Evaluation of DNA fragmentation of freeze-dried mouse sperm using a modified sperm chromatin structure assay

Freeze-dried sperm is applicable to the storage and transport of genetic material. We recently reported that freeze-dried mouse sperm required temperatures lower than −80 °C for long-term preservation and concluded that it was necessary to explore freeze-drying conditions before long-term preservation of sperm becomes viable. In the current study, we determined the percentage of sperm with elevated levels of DNA fragmentation using a sperm chromatin structure assay (SCSA), a technique not previously reported for the evaluation of freeze-dried mouse sperm. We applied SCSA to mouse sperm freeze-dried under four conditions (various combinations of primary drying pressure of 0.04 and 0.37 hPa and storage temperatures of 4 and −80 °C) and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. The DNA fragmentation index values under the four conditions determined by SCSA had good correlation with the developmental rate to the blastocyst stage of embryos from ICSI with freeze-dried mouse sperm. We concluded that the SCSA method applied to freeze-dried mouse sperm after storage will lead to not only clarification of the developmental rate derived from ICSI using freeze-dried sperm but also to improvements in the freeze-drying and storage processes.     

ATP-dependent movement of myosin in vitro: characterization of a quantitative assay

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP- dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP- dependent movement of myosin molecules on actin.     

Interactions of aged gametes: In vitro fertilization using in vitro-aged sperm and in vivo-aged ova in the mouse

A study of varying combinations of in vitro-aged sperm and in vivo-aged ova at 3 hr intervals from 0–24 hr resulted in failures at different steps of the fertilization process during in vitro fertilization of mouse ova. Significant decreases caused by sperm aging, ova aging, and sperm × ova aging interaction were found in sperm penetration. Pronuclear formation was not affected by sperm aging and was enhanced by ova aging, and there was a significant effect of sperm × ova aging interaction. Sperm aging significantly influenced the prometaphase stage of the fertilization process. Therefore, it is suggested that the detrimental fertilization effects resulting from aging gametes are due to different mechanisms in sperm and ova, that these mechanisms are affected at different times, and that they affect different steps in the fertilization process.     

Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose

A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.     

Possible involvement of a sialylated component of the sperm plasma membrane in sperm-zona interaction in the mouse

We have studied the molecular mechanisms of gamete interaction in vitro in the laboratory mouse, Mus musculus. In particular, we were interested in whether this interaction is similar to a lectin-hapten-mediated process. Inhibition of sperm-zona binding was examined using various concentrations (.25 mM to 50 mM) of different sugars (sialic acid α-methylmannose, glucose, fucose, galactose, and N-acetyl-glucosamine). Sperm-zona binding was significantly decreased when eggs were pretreated with 10 mM of sialic acid or α-methylmannose but not by other sugars tested. Furthermore, treatment of capacitated sperm with neuraminidase destroyed their ability to bind and fertilize eggs. We have also used a specific lectin for sialic acid from the horseshoe crab (Limulus polyphemus) to agglutinate mouse sperm. The lectin (.120 ng/ml) mediated agglutination of mouse sperm (10⁵ sperm/ml) was routinely observed to increase from a 10% agglutination immediately following their isolation from the epididymis to 100% agglutination 90 minutes later. Collectively, these results suggest the appearance of specific sugar moieties on the surface of the sperm plasma membrane which, in this particular species of mouse, are sialylated glycoproteins acting as ligands for specific receptors on the surface of the egg. These are the first data to indicate that sperm-egg recognition and attachment is a lectin-hapten-mediated process in the mouse.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.     

Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro

In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.     

Establishment of a short-term in vitro assay for mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.     

Gold nanoparticles disturb nuclear chromatin decondensation in mouse sperm in vitro

The effect of gold nanoparticles on mouse epididymal sperm has been studied using the model system of nuclear chromatin decondensation in vitro. It is shown that the treatment of gametes, preliminary membrane-freed by sodium dodecyl sulfate, in the mediums containing gold nanoparticles (with diameter ∼2.5 nm) in concentrations 1.0 × 10¹⁵ or 0.5 × 10¹⁵ particles/ml and following incubation in dithiothreitol solution (DTT) resulted in failure of chromatin decondensation process and nucleus structure. We conclude that gold nanoparticles possess spermatotoxicity. The mechanism of cytotoxic effect of gold nanoparticles may be related with their interaction with molecules of double-helix DNA. The model system studied in this research is applicable for further investigations of cytotoxic effects of nanoparticles of different origin and made of different metals.     

Modification of the quantitative DNA assay using adriamycin.

A modification of the adriamycin quantitative assay for DNA is presented which is sensitive to 1–20 μg DNA. The assay is simple, rapid, and highly reproducible but, contrary to previous claims, not specific for DNA, RNA interferes with the assay while protein does not. The mechanism and sensitivity of the assay is interpreted with reference to published data on adriamycin DNA binding.     

Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZP1, ZP2 and ZP3, having apparent molecular weights of 200,000, 120,000 and 83,000, respectively Bleil and Wassarman, 1978, Bleil and Wassarman, 1980a, Bleil and Wassarman, 1980b. In this investigation, ZP1, ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2-cell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2-cell embryos, reduced binding of sperm to as little as 10% of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-cell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-cell embryos to affect sperm binding. In no case did purified ZP1 and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm.     

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.     

Genetic influences on mouse sperm capacitation in vivo and in vitro

Intial in vivo studies were performed to observe the proportion of eggs fertillized at specific intervals after natural mating and ovulation in our research mouse colony. Proestrous females of the C57BL/10Wt, SJL/Wt inbred strains and the F₁ hybrid cross (B10 × SJL or reciprocals) were paired in the after-noon with males of their respective strain and examined for vaginal plugs at the midpoint of the dark period (2400 hours). Oviducts were periodically collected from mated females, and ovulation was first observed at 4, 5.2, and 3 hours after 2400 hours in the B10, SJL, and F₁ hyrid, respectively. The clutch of eggs from each ovulating female, was placed in culture, and cleavage oviduct lavage verifying female mating was placed in culture, and cleavage was used as the criterion for fertilizaition. Fifty percent of the eggs were fertilized 2.2, 5.0, and 2.5 hours after ovulation in B10, SJL, and F₁ hybrid females, respectively. Because twice the legth of time was required to fertilize a similar proportion of eggs from the SJL strain as the F₁ hybrid, these two strains were used for determining their rate of fertilization under more fully controlled conditions in vitro. Forty-nine percent of F₁ hybrid eggs were fertilized after 4 hours incubation with SJL epididymal sperm, whereas 53% fo SJL and 56% of F₁ hybrid eggs were fertilized after only 2 hours incubation with F₁ hybrid epididymal sperm. Thus, using sperm from these two mouse strains, the amount of time required to fertilize approximately 50% of the eggs within a clutch both in vivo and vitro was very similar. These observations demonstrte teh validity of using this in vitro system for fertilization studies and confirm that the temporal events in sperm capacitation and egg penetration are dependent on the genotype of the sperm. Similarities in fertilization rates at specific times after ovulation or insemination in vitro imply that the initiationof sperm capacitation in vivo occurs near the time of ovulation and several hours after mating. We tentatively suggest that follicular fluid may be required for completion of mouse sperm capacitaiton in vivo.     

Beyond the mouse model: Using Drosophila as a model for sperm interaction with the female reproductive tract

Although the fruit fly, Drosophila melanogaster, has emerged as a model system for human disease, its potential as a model for mammalian reproductive biology has not been fully exploited. Here we describe how Drosophila can be used to study the interactions between sperm and the female reproductive tract. Like many insects, Drosophila has two types of sperm storage organs, the spermatheca and seminal receptacle, whose ducts arise from the uterine wall. The spermatheca duct ends in a capsule-like structure surrounded by a layer of gland cells. In contrast, the seminal receptacle is a slender, blind-ended tubule. Recent studies suggest that the spermatheca is specialized for long-term storage, as well as sperm maturation, whereas the receptacle functions in short-term sperm storage. Here we discuss recent molecular and morphological analyses that highlight possible themes of gamete interaction with the female reproductive tract and draw comparison of sperm storage organ design in Drosophila and other animals, particularly mammals. Furthermore, we discuss how the study of multiple sperm storage organ types in Drosophila may help us identify factors essential for sperm viability and, moreover, factors that promote long-term sperm survivorship.