Biomedical subjects

S P Flaherty

Publications and source records attributed to S P Flaherty.

At least 37 records · Page 2Linked to original sources

PubMed1994

Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence in-situ hybridization.

The aim of this study was to evaluate the efficacy of enriching X-bearing human spermatozoa using 12-step (25-80%) discontinuous Percoll gradients. X- and Y-bearing spermatozoa were simultaneously identified in neat semen (controls) and in 80% Percoll fractions from the same samples using double-label fluorescence in-situ hybridization and chromosome-specific DNA probes. Hybridization and labelling efficiencies of 95-99% were obtained in all samples. The mean ratio of X- to Y-bearing spermatozoa in the controls was 49.0:48.2, whereas there was a significant enrichment (P < 0.0001) of X-bearing spermatozoa in the Percoll fractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratio varied from 1.1-1.5 in individual Percoll samples. There were no significant differences in the proportions of aneuploid spermatozoa (XX, YY, XY) between the control and Percoll fractions. We conclude that 12-step discontinuous Percoll gradients enrich X-bearing spermatozoa, but the degree of enrichment is insufficient for use in preconceptional sex selection.

Aneuploidy↗

PubMed1994

The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa.

The usefulness of sub-zonal injection (SZI) for the treatment of severe male factor infertility has been restricted by low and unpredictable fertilization rates and the high risk of polyspermy after the injection of multiple spermatozoa. In this prospective study, we have evaluated whether sperm morphology and the percentage of acrosome-reacted spermatozoa at the time of injection can be used to predict SZI fertilization outcomes. Populations of motile spermatozoa equivalent to those injected were collected from the medium/oil interface immediately after SZI of each cohort of oocytes. Morphology was assessed using the World Health Organization 1987 criteria and the acrosomal status of spermatozoa was determined after staining with rhodamine-conjugated Pisum sativum agglutinin. A fertilization index (FI) was calculated to express the actual fertilizing potential of the spermatozoa injected. In all, 67 patients underwent 72 SZI cycles. The overall fertilization and polyspermy rates were 36 and 47% respectively, and a clinical pregnancy rate per transfer of 22% was achieved. Linear regression analysis demonstrated a statistically significant relationship between morphology and the FI (r = 0.506, P < 0.0001). Patients with < or = 10% normal morphology always had a FI < or = 10%, and this was reflected by low fertilization and polyspermy rates and the high number (32%) of cycles with complete failure of fertilization in this group. In patients with > 10% normal morphology, there were two patterns: low (< or = 10% FI) or high (> or = 10% FI) fertility. This was evident in the fertilization (23 and 85%, respectively), and polyspermy (25 and 68%, respectively) rates of these two patient sub-groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗

PubMed1994

A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47,XYY male.

Two semen samples from a 47,XXY male were examined using chromosome-specific DNA probes and fluorescent in situ hybridization (FISH) to determine the distribution of sex chromosomes and an autosome (chromosome 17) in the sperm. A motile population of sperm was also prepared from one sample using the swim-up technique to compare the motile and total sperm populations. Chromosomes were localized using single FISH and a biotinylated chromosome 17 probe (TR17), or double FISH using a biotinylated X chromosome probe (TRX) and a digoxigenin-labelled Y chromosome probe (HRY). Labelling efficiencies were 95-98%. Ploidy levels were estimated by measurement against a microscope eye-piece graticule. The overall ratio of X- to Y-bearing sperm was 47% to 48.4% in the neat samples, and 48.4% to 45.3% in the swim-up fraction. Neither of the ratios was significantly different from 1:1. The frequencies of monosomic and disomic (but otherwise haploid sperm) were not different from the frequencies we observed in normal donors. In contrast, the frequencies of both diploid and tetraploid cells were increased in the neat samples of the XYY male. In the swim-up fractions, however, none of these parameters differed from those of ten normal semen donors. These results support the hypothesis that the extra Y chromosome in XYY men is eliminated during spermatogenesis.

Adult↗

PubMed1993

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization.

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.

Adult↗

PubMed1993

Biotinylation of proteins on the surface of zona-free mouse oocytes.

In this study, we have labelled proteins on the surface of unfertilized, zona-free mouse oocytes using a nonisotopic biotinylation procedure. The zona pellucida was weakened by brief incubation in chymotrypsin and removed by mechanical pipetting through a narrow-bore glass pipette. Surface proteins were labelled using sulfo-NHS-biotin (sulfosuccinimidobiotin), a water-soluble, membrane-impermeable biotinylation reagent. The distribution of biotinylated proteins on the oocyte surface was assessed by fluorescence microscopy using streptavidin-FITC. Bright fluorescence was noted on the surface of the oocyte, except in a circular region overlying the meiotic spindle where the fluorescence was weak or absent. The intensity of fluorescence was markedly reduced by incubation of biotinylated oocytes in trypsin (1 mg/ml) or chymotrypsin (2 mg/ml), and in vitro fertilization experiments showed that biotinylation did not compromise the fertilizability of the oocytes. The biotinylated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using streptavidin-HRP and enhanced chemiluminescence (ECL) detection. The most prominent biotinylated proteins were of M(r) 82 and 69 kD, but other major proteins of M(r) 93, 78, 61, 52, 49, 40, 28, and 22 kD were detected, as well as 14 minor proteins of M(r) 18-100 kD. The major bands could be detected in fewer than 50 oocytes. This biotinylation procedure is fast, versatile and sensitive, and it is therefore an excellent tool for studying proteins exposed on the surface of mammalian oocytes and embryos.

Animals↗

PubMed1993

A role for the WH-30 protein in sperm-sperm adhesion during rouleaux formation in the guinea pig.

Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic cross-bridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 micrograms/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (whole-head pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in the molecular interactions that maintain guinea pig sperm in rouleaux.

Animals↗

PubMed1993

Prediction of in vitro fertilization rates from semen variables.

OBJECTIVE: To assess the value of semen variables for predicting fertilization rates. DESIGN: Measures of the fresh semen and the motile sperm fraction used for insemination were related to the fertilization rate by multiple regression analysis. The regression model was then used to construct a two-dimensional clinical chart. SETTING: University-affiliated reproductive medicine unit. PATIENTS: The results of 294 IVF cycles were analyzed retrospectively. Selection criteria were: [1] first cycle of IVF; [2] tubal and/or male factor infertility; and [3] four or more oocytes inseminated. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The fertilization rate was related to measured variables of the fresh semen and the motile sperm fraction used for insemination. Fertilization rate was categorized as poor (< 35%) or acceptable (> or = 35%). RESULTS: Multiple regression analysis demonstrated a strong correlation between the fertilization rate and the combined indexes of percentage normal morphology and grade of motility in the fresh semen and percentage progressive motility in the motile sperm fraction. A two-dimensional chart that expressed these relationships was constructed. Its accuracy of prediction was 77% for poor fertilization and 95% for acceptable fertilization. CONCLUSIONS: The fertilization rate is strongly correlated with percentage normal sperm morphology in the fresh semen and the percentage progressive motility in the motile sperm fraction used for insemination. The clinical chart provides a simple but powerful tool for predicting fertilization outcome.

Confidence Intervals↗

PubMed1993

Detection of X- and Y-bearing human spermatozoa after motile sperm isolation by swim-up.

OBJECTIVE: To assess the ratio of X- to Y-bearing human spermatozoa in motile fractions isolated by the swim-up technique. DESIGN: The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in motile fractions isolated from the same samples by swim-up. X- and Y-bearing sperm were simultaneously identified using chromosome-specific DNA probes and double fluorescence in situ hybridization. SETTING: Hospital-based university department. PARTICIPANTS: Ten healthy donors with normal semen characteristics. MAIN OUTCOME MEASURES: The distribution of haploid cells (X or Y), normal size cells with two sex chromosome (XX, YY, or XY), and large cells containing two (XX, YY, or XY) or four (XXYY) sex chromosomes were measured in neat semen samples and in motile fractions prepared by swim-up. RESULTS: Overall, 95% of sperm in the neat semen and swim-up fractions were labeled with the probes. The ratios of X- to Y-bearing sperm were 47.3:46.9 (neat semen) and 48.4:47.1 (swim-up fractions), which were not significantly different from a 1:1 ratio. The frequencies of sperm with normal size nuclei and two sex chromosomes (XX, YY, or XY) in the swim-up fractions were not significantly different from the controls, but there was a significant reduction in the proportion of cells with large nuclei and two (XX, YY, or XY) or four (XXYY) sex chromosomes in the swim-up fractions. CONCLUSIONS: The swim-up technique does not selectively enrich either X- or Y-bearing sperm. Because the isolation of motile spermatozoa is an important procedure for routine IUI, IVF-ET, and GIFT, the results of this study are important reassurance that the sex ratio is not altered by this method of sperm preparation.

Cell Nucleus↗

PubMed1993

Proteases are not involved in the membrane fusion events of the lysolecithin-mediated guinea pig sperm acrosome reaction.

The guinea pig sperm acrosome reaction is characterized by a complex temporal and structural pattern of membrane fusions. In this study, we have used specific protease inhibitors to determine if proteases regulate this pattern of membrane fusions during the lysolecithin-mediated guinea pig sperm acrosome reaction. Inhibitors were chosen so as to cover a wide range of different types of proteases, and all were used at the highest concentration that did not adversely affect sperm motility. Of the eight inhibitors tested, leupeptin, soya bean trypsin inhibitor (SBTI), p-aminobenzamidine (pAB) and nitrophenyl p'-guanidino benzoate (NPGB) inhibited completion of the acrosome reaction, while diethylenetriaminepentaacetic acid (DTPA), phosphoramidon, bestatin and pepstatin had no effect. Sperm that had been acrosome-reacted in the presence of each inhibitor were examined by transmission electron microscopy to assess whether the inhibitors altered the pattern of membrane fusions during the acrosome reaction. DTPA, phosphoramidon, bestatin and pepstatin had no effect on membrane fusion or matrix dispersal. Serine protease inhibitors such as leupeptin, SBTI, pAB and NPGB prevented complete dispersal of the acrosomal matrix and completion of the acrosome reaction, but did not alter the temporal sequence or structural pattern of membrane fusions. The undispersed matrix was present along the dorsal and ventral aspects of the apical segment and throughout the principal segment. We conclude that proteases are not involved in regulating the temporal and structural pattern of membrane fusions which occurs during the lysolecithin-mediated acrosome reaction of guinea pig sperm.

Acrosome↗

PubMed1992

Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.

Aneuploidy↗

PubMed1991

Ultrastructural analysis of the acrosome reaction in a population of single guinea pig sperm.

Cauda epididymal guinea pig spermatozoa are arranged in rouleaux, with the sperm heads stacked one on top of the other; the plasma membranes over the apical segment of the acrosomes of adjacent sperm are linked and form non-fusigenic "junctional" zones. A complex structural and temporal sequence of membrane fusions occurs during the acrosome reaction of guinea pig sperm in rouleaux. In this study, we have devised a procedure for dispersing the rouleaux and isolating a population of single, motile guinea pig sperm, and have investigated the ultrastructural features of the acrosome reaction in single sperm to determine if the pattern of membrane fusions is different from sperm in rouleaux. The rouleaux were dispersed using trypsin, and damaged cells were removed by passing the sperm suspension through a glass bead column; a population of 70-90% motile, acrosome-intact, single sperm was obtained. Sperm were then induced to undergo lysolecithin-mediated, "synchronous" acrosome reactions, and processed for transmission electron microscopy. The acrosome reaction involved a complex sequence of membrane fusions between the plasma membrane (PM) and outer acrosomal membrane (OAM). On the convex surface of the apical segment, sheets of hybrid membrane and parallel arrays of hybrid membrane tubules formed; filaments were associated with the luminal surface of the residual OAM in these regions. Hybrid membrane vesicles were produced on the concave surface of the apical segment, but fusion was delayed relative to the convex surface. In the principal segment, branching arrays of hybrid membrane tubules formed and later vesiculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗

PubMed1991

A prospective trial of intrauterine insemination of motile spermatozoa versus timed intercourse.

STUDY OBJECTIVE: The efficacy of intrauterine insemination (IUI) of selected motile sperm. DESIGN: Prospective randomized sequential alternating cycle trial comparing IUI with luteinizing hormone (LH)-timed intercourse. SETTING: Clinical infertility service. PATIENTS: Couples selected included unexplained infertility (n = 73), cervical mucus hostility (n = 24), moderate semen defect (n = 110), and severe semen defect (n = 78). Two hundred eighty-five couples undertook 600 IUI cycles and 505 LH-timed intercourse. RESULTS: Overall, IUI was slightly more effective than LH-timed intercourse with a pregnancy rate of 6.2% versus 3.4% per cycle. When individual categories were considered only, IUI for severe semen defect was significantly better (5.6% versus 1.3%, P less than 0.05). The first IUI cycle was more effective when compared with both subsequent IUI cycles and the initial LH-timed cycle. Overall, 74% (27/37) of IUI pregnancies occurred in the first cycle. CONCLUSIONS: Compared with LH-timed intercourse, IUI provided little or no improved expectation of pregnancy but was beneficial in couples with severe semen defect. The occurrence of pregnancy was limited per cycle and confined essentially to the initial cycle of treatment. Continued IUI is considered to be unrewarding.

Cervix Mucus↗

PubMed1989

Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents.

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus-oocyte complexes were obtained from two species that possess a three-hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron-dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 microns) in P. australis, which has a three-hooked sperm head, and thickest (11.4 microns) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus-oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three-hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.

Animals↗

PubMed1988

Membrane domains in guinea pig sperm and their role in the membrane fusion events of the acrosome reaction.

In this study, we have examined the structure of domains of the periacrosomal plasma membrane (PM) and outer acrosomal membrane (OAM) of guinea pig sperm and defined their fate during the membrane fusion events of the acrosome reaction. Cauda epididymal sperm were arranged in rouleaux, joined by periacrosomal PM "junctional" zones; in these zones, the PMs were linked by cross bridges formed from a paracrystalline glycocalyx. Bridging elements linked the PM to the OAM on the ventral (concave) but not dorsal (convex) aspect of the apical segment. Parallel filaments were associated with the luminal face of the OAM overlying the dorsal surface of the apical segment. Sperm were induced to undergo a "synchronous" acrosome reaction after preincubation in Ca2+-free medium containing lysolecithin, by the addition of Ca2+. Fusion between the OAM and PM occurred at the boundaries but not within the PM "junctional" zones over the apical segment. In nonjunctional regions on the dorsal surface of the apical segment, sheets of unfenestrated hybrid membranes and parallel arrays of hybrid membrane tubules formed, while branching arrays of hybrid membrane tubules and vesicles were observed on the ventral surface. In the principal segment, networks of branching hybrid membrane tubules initially formed but later transformed into vesicles. Hence, the lysolecithin-mediated guinea pig sperm acrosome reaction involves a complex sequence of membrane fusions, which differs in domains of the periacrosomal PM and OAM. Stable nonfusigenic domains are present in both the PM and OAM of the apical segment; membrane-associated assemblies may maintain these domains and may also provide direction to some of the membrane fusion events of the acrosome reaction.

Acrosome↗

PubMed1988

Localization of actin in human, bull, rabbit, and hamster sperm by immunoelectron microscopy.

Mammalian spermatozoa have previously been shown to contain actin, but its subcellular localization and function have not been elucidated. In this study, actin has been localized at the ultrastructural level in human, bull, rabbit, and golden hamster spermatozoa by a monoclonal antiactin antibody and a preembedding immunogold labeling technique. Specific labeling was localized 1) around the connecting piece in the neck region of sperm from all four species, although a species-specific pattern was evident; 2) on the external surface of the fibrous sheath of human sperm; 3) in the perinuclear space underlying the postacrosomal sheath of bull and rabbit sperm; and 4) between the plasma membrane and outer acrosomal membrane along the concave margin of the hamster sperm head. SDS-PAGE and Western blots immunostained with the monoclonal antibody confirmed the presence of actin in SDS extracts of Percoll-purified sperm from each species.

Actins↗

PubMed1987

Further ultrastructural observations on the sperm head of the plains mouse, Pseudomys australis (Rodentia: Muridae).

Ultrastructural and histochemical techniques have been utilized to study selected aspects of the fine structure of the three-hooked sperm head of the plains mouse, Pseudomys australis. The peripheral layer of the two ventral hooks was found to consist of a continuation of the postacrosomal dense lamina. Parallel ridges connected the dense lamina and plasma membrane in the postacrosomal region and ventral hooks. Both these regions stained intensely with silver nitrate. The distribution of actin filaments in the hooks was investigated using NBD-phallacidin. Fluorescence was more intense in the apical regions of the ventral hooks, and two bands of fluorescence extended caudally from their base. It was also shown that the equatorial segment of the acrosome extended onto the dorsal hook. The structural features of the three hooks are discussed in relation to their possible functional significance.

Actins↗

PubMed1987

Formation of the ventral hooks on the sperm head of the plains mouse, Pseudomys australis.

The sperm head of the plains mouse, Pseudomys australis, has three curved hooks projecting from its anterior margin. The two ventral hooks have previously been shown to consist largely of an extension of the subacrosomal material. To characterize further the structure and composition of the ventral hooks, we have examined their formation during spermiogenesis using transmission electron microscopy, silver staining, and actin localization with NBD-phallacidin. The ventral hooks develop as an extension of the perinuclear space and postacrosomal dense lamina on the anteroventral margin of the sperm head. Bundles of 6-nm-thick filaments appear in the core of each hook; these are probably actin filaments based on staining of the hooks with NBD-phallacidin. Just prior to spermiation, electron-dense material condenses in the core of the ventral hooks and concurrently in the perinuclear space in the remainder of the sperm head. The two ventral hooks thus appear to consist of a core of perinuclear material and actin filaments, which is enclosed by a continuation of the postacrosomal dense lamina.

Actins↗

PubMed1987

Cytoskeletal assemblies of mammalian spermatozoa.

It is becoming increasingly evident that the different domains of the mammalian spermatozoa possess distinct cytoskeletal assemblies. In this paper we have discussed three assemblies, found in the acrosomal, postacrosomal, and midpiece segment, respectively. Each has a distinct substructure and associates with specific sperm-membrane systems. Their protein compositions are currently unidentified, and they may be comprised of sperm-specific polypeptides. Analysis of their formation and fate during sperm-egg interaction should provide valuable insight into their role in the development of cell polarity and in the membrane-mediated steps of fertilization.

Acrosome↗