Milk amyloid A: correlation with cellular indices of mammary inflammation in cows with normal and raised serum amyloid A.

This study compared concentrations of amyloid A in bovine milk with the cell-based indicators of intramammary inflammation, somatic cell count and California Mastitis Test. Mammary quarter data pertaining to 180 cows were categorised according to concentrations of serum amyloid A in the cow of origin. Ranked correlation, ranked regression and receiver operator characteristics all demonstrated acceptable agreement between milk amyloid A concentrations and cell-based indices. There were some indications of reduction in this agreement, in cows with raised concentrations of serum amyloid A. However, there were also indications that serum amyloid A did not significantly influence milk amyloid A. The results of the current study indicate that milk amyloid A exhibits good correlation with established cell-based indicators of intramammary inflammation.

Myocarditis in sibling boxer puppies associated with Citrobacter koseri infection.

Two sibling Boxer puppies presented with severe suppurative myocarditis in the absence of additional disseminated suppurative foci. The identification of gram-negative bacteria within areas of myocarditis in both puppies and the pure growth of large numbers of Citrobacter koseri from the myocardial lesions in one of the dogs were consistent with a bacterial etiology. The fact that C. koseri is an opportunist pathogen suggested intercurrent immunosuppression. The finding of a concomitant bacterial myocarditis in two canine siblings is novel. The case is also unusual in that syncope could be related to the myocardial injury.

Immune response of male baboons to testis-specific LDH-C(4).

Four sexually mature male baboons (Papio sp.) were immunized with a chimeric peptide containing a B-cell epitope of the testis-specific lactate dehydrogenase (LDH-C(4)) and a promiscuous T-cell epitope of tetanus toxin. LDH-C(4) is the testis-specific isozyme of lactate dehydrogenase, and antibodies to this protein reduce fertility significantly in female nonhuman primates. Animals were immunized on Day 0 and received booster injections at Days 29, 61, and 344 after priming. Serum specific antibodies were determined at regular intervals during the initial 6 months and after the last booster. Testis biopsies were taken at Days 61, 127, and 183 after the primary immunization. Sperm-zona binding was assessed prior to and three times after the last booster. The present study demonstrated that this epitope of LDH-C(4) did not cause autoimmune disease and that sperm from these immunized males had a diminished zona binding capacity. These results suggest that a safe male immunocontraceptive based on development of anti-sperm antibodies may be feasible.

Macrophage secretory products and sperm zona pellucida binding.

OBJECTIVE: To determine if exposure of human gametes to macrophage secretory products reduces sperm binding to the zona pellucida, and to determine which cytokine(s) may be responsible for this effect. METHODS: A human macrophage cell line was cultured and either activated with lipopolysaccharide for 2 hours and then washed or left unactivated. Culture-conditioned media from activated or unactivated cells was used in hemizona assay. Hemizonae were incubated with sperm suspended in culture medium from either unactivated macrophages or activated macrophages, with the matching hemizona incubated with sperm suspended in control medium. Matching hemizonae were incubated with sperm suspended in unactivated macrophage medium paired with sperm suspended in activated macrophage culture medium. Conditioned medium from activated macrophages was found to have elevated levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, and transforming growth factor-beta, therefore, gametes were also exposed to these cytokines followed by the hemizona assay. After each incubation, the number of sperm tightly bound to the outer surface of each hemizona was determined. RESULTS: Exposure of gametes to activated and unactivated macrophage culture-conditioned media significantly decreases sperm binding to the zona pellucida, with medium from activated macrophages inducing the greatest effect (P < .05). Exposure of sperm to TNF-alpha significantly impaired sperm binding (P < .05), whereas other cytokines tested had no effect. CONCLUSION: These results suggest that macrophage secretory products in the basal and activated state may be a factor in endometriosis-associated infertility through the interference of sperm binding to the zona pellucida, and that TNF-alpha is a key cytokine responsible for this effect.

Differential binding of X- and Y-chromosome-bearing human spermatozoa to zona pellucida in vitro.

The sex of human offspring has been associated with the day in the mother's menstrual cycle on which insemination occurs, with male zygotes being formed earlier in the fertile period than female zygotes. Using an in vitro environment designed to mimic the in vivo milieu, we tested the hypothesis that Y-chromosome-bearing spermatozoa survive functionally longer than X-chromosome-bearing spermatozoa, and that this differential functional survival is a contributing factor to the in vivo phenomenon. Donor semen was processed by swim-up and incubated at 37 degrees C in culture medium for 0, 24 and 48 h, with human zona pellucida (hemizona, HZ) being used to select functional spermatozoa. A second set of in vitro storage conditions, 4 degrees C in test-yolk refrigeration buffer, was used to determine whether changing the incubation conditions alters the process. The sex chromosome of the spermatozoa was determined using fluorescent in situ hybridization (FISH). For spermatozoa incubated at 37 degrees C, the percentage of functional (HZ bound) Y-bearing spermatozoa was significantly increased (P < 0.05) at 48 h (55.4 + 2.9%) but not at 0 h (50.5 + 0.7%) or 24 h (52.8 + 3.1%) compared to swim-up spermatozoa (50.6 + 0.3%). No difference in the percentage of functional Y-spermatozoa was observed at any time-point with storage at 4 degrees C in refrigeration buffer. Thus, we demonstrated that significantly more Y-bearing spermatozoa were capable of zona binding than X-bearing spermatozoa at 48 h at 37 degrees C incubation, with an observed Y : X ratio of 1.15 for these zona-bound spermatozoa compared to 1.02 for post-swim-up spermatozoa. We conclude that a differential functional survival appears to exist between X-bearing and Y-bearing spermatozoa, with the latter exhibiting a longer functional survival under in vitro conditions.

Evaluation of the effect of a cervical cap device on sperm functional characteristics in vitro.

Intracervical insemination continues to be employed for homologous and donor insemination in natural and stimulated cycles. Efficacy studies for potential fertility involve in vivo assessment; however, in vitro testing of particular sperm function(s) critically involved in fertilization is an important component of such evaluation. We report here on the in vitro evaluation of the effects of the silicone Veos cervical cap (Veos, London, UK) on sperm function. Donor semen was exposed to the Veos cervical cap or a sterile 15-cc centrifuge tube (control), or treated with the spermicide nonoxynol-9 (5 mg x ml(-1) in saline) for 4 h at 37 degrees C and 5% CO2 in water-saturated air. After exposure, motility characteristics, both in semen and in spermatozoa processed by standard swim-up procedure, cervical mucus penetration and sperm-zona pellucida interaction using the hemizona assay were assessed. Results indicated that exposure to the Veos cervical cap had no effect on either sperm motility characteristics or sperm-zona pellucida interaction. A small but significant difference was observed for cervical mucus penetration (P = 0.05); however, for both the control and treated groups, vanguard spermatozoa exceeded manufacturer's guidelines for a normal test, a penetration distance of > or = 30 mm. As expected, nonoxynol-9 was a potent inhibitor of sperm function. Lack of adverse effects on in vitro spermatozoa functional characteristics after exposure to the silicone Veos cervical cap supports its addition to the repertoire of fertility treatment modalities when cervical insemination is indicated.

Progesterone-induced calcium influx in cynomolgus monkey (Macaca fascicularis) spermatozoa.

For in vitro capacitation to occur in cynomolgus monkey (Macaca fascicularis) spermatozoa, there is an absolute requirement for exogenous stimulation with the sperm activators, caffeine (1 mM) and db-cyclic adenosine monophosphate (dbcAMP) (1 mM), which are known to induce capacitation-related hyperactivated motility. Tyrosine phosphorylation of sperm tail proteins is an integral component of this caffeine- and dbcAMP-stimulated hyperactivated motility. In both capacitated and noncapacitated human spermatozoa, progesterone (P4) has been reported to elicit an immediate, potent increase in intracellular calcium ion concentrations [Ca2+]i. The objective of this study was to examine the effects of progesterone on requisite events in macaque fertilization, including [Ca2+]i, hyperactivated motility, and the concomitant tyrosine phosphorylation of sperm tail (STTP) proteins after treatment with caffeine and dbcAMP. The effect of 1 microM of progesterone on [Ca2+]i was determined by spectrofluorometry with the fluorescent indicator, fura-2/AM, on hyperactivated motility using computer analysis (HTM-IVOS) with the sorting criteria lateral head amplitude (> or = 8.0 microm), curvilinear velocity (> or = 150 microm/s), linearity (< or = 60%), and on STTP by immunocytochemistry. The results showed that progesterone elicited a significant increase in [Ca2+]i in caffeine- and dbcAMP-activated macaque sperm with maximal stimulation at 30 minutes after activation. The response in nonactivated sperm was dramatically reduced compared with the response in activated sperm. Basal [Ca2+]i increased as a function of time in both activated and nonactivated control sperm although basal levels were significantly increased in activated sperm. Progesterone stimulation resulted in a small but significant increase in both hyperactivation and STTP when sperm were first pretreated with caffeine and dbcAMP. Our results provide evidence that macaque sperm activation with caffeine and dbcAMP is required for a progesterone-elicited response, which results in calcium influx, hyperactivated motility, and sperm tail tyrosine phosphorylation.

Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound.

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.

Baboon spermatozoa-zona pellucida binding assay.

This study developed a baboon in vitro system that allows transport of sperm from a treatment facility to an off-site location for subsequent evaluation of sperm functional capacity. We further described a sperm functional assay that evaluates baboon sperm binding to homologous zona pellucida, a baboon hemizona assay (HZA). Semen samples were collected from baboons via electroejaculation directly into refrigeration transport buffer. Postshipment semen characteristics were analyzed and each specimen prepared for assessment of sperm-zona pellucida interaction. Optimization of the baboon HZA included determination of the relationship between motile sperm concentration and zona pellucida binding. The effect of the sperm activators, caffeine and dbcAMP, on computerized sperm motion characteristics and HZ binding was also determined. A significant motile sperm concentration dependent increase was observed in sperm-zona pellucida binding. Maximal binding was observed at approximately 1-2 million motile sperm/mL. Treatment with the sperm activators, caffeine and dbcAMP, resulted in a significant increase in sperm progressive motility, straightline velocity (VSL), and amplitude of lateral head displacement (ALH), p <0.05 and a highly significant increase in curvilinear velocity (VCL), p <0.01. Treatment with caffeine and dbcAMP was not an absolute requirement for sperm-zona pellucida binding, inasmuch as binding did occur in the absence of activators. However, treatment with the two activators, caffeine and dbcAMP, resulted in a highly significant increase in HZ binding, p <0.0001. This system allows for the short-term maintenance of baboon sperm in a semiquiescent state until stimulation with the activators, caffeine and dbcAMP. It further provides a novel approach to delineating a contraceptive regimen's or agent's (ie, sperm vaccine) impact on specific cellular events occurring in the male gamete during fertilization.

Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey (Macaca fascicularis) spermatozoa.

Capacitation and capacitation-related hyperactivated motility do not occur spontaneously in cynomolgus monkey (Macaca fascicularis) spermatozoa; instead, both have an absolute requirement for exogenous stimulation with caffeine and dibutyryl (db)cAMP. In the present study, we 1) defined sorting criteria for automated analysis of macaque sperm exhibiting hyperactivated motility (HA) and 2) investigated protein tyrosine phosphorylation involvement in dbcAMP- and caffeine-stimulated capacitation and HA. Motion characteristics were assessed by computer-assisted motion analysis. Tyrosine phosphorylation of sperm tail proteins was determined by immunocytochemistry with PY-20 antiserum. Automated sorting criteria for HA were curvilinear velocity (VCL) >/= 150 microm/sec; amplitude of lateral head displacement (ALH) >/= 8.0 microm, and linearity (LIN)

Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the human epididymis.

OBJECTIVE: To determine the regional distribution and relative expression of 5alpha-reductase type 1 and type 2 mRNA within the human testis and regions of the epididymis. DESIGN: Prospective observational study. SETTING: University academic medical center. PATIENT(S): Two young adult male organ donors. INTERVENTION(S): None MAIN OUTCOME MEASURE(S): The distribution of 5alpha-reductase type 1 and type 2 mRNA in the testis and regions of the epididymis was detected by Northern blot analysis. The relative abundance of each 5alpha-reductase mRNA was evaluated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in which cyclophilin mRNA, a house-keeping gene product, was coamplified as the reference standard. RESULT(S): Northern blot analysis revealed the 5alpha-reductase type 2 transcript in the midcaput, distal caput, corpus, and proximal cauda of the epididymis, but the transcript was undetectable in the testis, proximal caput, and distal cauda region. No transcript for the type 1 isozyme was detected by Northern blot. The more sensitive RT-PCR showed low levels of type 1 mRNA in the testis and epididymis, with the highest abundance in the proximal caput. Type 2 mRNA of 5alpha-reductase was most abundant in the midcaput, was decreased in the more distal regions, and was more abundant than type 1 mRNA in all epididymal regions except for the proximal caput. CONCLUSION(S): Both 5alpha-reductase type 1 and type 2 mRNAs are present in the human epididymis. The type 2 isozyme mRNA is predominant, being more highly expressed than the low-abundance type 1 mRNA.

Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the nonhuman primate (Macaca fascicularis) epididymis.

The 5alpha-reduced metabolites of testosterone, including dihydrotestosterone, are considered the primary regulators of epididymal function. Two genes encode two 5alpha-reductase isozymes. We examined 5alpha-reductase type 1 and type 2 mRNA tissue distribution and relative abundance in cynomolgus monkey (Macaca fascicularis) testicular and epididymal tissues using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA extracted from monkey tissues including the testis (T) and the proximal caput (PCp), the caput (Cp), the midcorpus (Co), and the distal cauda (Cd) epididymis was reverse transcribed to produce cDNAs. 5alpha-reductase type 1 and 2 cDNAs were subsequently coamplified with the housekeeping gene, cyclophilin, in a PCR spiked with 33P-dCTP. Relative abundance was reported as the cpm ratios of type 1 or type 2/cyclophilin mRNA. Semiquantitative RT-PCR results indicated that type 1 mRNA was most abundant in the testis (0.48 +/- 0.06) and significantly decreased distally along the monkey epididymis (PCp: 0.29 +/- 0.04; Cp: 0.29 +/- 0.04; Co: 0.21 +/- 0.03; Cd: 0.07 +/- 0.01) (P < 0.001). Type 2 mRNA was undetectable in the testis but was present throughout the epididymis at uniform levels (PCp: 1.6 +/- 0.2; Cp: 1.4 +/- 0.3; Co: 1.6 +/- 0.2; Cd: 1.5 +/- 0.2). These data demonstrate that 5alpha-reductase type 1 mRNA is differentially expressed but of low abundance along the nonhuman primate epididymis, whereas 5alpha-reductase type 2 gene expression is uniform.

Effects of caffeine and dbcAMP on zona pellucida penetration by epididymal spermatozoa of cynomolgus monkeys (Macaca fascicularis).

Spermatozoa mature during epididymal transit, acquiring the abilities to swim progressively, fertilize oocytes, and produce viable offspring. In this study, we investigate the capacity of spermatozoa retrieved from the midcorpus and distal cauda regions of the epididymis of the cynomolgus monkey to penetrate homologous zona pellucida. Successful in vitro fertilization by ejaculated macaque sperm is dependent upon the addition of caffeine and dbcAMP. Therefore, the effect of these cyclic nucleotide mediators was also examined in this study. Results of sperm motion analysis indicate no difference in baseline values (without stimulators) for any motion parameter. With the addition of caffeine and dbcAMP, curvilinear velocity significantly increased only for the distal cauda sperm (P = 0.05). Amplitude of the lateral head displacement was significantly increased for distal cauda sperm (P < 0.01); although elevated above baseline, the increase observed after activation by corpus sperm was significantly lower than that achieved by cauda sperm (P < 0.05). The addition of caffeine and dbcAMP was an absolute requirement for zona penetration by both midcorpus and distal cauda sperm. With activation, zona penetration was significantly decreased for corpus sperm compared to cauda sperm (P < 0.001). These results suggest that cynomolgus monkey sperm reaching the midcorpus region of the epididymis have not completed all of the maturational changes requisite for successful fertilization; this immaturity is evidenced by decreased sperm motion and by impedance at the level of zona penetration.

Prediction of ovarian response to exogenous gonadotropin stimulation: utilization for collection of primate oocytes for fertilization in vitro.

The predictive value of 2 tests for ovarian response to controlled ovarian hyperstimulation in the cynomolgus monkey model was evaluated. The tests utilized were: 1) the cycle Day 3 (Day 1 = onset of menses) FSH value and 2) the acute estradiol (E(2)) response to a GnRH agonists (GnRHa) administered on Day 3. Both tests were performed during the cycle preceding control ovarian hyperstimulation. Subsequently, monkeys (n = 26) were stimulated with Metrodin(T) (Days 2-6, 25IU/d) and Pergonal(T) (Day 7 to hCG administration, 25IU/d). Laparoscopic oocyte retrieval was performed 32 to 34 after hCG administration. Analysis of the data revealed that Day 3 FSH values could not predict whether an animal would respond well to control ovarian hyperstimulation in a subsequent cycle (P = 0.77). However, the E(2) change 24 h post-GnRHa administration was significantly greater for animals responding well to control ovarian hyperstimulation compared with the animals deleted after 6 d of stimulation (P = 0.042). The mean change in E(2) levels in animals taken to aspiration was 97.8 pg/ml compared with only 21.6 pg/ml for the deleted animals. This differential response of E(2) production after GnRHa treatment was used to correctly identify (by discriminant analysis) 78% of the animals subsequently deleted for poor response. Thus, the increase in serum E(2) level after GnRHa, but not the basal FSH level, was found to be predictive of ovarian response to stimulation in the cynomolgus monkey.

Mechanism for species-specific induction of Leydig cell tumors in rats by lansoprazole.

Lansoprazole is a substituted benzimidazole which inhibits gastric acid secretion by inhibiting the hydrogen-potassium ATPase (proton pump) in the parietal cell. The finding of Leydig cell hyperplasia and Leydig cell tumors in 2-year oral studies in Sprague-Dawley rats but not in CD-1 mice prompted investigative studies to determine the mechanism for the Leydig cell changes. hCG challenge studies in Sprague-Dawley rats revealed decreased testosterone responsiveness in rats treated orally for 1 or 2 weeks with lansoprazole. After 4 weeks of daily oral treatment increases in serum LH and decreases in serum testosterone were detected within a few hours after dosing. In a study where 9-month-old male F344 rats were given testosterone supplementation via Silastic implants and then treated with lansoprazole for 6 months, a high incidence of Leydig cell tumors was seen in lansoprazole-treated, unsupplemented rats, whereas no Leydig cell tumors were seen in testosterone supplemented rats. This implied that reduction of the normal feedback inhibition at the level of the hypothalamus and/or pituitary due to reduced testosterone levels, thus giving rise to elevated levels of LH, was involved in the induction of Leydig cell tumors by lansoprazole. In vitro studies with Leydig cells from rats using various stimulators and precursors of testosterone biosynthesis demonstrated that the most sensitive site for inhibition of testosterone synthesis by lansoprazole is the transport of cholesterol to the cholesterol side chain cleavage enzyme. The IC50s for inhibition of LH or hCG-stimulated testosterone synthesis in Leydig cells from rats, mice, and monkeys were 11-12, 8, and 24.7 micrograms/ml, respectively. In vitro studies with metabolites of lansoprazole revealed that three metabolites were more potent inhibitors of testosterone synthesis than the parent drug, two of them being at least 10 times more potent. These metabolites are present in rats at substantial levels but are undetectable in humans. The lack of induction of Leydig cell tumors in mice, lower sensitivity of primate Leydig cells, and the absence of testosterone synthesis-inhibiting metabolites in man suggest that Leydig cell tumors found in rats represent a species-specific sensitivity and does not imply a risk for clinical use in man.

Estrogen and progesterone receptor mRNA are expressed in distinct pattern in male primate reproductive organs.

PURPOSE: The role(s) of estrogens (E) and progesterone (P) in male reproductive physiology remain unclear. Estrogens are used in the treatment of prostatic cancer. Progestins have been used to control excessive sexual behavior in men, and proposed as a male contraceptive. Previous immunohistochemical studies have shown that E receptors (ER) are present in the reproductive tract of male nonhuman primates. METHOD: We examined the expression pattern of ER and progesterone receptor (PR) mRNA in adult primate male reproductive tract. mRNA was extracted from male pituitary, testis, prostate and different regions of the epididymis of three intact adult cynomolgous monkeys. Ovarian, myometrial and spleen mRNA were used as controls. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify ER and PR mRNA; beta-actin mRNA was used as a reference. Primers for ER, PR and beta-actin were designed using the most conserved areas in the corresponding human cDNA sequences, and the identity of the PCR products was verified using Southern hybridization. Semiquantitative analysis of ER and PR mRNA content in different parts of the male reproductive tract was carried out by spiking the PCR reaction with 33P-dCTP, and amplifying the samples for 20 cycles with the beta-actin primers, whereas 30 cycles were used for ER and PR. RESULTS: The results are expressed as cpm ratios of ER or PR/beta-actin. All the male reproductive organs studied revealed a strong signal for ER and PR mRNA. The results of the semiquantitative analysis indicate that the expression of both ER and PR was highest in testis (mean +/- SE 6.4 +/- 1.3 and 0.5 +/- 0.1, respectively). The mean figures for prostate were 0.5 and 0.4, respectively. The mean content of ER and PR in the different areas of epididymis was 0.5 and 0.1, respectively. The epididymal ER mRNA was highest in the corpus region (ER/beta-actin 0.7), the ratio being 0.4 for the caput and cauda regions. The expression pattern of PR mRNA was different, and the caput of epididymis being the most intense (0.2). Surprisingly, the pituitary content of ER and PR mRNA was close to that seen in the ovary, the mean +/- SE values being 7.6 +/- 0.5 and 1.3 +/- 0.1, respectively. CONCLUSIONS: We, therefore, conclude that male monkey reproductive tract contains mRNA for ER and PR, and there appears to be regional variation in their expression. Thus the role(s) of Es and P in male reproductive physiology, specifically in sperm maturation, warrants further investigations.

Surveillance of waterborne disease in England and Wales.

A pilot scheme, designed to improve the information on waterborne disease available nationally, was set up in five health regions from October 1991 to March 1992. Consultants in communicable disease control were asked to report each month on confirmed and suspected cases of waterborne disease, and microbiological and other contamination incidents. Twelve events were reported to the PHLS Communicable Disease Surveillance Centre (CDSC) in six months: five involved human illness and seven were contamination incidents. Six other events were reported to CDSC from regions that did not take part in the scheme. The total number of events reported was small and epidemiological evidence that linked disease with water consumption was often weak or absent. Nevertheless, the scheme provided valuable information on events associated with water and would prove useful if it were established nationally, linked with guidance on the investigation of incidents.

Maturation antigens present in epididymis of cynomolgus monkeys (Macaca fascicularis).

PROBLEM: The present study was carried out to evaluate the changes in morphology and motility of spermatozoa retrieved from different regions of the epididymis of the cynomolgus monkey. The role of the epididymis in sperm maturation is assessed by assaying protein synthesis within different regions of the epididymis and by correlating these with changes in spermatozoal membrane surface components. METHOD: Spermatozoa retrieved from proximal caput (CP), midcorpus (CO), and distal cauda (CD) were assessed by morphological evaluation and computerized motion analysis. Membrane surface proteins of spermatozoa of different epididymal regions were extracted and separated on SDS-PAGE. Protein synthesis of different regions of the epididymis were assayed in vitro by [35S]-methionine incorporation. RESULTS: Spermatozoa obtained from different regions of the epididymis differed morphologically only in the location of the cytoplasmic droplet. Specifically, from caput to corpus to cauda, sperm steadily exhibited a more distal cytoplasmic droplet. When the motion parameters of velocity and amplitude of the lateral head were examined, CP spermatozoa were not progressively motile, and poor duration of movement was most noticeable for CO spermatozoa compared with CD spermatozoa. Membrane extracts from CP, CO, and CD epididymal monkey spermatozoa differed in only several protein bands. Three major polypeptide bands (19, 30, and 60 kD) that were absent from CP sperm were present in CO and CD sperm, with the latter showing increased intensity. Several polypeptides were lost from the sperm during epididymal transit: a 25-kD band was lost in CD sperm; and bands at 27 kD, 50-52 kD, and 90 kD were only present for CP sperm. Additionally, regional differences exist for proteins secreted by the cynomolgus monkey epididymis. Proteins (15, 25 kD) were only secreted in the CP region; a 38-kD protein increased in intensity from the CP to CD regions, whereas a 21-kD protein was absent from CD-secreted medium. CONCLUSION: These preliminary findings permitted the identification of several "maturational antigens" for cynomolgus monkey spermatozoa. Further characterization of these antigens that are modified during epididymal transit is warranted to determine their significance in the acquisition of progressive motility and fertilizing ability by epididymal spermatozoa.

Does endothelin-1 affect human spermatozoa function?

PROBLEM: It has been reported that massive amounts of immunoreactive endothelins (ETs) exist in human seminal plasma. However, the physiological role of ETs in seminal plasma remains to be determined. We speculated that ETs might affect sperm function. METHOD: The present study was designed to investigate the effect of endothelium-1 (ET-1) on: (a) sperm motion parameters, (b) hyperactivated motility, (c) sperm-zona pellucida-binding capacity, (d) sperm-oocyte penetration capacity, (e) acrosome reaction and its prerequisite, an increase in intracellular Ca2+ concentration ([Ca2+]i), and to examine (f) the presence of binding sites for ET-1 in human sperm. Forty-six semen ejaculates from 14 fertile men were assessed under capacitating conditions after separation of the motile sperm fraction by wash and swim-up. RESULTS: ET-1 (1 micron) exhibited significant stimulatory effects on sperm velocity at 30 min (P = 0.01), amplitude of lateral head displacement (ALH)max (P = 0.05), and ALHmean at 60 min (P = 0.04) in some samples (n = 10). However, these effects were not observed in experiments using a larger number of samples (n = 39). ET-1 had no effect on hyperactivated motility of sperm at 30 min to 24 h. Neither ET-3 nor IRL 1620, a selective ETB receptor agonist, affected sperm motion parameters or hyperactivated motility. ET-1 did not affect sperm-zona-binding capacity, sperm-oocyte penetration capacity, acrosome reaction, or [Ca2+]i of sperm. Specific binding sites for ET-1 were not detected on the cell surface of human sperm. CONCLUSIONS: Although ET-1 is present in massive amounts in human seminal plasma and may have the capacity to alter the quality of motile sperm in some samples, a physiological role of ET-1 in the modulation of the function of mature, ejaculated sperm still remains unknown.