Showing papers on "Sperm published in 1994"

Associations between Body Size, Mating Pattern, Testis Size and Sperm Lengths across Butterflies

Abstract: This paper investigates mechanisms of sperm competition by comparing reproductive characteristics across 74 butterfly species. Testis size scales with body size and, after controlling for this allometry, relative testis size increases with risk of sperm competition, as defined by female mating frequency. Both eupyrene (fertilizing) and apyrene (non-fertile) sperm lengths correlate positively with body size. After controlling for body size, relative eupyrene sperm lengths are greater in species where males experience higher risks of sperm competition. These results suggest that sperm competition in butterflies selects for increased investment in spermatogenesis, and specifically longer fertilizing sperm. Because longer sperm may be faster and more powerful, eupyrene sperm may therefore compete energetically, and are not selected to be minimally sized to maximize numbers for a purely raffle-based sperm competition mode. Apyrene sperm lengths are not affected directly by risk of encountering rival sperm. Instead, apyrene sperm show closer associations with body size which, if female tract morphometry correlates with body size, is consistent with the hypothesis that apyrene sperm retard female sexual receptivity by moving while in storage.

Mesoscale sperm handling devices

Abstract: Devices and methods are provided for the clinical analysis of a sperm sample. The devices include a solid substrate, typically on the order of a few millimeters thick and approximately 0.2 to 2.0 centimeters square, microfabricated to define a sample inlet port and a mesoscale flow channel extending from the inlet port. In one embodiment, a sperm sample is applied to the inlet port, and the competitive migration of the sperm sample through the mesoscale flow channel is detected to serve as an indicator of sperm motility. In another embodiment, the substrate of the device is microfabricated with a sperm inlet port, an egg nesting chamber, and an elongate mesoscale flow channel communicating between the egg nesting chamber and the inlet port. In this embodiment, a sperm sample is applied to the inlet port, and the sperm in the sample are permitted to competitively migrate from the inlet port through the channel to the egg nesting chamber, where in vitro fertilization occurs. The devices of the invention may be used in a wide range of applications in the analysis of a sperm sample, including the analysis of sperm morphology or motility, to assess sperm binding properties, and for in vitro fertilization.

A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.

Abstract: A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.

Human oocyte activation after intracytoplasmic sperm injection

Abstract: Oocyte activation is a series of events triggered by the fertilizing spermatozoon and necessary for the beginning of the embryonic development. Calcium plays a pivotal role in this process. Here we used confocal laser scanning microscopy to examine the changes in the concentration of intracellular free calcium ([Ca2+]i) in human oocytes after intracytoplasmic sperm injection (ICSI). The first considerable but short (< 2 min) increase in [Ca2+]i was detected immediately after the penetration of the microinjection needle into the ooplasm. This rise by itself did not provoke oocyte activation and was also obtained after the injection of medium without spermatozoa. After a lag period of 4-12 h, oocytes that were subsequently activated initiated a second period of [Ca2+]i changes. These changes were sperm-dependent and followed one of two alternative patterns, a non-oscillatory one and an oscillatory one. The non-oscillatory pattern resembled the changes described previously during parthenogenetic activation of mammalian oocytes. The oscillatory pattern was similar to the changes accompanying normal fertilization in different mammalian species. It is concluded that the initial [Ca2+]i rise provoked by the ICSI procedure is not responsible for oocyte activation, and that a release of a sperm factor(s) is required to initiate this process.

A free radical theory of male infertility

Abstract: There is a growing body of evidence to indicate that a significant factor in the aetiology of male infertility involves a loss of sperm function as a consequence of oxidative stress. This stress originates from the excessive generation of reactive oxygen species by the spermatozoa and results in the peroxidation of unsaturated fatty acids in the sperm plasma membrane. It is possible that reactive oxygen species originating from infiltrating leucocytes could also stress the spermatozoa although the protective properties of seminal plasma would render this unlikely in vivo. Whatever the source of the reactive oxygen species, the lipid peroxides thereby generated exhibit powerful negative correlations with the movement characteristics of the spermatozoa and their capacity for sperm-oocyte fusion. These findings should have important implications for the development of rational techniques for the diagnosis and treatment of male infertility.

Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation.

Abstract: In human in vitro fertilization (I.V.F.), it was first assumed that all the embryos obtained had the same developmental potential whatever the quality of sperm. However, this has not been confirmed. We have used the coculture technique and determined the blastocyst formation rate in three groups of patients: group 1: patients with normal sperm count (> 20 x 10(6)/ml), motility (> 30%), and morphology (> 50%); group 2: patients treated by I.V.F. with frozen donor sperm; group 3: patients with severely impaired sperm quality (< 3 x 10(6) forward motile and morphologically normal spermatozoa per ml). In group 1, we found a strong correlation between cleavage rate and blastocyst formation rate (P < 0.0001) with a blastocyst formation rate comprised between 40% and 50%. This was not true for the two other groups for which the overall number of blastocysts obtained and the number of patients having at least one blastocyst were severely reduced (P < 0.0001). These data are discussed in terms of DNA quality, timing of formation of the pronuclei, and delays in cell cycles at the time of genomic activation. These observations lead to a new approach to the study of fertilizing ability of poor quality sperm. It may help in the decision as to whether couples treated for male infertility should be excluded from I.V.F. protocols.

Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide.

Abstract: A new membrane-permeant DNA stain, SYBR-14, was used in combination with propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR-14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR-14 resulted in the nuclei of living sperm fluorescing bright green. Aliquots containing nearly all living bovine sperm were prepared using glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20.7, and 1.4 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES-0.1% bovine serum albumin and incubated for 0, 3, 6, and 24 hours at 36 degrees C to assess changes in cell viability. As cell death occurred during this incubation period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR-14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of these samples, which had been processed and stored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.

Birth of normal young after electrofusion of mouse oocytes with round spermatids

Abstract: Normally, round spermatids, the youngest male germ cells with a set of haploid chromosomes, cannot fertilize mature oocytes. However, when mouse spermatids were fused with oocytes, some of the resulting zygotes developed into normal fertile mice of either sex. This demonstrates that the nuclei of spermatids can provide the paternally imprinted chromosomes needed for full embryonic development and also that the complex postmeiotic modifications involved in sperm formation in the testis and their maturation in the epididymis merely serve to facilitate natural delivery of the paternal genome. This finding may find an application in the treatment of male infertility due to defective spermiogenesis/sperm maturation.

Male Gametic Strategies: Sperm Size, Testes Size, and the Allocation of Ejaculate Among Successive Mates by the Sperm-Limited Fly Drosophila pachea and Its Relatives

Abstract: The gametic strategy of males comprises the amount of energy invested per sperm, the total amount invested in sperm production, and the pattern of sperm allocation among successive reproductive bouts. All of these variables were measured for each of the four species constituting the nannoptera species group of the Drosophilidae. Extreme interspecific variation was identified for all variables and enigmatic male reproductive strategies, including submaximal insemination of females, partitioning of ejaculate among successive mates, and production of few large sperm, were observed. Variation among species in female remating behavior was found to occur concomitantly with male remating behavior, probably because of female fertility demands. Relationships among testes size, sperm size, sperm numbers, and mating systems in these fruit flies are examined. These relationships are not consistent with patterns identified in studies of vertebrate taxa and suggest fundamental differences between vertebrates and invert...

Intracytoplasmic sperm injection.

Abstract: Intracytoplasmic sperm injection (ICSI) involves the insertion of a single spermatozoon into the oocyte, bypassing all the egg coat penetration and gamete fusion steps characteristic of natural fertilization. This was first achieved in the sea urchin, in the mouse, and later in hamster eggs. This micromanipulation approach was also plagued by oocyte injury and lysis, with only about 30% of injected mouse eggs surviving the procedure. Because the sperm-egg fusion step is bypassed in ICSI, male pronucleus development generally required oocyte activation in most species tested. This was achieved by the vigorous suction of ooplasm prior to sperm nucleus insertion or by exposure to A23187. The first ICSI offspring were obtained in the rabbit following the transfer of sperm-injected eggs into the oviduct of a pseudopregnant female, with a bovine live birth reported soon thereafter. Although applied to human gametes some years earlier, the first human pregnancies from ICSI was established only in 1992, and since then, hundreds of thousands of ICSI babies have been born. ICSI has made conception and parenthood possible for couples with many forms of male factor infertility and at rates similar to those in patients treated by standard IVF with apparently normal gametes.

Leukocytic infiltration into the human ejaculate and its association with semen quality, oxidative stress, and sperm function

Abstract: Immunocytochemical techniques have been used to monitor the size and composition of the leukocyte population in unfractionated human semen samples and sperm populations generated by Percoll gradient centrifugation. The characteristics of the leukocyte population have then been related to the quality of the semen profile, the production of reactive oxygen species, and the functional competence of the spermatozoa. A majority (97%) of the ejaculates examined contained leukocytes, and in 82.4% the major cell type was the granulocyte. Small numbers of T cells, B cells, and monocytes/macrophages could also be found in 62%, 43%, and 21% of samples, respectively, and patients were occasionally identified in whom one of these cell types became the predominant leukocyte species. Although a subpopulation of patients was identified in whom the infiltration of multiple leukocyte species was positively correlated with the concentrations of spermatozoa and precursor germ cells in semen, in general, the presence of leukocytes, to the point of leukocytospermia, did not significantly influence any component of the semen profile. Similarly, the fertilizing potential of the washed spermatozoa, as assessed by in vitro tests of the acrosome reaction and sperm-oocyte fusion, was not correlated with the concentration of seminal leukocytes. In contrast, the carryover of leukocytes into the washed sperm preparations profoundly influenced the fertilizing potential of the spermatozoa via mechanisms that were associated with the production of reactive oxygen species. These results have implications for the diagnostic significance of leukocyte contamination in the context of male infertility and assisted conception.

Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions.

Abstract: In this study, we have examined the relationship between creatine phosphokinase (CPK), a biochemical measure of human sperm quality (Huszar et al., 1988a,b, 1990; Huszar and Vigue, 1994), and a marker for the presence of residual cytoplasm in human spermatozoa, glucose-6-phosphate dehydrogenase (G6PDH). We then determined whether the diagnostic potential of these enzymes was related to the capacity of the sperm suspensions to generate reactive oxygen species (ROS) and/or the presence of leukocytes and precursor germ cells. Across the data set as a whole, G6PDH and CPK were highly correlated with each other and, to a lesser extent, with the generation of ROS. Contamination of the sperm suspensions with leukocytes might have contributed to these associations, since the presence of such cells was also significantly correlated with CPK, G6PDH, and ROS. However, even after the leukocytes had been carefully removed, G6PDH was still highly correlated with CPK (r = 0.794), indicating that both criteria were providing similar information of the cytosolic component of human sperm suspensions. In the absence of leukocyte contamination, CPK and G6PDH activities were also correlated with the presence of precursor germ cells, and this association may, in part, explain the diagnostic value of these criteria. An additional component of their prognostic value may be reflected in the statistically significant association observed between G6PDH activity and ROS generation. A possible mechanism for such an association is suggested, which should be amenable to experimental verification.

Sperm Economy in a Coral Reef Fish, Thalassoma Bifasciatum

Abstract: In many coral reef fishes, males face the evolutionary problems of how to allocate sperm among frequent, daily mating in order to maximize the number of eggs they fertilize. A method is developed and tested for collecting and counting the number of sperm and eggs released during separate spawns of the coral reef fish Thalassoma bifasciatum. The method was used to examine the pattern of sperm allocation for pair— and group—spawning males. The number of sperm released by pair—spawning males varied positively with the number of eggs released by their female mates and with female body size. The data suggest that males economize on sperm release by providing the minimum amount of sperm needed to fertilize the egg clutch of the female partner. Fertilization efficiency (the number of eggs fertilized by a given number of sperm) was higher with large females than it was with small females. Males differed significantly among themselves in sperm output per spawn, with some males consistently releasing more sperm than other males with same—sized females. Male—male differences were not due to differences in male body size, quality of the spawning site, or the apparent degree of water movement. The number of sperm released per spawn did not decline throughout the daily spawning period, a pattern that disproved one but not all possible patterns of sperm depletion. In group spawns, the total number of sperm released and the number released per male were respectively 50 and 6 times the number released in pair spawns, on average. Both of these measures increased significantly with the clutch size of the spawning female. Overall, sperm production is probably sufficiently costly that males have been selected to allocate sperm carefully among their frequent daily spawns.

Sperm displacement without sperm transfer in drosophila melanogaster.

Abstract: In this paper we show that when Drosophila melanogaster females are mated twice, the semen of the second male causes a reduction of the effective number of resident sperm from the previous mating. This is demonstrated by two different kinds of experiments. In one set of experiments, mated females were remated to two different kinds of sterile males, one with normal semen and the other with deficient semen. The effect on the resident sperm was determined from the number of remaining progeny after mating to the sterile male, with the result that the normal semen reduced the amount of resident sperm in comparison with matings to the males with deficient semen. The second set of experiments employed interrupted matings. These experiments were based on the observation that semen is delivered before sperm during the first 5 min of copulation. The second matings were interrupted instantly, 2 min, and 4 min after the initiation of copulation. Compared to the instant interruptions, the two later interruptions had the effect of reducing the amount of resident sperm. The results of these two experiments clearly indicate that a sperm-incapacitation process plays a role in the well-documented phenomenon of sperm displacement (last-male advantage) in this species. Such a process could play a role in sperm displacement in the many cases where the mechanism is unknown.

Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction

Abstract: Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening.

Sperm-egg recognition in the mouse : characterization of sp56, a sperm protein having specific affinity for ZP3

Abstract: Recognition between mammalian gametes occurs when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding eggs. ZP3, one of three glycoproteins in the ZP, is the egg protein recognized by sperm. A mouse sperm surface protein, sp56 (M(r) = 56,000), has been identified on the basis of its specific affinity for ZP3 (Bleil, J. D., and P. M. Wassarman. 1990. Proc. Natl. Acad. Sci. USA. 87:5563-5567). Studies presented here were designed to characterize mouse sperm sp56 and to further test whether or not this protein specifically recognizes ZP3. sp56 was purified by both ZP3 affinity chromatography and by ion exchange chromatography followed by size-exclusion chromatography. The purified native protein eluted from size-exclusion columns as a homomultimer (M(r) approximately 110,000). Each monomer of the protein contains intramolecular disulfide bonds, consistent with its extracellular location. Immunohistochemical and immunoblotting studies, using monoclonal antibodies, demonstrated that sp56 is a peripheral membrane protein located on the outer surface of the sperm head plasma membrane, precisely where sperm bind ZP3. Results of crosslinking experiments demonstrated that the ZP3 oligosaccharide recognized by sperm has specific affinity for sp56. Collectively, these results suggest that sp56 may be the sperm protein responsible for sperm-egg recognition in the mouse.

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte

Abstract: By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.