Biomedical subjects

M C Mahony

Publications and source records attributed to M C Mahony.

14 recordsLinked to original sources

PubMed1996

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.

Animals↗

PubMed1995

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.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1995

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.

Actins↗

PubMed1993

The specificity of human spermatozoa/zona pellucida interaction under hemizona assay conditions.

The objective of this prospective study was to evaluate the specificity of human sperm/zona pellucida interaction under hemizona assay (HZA) conditions in experiments with gametes from the same and different species. Human, cynomolgus monkey and hamster oocytes were used after salt-storage. Oocytes were bisected into matching hemizonae by micromanipulation and used in the HZA. Semen was obtained from healthy men (donors) and male cynomolgus monkeys and prepared by wash and swim-up. Sperm binding to matching hemizonae was assessed (tight binding) after 4-h coincubation in the HZA in homologous and interspecies experiments. Acrosome reaction was evaluated in the sperm droplets using FITC-PSA and on the hemizonae using the T-6 monoclonal antibody. On human hemizonae, the number of tightly bound sperm for human and monkey were 93.2 +/- 15.8 and 3.9 +/- 1.3, respectively (P < 0.001). On monkey hemizonae, the number of tightly bound sperm for monkey and human were 126.0 +/- 34.8 and 2.8 +/- 1.6, (P = 0.02) respectively. On hamster hemizonae, there was negligible binding of human and monkey sperm. There was a significantly higher incidence of acrosome reacted sperm on the zona pellucida in homologous compared to heterologous experiments. These results demonstrate a high species-specificity of human gamete functions under HZA conditions, providing further support for the use of this bioassay in infertility and contraception testing.

Acrosome↗

PubMed1993

Fucoidin binding activity and its localization on human spermatozoa.

We previously reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits: (1) tight binding of human sperm to human zona pellucida in vitro and (2) stimulation of the acrosome reaction by acid solubilized human zona pellucida. Here, we determined fucoidin binding activity on human spermatozoa and its localization on both live and permeabilized human sperm populations. A typical binding curve was demonstrated with biotinylated fucoidin. In competitive inhibition assays with unlabelled fucoidin or human sperm membrane extracts, IC50's were 4.0 micrograms/ml and 31.4 micrograms/ml, respectively. Fucoidin binding was localized over the acrosomal region of methanol-fixed human sperm and this pattern of binding significantly decreased from 92 +/- 3% to 74 +/- 6% with calcium ionophore A23187 treatment (p < 0.01). Binding of fucoidin-coated beads to live (non-permeabilized) human sperm was less than 1%. Addition of the detergent, Triton-X, to permeabilize sperm membranes resulted in a significant increase in binding (p = 0.001). These results provide evidence for the presence of a fucoidin binding compound in human spermatozoa that is localized to the membranes of the acrosomal region and can be extracted by a mild detergent extraction. Absence of binding by fucoidin to intact but not permeabilized spermatozoa suggests that the heteropolysaccharide binds to a receptor within the acrosomal matrix. However, further investigation is warranted to determine whether a fucoidin binding site is present both at the sperm's surface for the initial contact with the zona pellucida, and also for secondary binding after exposure of the acrosomal membranes.

Acrosome↗

PubMed1993

Functional and morphological features of spermatozoa microaspirated from the epididymal regions of cynomolgus monkeys (Macaca fascicularis).

In most species examined to date, spermatozoa mature during transit through the epididymis to become functionally competent to swim progressively, fertilize oocytes, and produce viable offspring. In the studies presented here, the status of spermatozoa obtained from the regions of the epididymis of the cynomolgus monkey (Macaca fascicularis) was examined. Epididymal fluid was collected by microaspiration from the caput, corpus, and cauda regions. The spermatozoa obtained were evaluated for morphology, motion parameters, and tight zona pellucida binding and were compared to spermatozoa collected by electroejaculation. Epididymal and ejaculated sperm differed morphologically only in the location of the cytoplasmic droplet. Motile sperm recovered by swim-up procedure exhibited a significantly higher proportion of sperm with more distal cytoplasmic droplets than the original aspirated samples (p < 0.02). Poor duration of movement was most noticeable for corpus spermatozoa, and to a lesser degree for cauda spermatozoa, when the motion parameters of velocity, linearity, and amplitude of the lateral head were examined. Zona pellucida binding was decreased only for sperm microaspirated from the caput region; sperm obtained from the corpus and cauda regions bound comparably to those collected via electroejaculation. These results suggest that by the time cynomolgus sperm reach the corpus region of the epididymis, they are functionally competent for tight zona binding. However, these nonhuman primate sperm may not complete the metabolic changes requisite for sustained sperm motility until reaching the most distal regions of the epididymis.

Animals↗

PubMed1991

Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa.

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.

Acrosome↗

PubMed1991

Evaluation of human sperm-zona pellucida tight binding by presence of monoclonal antibodies to sperm antigens.

Characterized WHO monoclonal antibodies (MAbs) to human sperm antigens were evaluated as to whether they inhibited sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the 26 MAbs tested, only one inhibited zona binding. The whole sperm-specific MAb inhibited zona binding by 70%. The MAb also caused strong agglutination. Two procedures, Sephadex column chromatography and papain digestion, were used to determine whether agglutination or steric hindrance was a factor in the capability of MAb to inhibit zona binding. However, inhibition remained comparable to previous results. The MAb did not prevent capacitation, nor calcium influx and the resulting increase in hyperactivated motility and acrosome reaction. Since its inhibitory influence is not due to agglutination factors, steric hindrance or prevention of normal pre-fertilization maturation, the MAb may be blocking a portion of the zona binding receptor and may be useful in elucidating sperm antigens important to sperm-egg interaction. The approach used in this study allows definition of sperm surface antigens involved in zona pellucida binding.

Antibodies, Monoclonal↗

PubMed1991

Inhibition of human sperm-zona pellucida tight binding in the presence of antisperm antibody positive polyclonal patient sera.

Patient sera previously characterized as containing high levels of IgG and IgA antisperm antibodies that bound to the sperm surface, most specifically the head region, were evaluated for their effect on sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the ten patient sera tested, 7 reduced zona binding by approximately half. Two of the most strongly inhibitory (greater than 70% inhibition) were examined for their effect on the prefertilization maturation of sperm. The patient sera did not affect sperm motion characteristics, or development of hyperactivated motility. However, in the presence of these sera some impedance was noted in calcium uptake after stimulation with human follicular fluid and in the acrosome reaction after calcium ionophore induction. Whether these two sera specifically affect sperm-zona pellucida binding or non-specifically affect the normal progression of capacitation remains to be eludicated.

Calcium↗

PubMed1991

Sites of antisperm antibody action.

Antisperm antibodies can affect sperm function in the cervical mucus, as they undergo capacitation, or during sperm-egg interaction. During capacitation, the sperm membrane destabilizes and antigens previously not available to antibodies may become exposed. Antibody attachment can reduce fertilization as measured by hamster egg preparation or the hemizona assay. A panel of monoclonal and polyclonal patient sera were tested for their ability to inhibit sperm-zona pellucida tight binding. We tested 25 monoclonals from a panel from the World Health Organization, but only nine were positive by indirect bead binding, indicating that the majority of the antibodies were not sperm surface antigens. The sera of 13 patients were used; three were negative and served as controls, nine had antibodies of the IgG isotype bound to 100% of the sperm heads examined, and seven also exhibited sperm specific IgA antibodies. Of the monoclonals tested, six inhibited zona binding by 60%. Sera of seven patients caused inhibition of greater than or equal to 50% zona binding; those from male patients caused the greatest inhibition. When further specific testing was conducted, one monoclonal caused the greatest inhibition of zona binding; sera of three patients inhibited zona binding by 70%. The antibodies, whether monoclonal or patient sera, were tested for their effect on capacitation. The ability to undergo hyperactivated motility after antibody exposure was assessed and no changes appeared due to antibody exposure. Serum from one of the patients appeared to affect sperm calcium influx to some extent. Sera from two patients appeared to reduce the number of spermatozoa capable of undergoing the acrosome reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination↗

PubMed1988

Evaluation of semen parameters by means of automated sperm motion analyzers.

Fresh semen specimens from 46 patients and donors were evaluated for concentration, motility, velocity, and linearity using two different commercially available computerized sperm motion analyzer systems. Although no significant differences in measurement of concentration or motility were observed, significant differences in velocity and linearity were recorded. Fourteen cryopreserved/thawed samples were assessed with the same set-up parameters as fresh specimens. When discrepancies between manual and computer counts were noted, the authors changed the set-up parameters and evaluated 33 additional specimens. Again, no differences in concentration and motility, but significant differences in velocity and linearity were observed. Interlaboratory results must be correlated and standardization of set-up parameters of various analyzers is essential.

Autoanalysis↗

PubMedDate not supplied

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.

Acrosome↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

Effect of fucoidin on human sperm-zona pellucida interactions.

The authors recently reported that fucoidin (a polymer of predominantly L-fucose sulfate) produced a strong, significant, and dose-dependent inhibition of sperm-zona binding under hemizona assay conditions. The current studies were designed to evaluate the mechanisms underlying this inhibitory activity. Using computerized semen analysis, the monoclonal anti-sperm antibody T-6 and indirect immunofluorescence technique, and the fura-2 indicator, no significant impact of fucoidin on sperm motion parameters, on the spontaneous acrosome reaction, or its prerequisite, the increase in calcium influx, were observed. Subsequently, a mild acid hydrolysis of the fucoidin molecule was performed, followed by sizing hydrolysates in a Biogel P2 column and separating fractions (n = 6). All fucoidin fragments significantly inhibited tight binding of human sperm to human zona pellucida under hemizona assay conditions (range, 56%-94%) when tested individually. These results provide further evidence that the effect of fucoidin is produced by a receptor-ligand association.

Analysis of Variance↗