Hyperactivation of mammalian sperm.

Mammalian sperm commonly show hyperactivated motility just before fertilization. The movement of hyperactivated sperm appears different in fluids of different viscosity and elasticity and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Hyperactivation may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none have yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, divergent pathways regulate the two events.     

Zona drilling enhances fertilization by mouse caput epididymal sperm

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.     

Hyperactivation of hamster sperm motility by temperature-dependent tyrosine phosphorylation of an 80-kDa protein.

Protein phosphorylation and dephosphorylation are believed to play key roles in regulation of sperm motility. Here we examine the effect of temperature on hamster sperm motility and protein tyrosine phosphorylation status. As in previous work, a decrease from 37 degrees C to 22 degrees C caused loss of hyperactivated motility. We now find that cooling also produces a dephosphorylation of several 48-80-kDa flagellar peptides. A return to 37 degrees C restored hyperactivation but resulted in rephosphorylation of only an 80-kDa protein. Conversely, hyperactivation and phosphorylation of the 80-kDa component were insensitive to incubation temperature for sperm incubated with the protein phosphatase inhibitor, calyculin A, or for sperm demembranated by detergent extraction. These results strongly indicate that the temperature-sensitive tyrosine phosphorylation status of an 80-kDa sperm flagellar peptide explains the sensitivity of hyperactivation to temperature.     

Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes

To study the repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes, the potentiating effects of 3 well-known repair inhibitors, arabinofuranosyl cytosine (ara-C), 3-aminobenzamide (3AB) and caffeine, on the frequency of induced chromosome aberrations were examined in eggs fertilized with X-irradiated sperm or in eggs irradiated with X-rays at the mature oocyte stage immediately before fertilization. Gametic treatment, fertilization and embryo culture were carried out in vitro. Ara-C treatment was done only in the pre-DNA replication period, while treatment with 3AB and caffeine was continuous from fertilization to the first-cleavage metaphase. The induction of chromosome aberrations by exposing sperm or oocytes to X-rays was remarkably potentiated by post-treatment incubation in the presence of each of the 3 inhibitors. This result indicates the possibility that X-ray damage induced in sperm or oocytes is reparable in the fertilized eggs and that various types of repair processes are involved.     

Physical characteristics of mouse sperm nuclei.

The nuclei of epididymal sperm, isolated from C57BL/6J and CBA/J inbred mice by their resistance to trypsin digestion, retain the shape differences of the intact sperm head. Various physical characteristics of these nuclei were measured and compared. The measurement of the projected dimensions of nuclei showed that the CBA nuclei are 13.5% longer than C57BL/6 nuclei (8.64 +/- 0.02 mum compared with 7.61 +/- 0.02 mum), 0.8% narrower (3.51 +/- 0.01 vs. 3.54 +/-0.01 mum) with 6.8% more area (22.34 +/- 0.10 vs. 20.91 +/- 0.09 mum2). However, the volumes of the nuclei as based on reconstructing calibrated electronmicrographs of serial sections of the nuclei indicated that CBA are about 7% smaller than C57BL/6 nuclei (3.72 +/- 0.08 vs. 4.01 +/- 0.03 mum3). The buoyant density of the CBA nuclei is 1.435 +/- 0.002 g/cm3 compared with 1.433 +/- 0.002 g/cm3 for the C57BL/6 nuclei as determined on linear CsCl and Renografin-76 density gradients and confirmed by a technique utilizing physiological tonicities. Therefore, the average mass of the CBA nuclei is less than that of the C57BL/6 nuclei (5.34 +/- 0.12 vs. 5.75 +/- 0.05 pg). The sedimentation velocities at unit gravity of nuclei from 11 inbred strains differ over a range of more than 6% with CBA nuclei sedimenting about 2.0% more slowly than C57BL/6 nuclei. We show that for these nuclei the sedimentation velocity can be related to their buoyant density, volume and a sedimentation shape factor. Within the errors of our measurements of these various characteristics, it was found that C57BL/6 and CBA nuclei have similar sedimentation shape factors. Therefore, the difference in sedimentation velocity between these nuclei appears to be primarily a result of differences in volume. The possible applications of these techniques to the physical separation of sperm are evaluated in the discussion.     

Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility.

The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.     

Quantitative fluorometry of abnormal mouse sperm nuclei.

An extensive quantitative analysis of deformed mouse spermatozoa was undertaken. Improvements over previous studies included the isolation and purification of sperm nuclei, a multifaceted analytical approach using several fluorochromes and the analysis of individual nuclei classified into shape categories. Malformed sperm nuclei in BALB/c mice could not be distinguished from normal ones in terms of total and basic proteins, sulfhydryl and disulfide group concentration, DNA concentration and chromatin organization. The shape of sperm nuclei is therefore probably determined by the manner in which the internal biochemical components are assembled.     

Hyperactivated sperm progress in the mouse oviduct.

Sperm from naturally mated mice were observed and videotaped moving within mouse oviducts. The typical pattern of sperm progress involved intermittently breaking free and swimming a short distance, then reattaching to the epithelium. The proportion of sperm that swam freely (were not attached to the epithelium) was calculated and analyzed for effects of oviductal region, ovulation status, and sperm location relative to the lumen. A significantly higher proportion of sperm were free in the ampulla than in the isthmus (26.3% +/- 0.8% vs. 11.8% +/- 1.0%; p less than 0.0001) and in post-ovulatory than pre-ovulatory (16.2% +/- 2.0% vs. 10.6% +/- 1.6%; p less than 0.05) oviducts. Flagellar curvature ratio values showed that free sperm (0.716 +/- 0.024) had more sharply curved tails than stuck sperm (0.782 +/- 0.013). While this difference is significant (p = 0.01), the effect of attachment status interacted significantly (p less than 0.05) with the oviductal region such that there was a greater difference in the isthmus than in the ampulla. Only sperm using the more curved tail beats of hyperactivation were seen to break free from the epithelium and to progress along the oviduct. These results indicate that hyperactivation plays a role in moving sperm out of the isthmic reservoir and to the site of fertilization.     

Methyl parathion-induced sperm shape abnormalities in mouse.

Metacid 50, the commercial grade of methyl parathion (O,O-dimethyl-O-4-nitrophenyl phosphorothionate), a commonly used organophosphorus insecticide, was tested for its genotoxicity in Swiss albino mice using the sperm abnormality assay. Sperms of albino mice were examined at two time intervals, 1 week and 5 weeks after a single acute oral treatment with the pesticide at four dose levels, viz., 75.0, 37.5, 18.75 and 9.375 mg/kg body weight corresponding to 1/2 LD50, 1/4 LD50, 1/8 LD50 and 1/16 LD50 values respectively. A dose-related statistically significant increase in the percentage of abnormal sperm observed indicates the genotoxic potency of methyl parathion.     

Life history of mouse sperm protein. Intratesticular stages

A basic protein fraction, migrating as a single band in acetic acid-urea gel, distinct from histones, was isolated from mouse sperm collected from vasa deferentia and caudae epididymides and was used to immunize female rabbits. The presence of antibodies to the mouse sperm protein (MSP) in the rabbit antisera was demonstrated by a cytoimmunofluorescence procedure using the cells of origin of the antigenic protein, the mature mouse sperm. The specificity of the antisera was verified by fluid and gel precipitation tests and by crossed immunoelectrophoresis. The latter procedure demonstrated the presence of two antigen-antibody systems, consonant with earlier reports that the basic chromosomal protein of mouse sperm is heterogeneous. MSP antigen in situ was recognized by the specific antibodies of the rabbit antisera only after the smear of mature sperm was treated with either of two reducing agents: 2-mercaptoethanol or dithiothreitol. However, when the immunofluorescence procedure was applied to untreated smears of mouse testicular cells, spermatids of all stages from 1 to 14-15 were positive, while spermatocytes, stage 16 spermatids and spermatozoa were negative. After treatment of testes smears with reducing agent, only spermatocytes remained negative. Those observations indicate the following: (a) MSP is immunogenic in a heterologous species; (b) its antigenic sites are detectable in spermatozoa and spermatids of all stages, but not in primary spermatocytes; (c) those antigenic sites become masked at about stage 15 of spermiogenesis and may be unmasked by treatment with a reducing agent. The interpretation is made, therefore, that one or more components of MSP are assembled at the beginning of spermiogenesis and undergo an alteration in the final intratesticular stage of spermatid maturation. That alteration may be presumed to be the formation of disulfide linkages between the cysteine residues.     

Selenoprotein P is required for mouse sperm development

Selenoprotein P (SEPP1), an extracellular glycoprotein of unknown function, is a unique member of the selenoprotein family that, depending on species, contains 10-17 selenocysteines in its primary structure; in contrast, all other family members contain a single selenocysteine residue. The SEPP1-null (Sepp1(-/-)) male but not the female mice are infertile, but the cellular basis of this male phenotype has not been defined. In this study, we demonstrate that mature spermatozoa of Sepp1(-/-) males display a specific set of flagellar structural defects that develop temporally during spermiogenesis and after testicular maturation in the epididymis. The flagellar defects include a development of a truncated mitochondrial sheath, an extrusion of a specific set of axonemal microtubules and outer dense fibers from the principal piece, and ultimately a hairpin-like bend formation at the midpiece-principal piece junction. The sperm defects found in Sepp1(-/-) males appear to be the same as those observed in wild-type (Sepp1(+/+)) males fed a low selenium diet. Supplementation of dietary selenium levels for Sepp1(-/-) males neither reverses the development of sperm defects nor restores fertility. These data demonstrate that SEPP1 is required for development of functional spermatozoa and indicate that it is an essential component of the selenium delivery pathway for developing germ cells.     

Microsatellite genotyping for genetic quality testing using sperm cells in the mouse.

We attempted to determine the number of sperm cells required for genotyping of one microsatellite marker. The crude genomic DNA extracted from about 760 or more sperm cells gave sufficient quantity of PCR product using a 20 microl-scale PCR. We also studied the effects of non-ionic detergents on extraction of crude sperm genomic DNA. PCR products amplified with the crude sperm genomic DNA extracted using the lysis buffer supplemented with non-ionic detergents showed much clear bands. In conclusion, our results suggest that a small part of the frozen sperm, which is less than 1/10 of the original volume (10 microl), provides sufficient quantity of template DNA for genetic quality testing.     

Localization of sphingosine kinase-1 in mouse sperm acrosomes.

Sphingosine kinase (SPHK) catalyzes sphingosine phosphorylation to form a bioactive lipid mediator, sphingosine-1-phosphate (S1P). In the current study, we report the presence of SPHK-1 in mouse spermatozoa. SPHK-1 was localized to the acrosomes of spermatozoa, and its expression was proven by RT-PCR and Western blot analysis. SPHK activity of mouse spermatozoa was 18.1 pmol/min/mg protein. Furthermore, we identified the presence of the S1P receptors S1P1, S1P2, S1P3, and S1P5, in mouse spermatozoa by RT-PCR. These results suggest that S1P produced by SPHK-1 would play a role in the acrosomal reaction through S1P receptors.     

Absence of selection against aneuploid mouse sperm at fertilization.

Is there selection against aneuploid sperm during spermatogenesis and fertilization? To address this question, we used male mice doubly heterozygous for the Robertsonian (Rb) translocations Rb(6. 16)24Lub and Rb(16.17)7Bnr, which produce high levels of sperm aneuploid for chromosome 16, the mouse counterpart of human chromosome 21. The frequencies of aneuploid male gametes before and after fertilization were compared by analyzing approximately 500 meiosis II spermatocytes and approximately 500 first-cleavage zygotes using fluorescence in situ hybridization with a DNA painting probe mixture containing three biotin-labeled probes specific for chromosomes 8, 16, and 17 plus a digoxigenin-labeled probe specific for chromosome Y. Hyperhaploidy for chromosome 16 occurred in 20.0% of spermatocytes and in 21.8% of zygotes. Hypohaploidy for chromosome 16 occurred in 17.0% and 16.7% of spermatocytes and zygotes, respectively. In addition, there was no preferential association between chromosome 16 aneuploidy and either of the sex chromosomes, nor was there an elevation in aneuploidy for chromosomes not involved in the Rb translocations. These findings provide direct evidence that there is no selection against aneuploid sperm during spermiogenesis, fertilization, and the first cell cycle of zygotic development.     

Sonication of mouse sperm membranes reveals distinct protein domains.

Molecular interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized. To begin to characterize sperm components that are involved in sperm-ZP interactions, we isolated and density fractionated sperm membranes. The membrane fractions recovered from a density fractionation protocol were characterized, and sonication was compared with vortexing for preparation of sperm membranes by examining the distribution of proteins in the membrane fractions obtained from these 2 protocols. Biochemical and microscopic analyses were used to determine the composition of the sonicated membrane fractions, and immunoblotting was used to identify fractions containing some of the previously suggested ZP3 receptors. Transmission electron microscopy revealed that bands 1-3 contained membrane vesicles and band 4 contained axonemal and midpiece fragments. SDS-PAGE revealed that bands 1 and 2 shared many proteins, but band 3 contained a number of unique proteins. Surface labeling with 125I demonstrated that bands 1 and 2 contained the majority of the sperm surface protein markers, whereas band 3 contained minor amounts of surface markers. Lectin-binding characteristics of sperm membrane glycoproteins were used to compare the relative distribution of glycosylated proteins in vortexed or sonicated membrane preparations. These characterizations indicate that sonication enhanced the differential distribution of sperm membrane proteins among the density fractions and suggests that this method is preferable for preparation of membrane fractions to be used for identification of proteins that mediate sperm-egg interactions.     

Chromosomal abnormalities and the morphology of mouse sperm heads.

In the mouse, numerous mutagens, teratogens and carcinogens have been shown to induce marked elevations in the fraction of sperm with head shape abnormalities. Since carcinogens and teratogens may act by causing genetic damage, a likely explanation of these results is that the sperm abnormalities are also caused by genetic damage. There are two more or less distinct classes of genetic damage, chromosomal aberrations and point mutations. In this paper, we provide evidence, that in general, chromosomal aberrations are not responsible for causing abnormally shaped sperm. Chromosomal aberrations could have caused abnormal sperm morphology in a number of ways. One possibility was that the mere presence of a translocated chromosome within the germ cell led to the malformation of the sperm head. A second possibility was that chromosomal imbalance, i.e., aneuploidy, duplications or deficiencies, within the spermatid or haploid cells caused abnormalities in shape. We tested these hypotheses by measuring the level of abnormally shaped sperm in mice homozygous and heterozygous for 24 various reciprocal and Robertsonian translocations. The diploid cells of these mice are known to be chromosomally balanced, containing translocated chromosomes. A predictable proportion of their gametes are, however, chromosomally unbalanced and carry translocated chromosomes. It was found that the levels of sperm abnormalities in these mice were convincingly unrelated to the levels predicted by any of the above hypotheses. Based on these results it seems that sperm abnormalities in mice are not due to the mere presence of translocated chromosomes in germ cells and also not due to chromosomal aneuploidy or duplication-deficiencies of chromosomal segments in the spermatid during development of the sperm.     

A shuttle system for the transfer of reducing equivalents in mouse sperm mitochondria.

Studies have been carried out on an $\text{in vitro}$ reconstituted system composed of mouse lactate dehydrogenase isozyme X or C4, branched chain amino acid aminotransferase, NAD, alpha-hydroxy isocaproate, glutamate and mouse sperm mitochondria. This system demonstrated capacity for the oxidation of extramitochondrial NADH. It is proposed that a branched chain alpha-hydroxyacid / amino acid shuttle for the transfer of reducing equivalents from cytosol to mitochondria may be functional in mouse spermatozoa.     

A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida

Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha- lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta- N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.     

Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.