fresh or frozen-thawed human sperm after a 10-minute exposure to synthetic FertPlus peptide at 0 (control), 80, 160, 320, 640, or 1280 pM, followed by 1:50 dilution for evaluation of binding. The criterion of response was mean percentage of sperm bound to a substrate prepared from chicken egg membranes after sperm were incubated for 60 minutes at 37 degrees C in substrate-coated wells of a sperm-binding assay plate. For each seminal sample, data were normalized against the percentage of sperm bound for control aliquots, providing values for relative binding. With fresh sperm, relative binding was increased (P < 0.01) by exposure of sperm to peptide, and the effect was especially obvious at 1280 pM. Higher doses were not tested. Collectively at three study sites, exposure of fresh sperm to 1280 pM peptide substantially increased (above 99% confidence interval; on the basis of duplicate control samples) percentage of sperm bound for 25 of 74 (34%) samples. For frozen-thawed sperm, exposure to 1280 pM peptide increased binding for 29 of 65 (45%) samples. We concluded that for >30% of men, exposure of their sperm to this synthetic fragment of prosaposin at 1280 pM increased binding of sperm to an egg membrane substrate similar to that offered by the zona pellucida.

Cytoplasmic extrusion and the switch from creatine kinase B to M isoform are completed by the commencement of epididymal transport in human and stallion spermatozoa.

Although in several species there is a relationship between epididymal sperm transport and fertility, in human in vitro fertilization (IVF), spermatozoa recovered from the caput epididymidis or even the rete testis are fertile. We studied two objective markers of sperm maturity in the sperm of men and stallions: creatine kinase (CK) concentrations, which are a measure of cytoplasmic retention in immature spermatozoa, and the ratio of CK-M and CK-B isoforms (% CK-M/[CK-M + CK-B]), which is proportional to the incidence of mature sperm. The CK markers and the fertilizing function are closely related: Immature sperm with cytoplasmic retention do not bind to the zona, because during cytoplasmic extrusion, the sperm plasma membrane is also remodeled. We examined whether changes in sperm CK values are still ongoing during epididymal transport, or if cellular maturation is completed prior to the arrival of sperm in the caput epididymidis. The incidences of mature sperm in human caput and corpus epididymidis (studied in six men with obstructive azoospermia of various pathogeneses) were (mean+/-SEM) 55.7+/-2.2 and 49.3+/-7.6%, respectively; and the sperm CK-M ratios in the caput epididymidis of three men were 72, 75, and 70%, values that are similar to those of ejaculated sperm. In four segments of the proximal and distal epididymis of three stallions (the origin of sperm was also verified by the position of the cytoplasmic droplet) and in ejaculate of five stallions, the incidences of mature sperm were 88.2+/-6.2, 89.0+/-6.7, 90.3+/-7.8, 87.6+/-5.9, and 86.7+/-0.8%, and the respective CK-M ratios were 75.0+/-8.7, 84.2+/-2.9, 87.9+/-1.2, 92.5+/-1.5, and 69.3+/-3.5%. There were no differences in the incidences of mature and immature spermatozoa or in CK-M ratios among sperm arising from the various epididymal regions or from the ejaculate in men or stallions. Thus, the cellular maturation events in sperm, as detected by the CK markers, are completed by the time the sperm commences epididymal transport. These findings are in agreement with the IVF fertility of sperm aspirated from the male reproductive tract. The data may also suggest that the primary role of sperm epididymal transport in men is to remodel the plasma membrane to enhance sperm functional integrity in the diverse environments of the male and female reproductive tracts prior to fertilization.

Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites. III. Guidelines for field studies of male reproductive disorders.

Exposures to environmental toxicants can have detrimental effects on several aspects of human male reproduction: fertility, sexual function, hormone status, and pregnancy/birth outcomes. However, no simple prescreening methods are available for reliably identifying potential hazards; questionnaires alone are relatively imprecise and inefficient in the absence of field data. Multidisciplinary field studies are required that include detailed exposure information, health and reproductive histories, physical examinations, semen analyses, and possibly, hormone analyses. Semen analysis is a critical component of field studies for evaluating two aspects of male reproduction: 1) changes in sperm or seminal content, which may be indicative of adverse effects on the male reproductive system with possible implications for fertility potential; and 2) defects in sperm DNA or chromosomes, which may be associated with subsequent changes in viability during embryonic development and health risks to the offspring. Semen analyses may be tiered: 1) initially, each semen study may include conventional semen assays (concentration, motility, and morphology) as well as specific biomarkers indicated by the health effect of concern in the study cohort: and 2) archived samples (i.e., frozen, videotaped, or smeared) may be utilized in later second-tier analyses to further characterize specific findings. Before initiating any field study, it is cost effective to critically evaluate the suitability of the cohort by confirming exposure and determining that there are adequate numbers of male participants in each exposure category. Such evaluations must be based on the statistical sensitivities of the specific tissue biomarkers and health endpoints for detecting changes. This article summarizes the components of the ideal field study and identifies research needs for improving field studies of male effects and for understanding the mechanisms of male reproductive toxicity. Several promising semen methods currently under development are also discussed.

Male reproductive effects of solvent and fuel exposure during aircraft maintenance.

Few studies have addressed the effects of mixed, low-level exposures to complex mixtures on a man's reproductive potential. In this prospective study, each subject was evaluated before first exposure and at 15 and 30 weeks after exposures had begun. A total of 50 men working on aircraft maintenance at an Air Force installation were included in the study. In addition, eight unexposed men were concurrently sampled. Industrial hygiene (IH) sampling and expired breath samples were collected for jet fuel as measured by total napthas, benzene--a component of jet fuel, 1,1,1-trichloroethane, methyl ethyl ketone, xylenes, toluene, and methylene chloride. Sperm production, structure, and function (sperm concentration, sperm motion, viability, morphology, morphometrics, and stability of sperm chromatin) were evaluated. Exposures were low. All mean IH measures were below 6 ppm, which is less than 10% of the Occupational Safety and Health Administration standard for all chemicals except benzene. Sheet metal workers had the highest mean breath levels for both total solvents (24 ppb) and fuels (28.3 ppb). For most sperm measures, mean values remained in the normal range throughout the 30 weeks of exposure. When jobs were analyzed by exposure groups, some adverse changes were observed. The paint shop group had a significant decline in motility of 19.5% at 30 weeks. Internal dose measures, however, did not show a significant association with spermatogenic changes.

Urinary 3-methylhistidine excretion and nitrogen balance in healthy and stressed premature infants.

Conventional methods of assessing adequate nutrition in children may be inaccurate or change too slowly to be helpful during acute illness. Techniques to measure protein synthesis or breakdown provide more accurate information. Three-methylhistidine (3MH), an unusual amino acid found in actin and myosin, is not further metabolized and is quantitatively excreted following muscle degradation. Urinary excretion of 3MH has, therefore, been proposed as a marker of protein catabolism. The ratio of 3MH to creatinine in 24-hr urine collectons was studied in 14 healthy or stressed premature infants, and compared to nitrogen balance (N bal), caloric intake and clinical course. There is a significant inverse correlation between 3MH/Cr ratio and N Bal (R = -.507). The mean 3MH/Cr ratio was 0.140 +/- 0.037 mu mole/mg (n = 37) in healthy growing premature infants and 0.296 +/- 0.160 (n = 26) in infants who were stressed and/or had inadequate nutrient intake. Healthy growing infants almost invariably had a ratio below 0.200. Serial determinations in three infants consistently showed a rise in the ratio to above 0.200 during periods of stress or decreased intake. The 3MH/Cr ratio may be a more sensitive indicator of metabolic status and may be useful clinically, especially in infants receiving total parenteral nutrition.