Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.

The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome.     

Influence of partial deletion of the Y chromosome on mouse sperm phenotype

Two congenic strains of mice (control, B10.BR/SgSn; mutant, B10.BR-Ydel/Ms with partial deletion of the Y chromosome) were examined. In control males, 22.6% of spermatozoa had abnormal heads; in mutant males, there were 64.2%, the most common being heads with flat acrosomes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of mature sperm proteins, followed by acrosin assay and acrosome silver staining, revealed a reduced concentration of acrosin in acrosomal caps in 35.8% of the spermatozoa in mutant males. Electron microscope analysis showed that some of the round, early spermatids in the mutants had normally formed acrosomal caps but lacked the proacrosomal granule and had no, or only scarce, acrosomal material. These observations indicate that formation of the acrosomal cap is controlled separately from the synthesis of the acrosomal material and suggest that some factors linked on the Y chromosome are involved in the control of acrosome development.     

Sperm migration and selection in the reproductive tract of female mice is mostly affected by male genotype

The aim of the present study is to assess the influence of male and female genotypes on the transport of sperm to the site of fertilization. We mated B10.BR, B10.BR-Ydel and BALB/c males with B10.BR and BALB/c females and then analyzed the quality and quantity of spermatozoa found five hours post coitus in three successive parts of the female reproductive tract. We found that B10.BR and B10.BR-Ydel spermatozoa are very effectively selected by the uterotubaljunction and other barriers of the female genital tract. On the contrary, severely deformed BALB/c spermatozoa appeared to be able to cross selective barriers and were present both in oviducts and in cumulus oophorus. It cannot be excluded that these morphologically abnormal male gametes take part in fertilization. B10.BR-Ydel spermatozoa were very rarely observed above the uterotubaljunction. This shows that in vivo they migrate with delay and with difficulties pass the border between uterus and oviducts. This finding is in agreement with previous in vitro analyzes, which revealed many irregularities in movement of B10.BR-Ydel spermatozoa. Sperm quality and quantity in the reproductive tracts of B10.BR and BALB/c females were convergent if they were mated with males belonging to one strain, proving that migration and selection of spermatozoa in the female genital tract depend mostly on male genotype.     

Effect of Y chromosome and H-2 complex derived from Japanese wild mouse on sperm morphology

Segregation of sperm abnormality level and H-2 haplotypes was investigated in F2 hybrid males obtained from reciprocal crosses involving two B10.congenic strains carrying H-2 and the Y chromosome of Japanese wild mice: B10.MOL-OHM (H-2wm4, 23.1% of sperm abnormalities) and B10.MOL-OKB (H-2wm8, 11.1% of sperm abnormalities). In both types of crosses mean levels of abnormal spermatozoa were significantly higher for males typed as H-2wm4/H-2wm4 than for heterozygous H-2wm4/H-2wm8 or homozygous H-2wm8/H-2wm8. These results suggest that the gene for high sperm abnormality is linked to H-2 complex of the B10.MOL-OHM strain.     

Linkage between sperm abnormality level and major urinary protein phenotype in mice

Segregation of sperm abnormality level and the pattern of major urinary proteins (MUPs) were investigated in F2 and B1 hybrid males obtained from crosses involving two contrasting inbred strains of mice: CBA/Kw (Mup-1a1a, 3.3% abnormal sperm) and C57BL/Kw (Mup-1b1b, 21.9% abnormal sperm). In the progeny of both crosses mean levels of abnormal spermatozoa were significantly higher for males typed as Mup-1b1b than for heterozygous Mup-1a1b males. Moreover, all F2 hybrid males showing very high percentages of abnormal sperm were Mup-1b1b homozygotes. Similarly, among B1 males with a high level of deformed spermatozoa, a statistically significant majority were Mup-1b1b genotypes. Our results suggest that at least two genes which influence sperm abnormality level are segregating in these crosses. Both appear to be recessive for high sperm abnormality level, and one shows weak linkage to Mup-1 on chromosome 4.     

The influence of the deletion on the long arm of the Y chromosome on sperm motility in mice

The multicopy region on the long arm of the mouse Y chromosome contains four known genes. There are evidences that deletions in this region lead to decrease of sperm quality in mutant mice. Male mice completely lacking this region are infertile. Here we report results obtained by using the computer assisted semen analysis system (CASA), describing the movement parameters of spermatozoa from mutant males with partial deletion on the long arm of the Y chromosome (B10. BR-Y(del)). First we have determined that genes necessary for spermiogenesis and located in this region are still active in mutants, than we have compared the sperm movement of mutants and control animals. This analysis revealed that the Yq deletion affects: velocity parameters (VAP, VCL, VSL), parameters describing sperm head activity during movement (ALH and BCF) and linearity (LIN) of movement. Our findings indicate that sperm movement is controlled by genes located in the long arm of the Y chromosome.     

The effects of deletions of the mouse Y chromosome long arm on sperm function--intracytoplasmic sperm injection (ICSI)-based analysis

In mouse and man, Y chromosome deletions are frequently associated with spermatogenic defects. XY(Tdy)(m1)qdelSry males have an extensive Yq deletion that almost completely abolishes the expression of two gene families, Ssty and Sly, located within the male-specific region of the mouse Y long arm. These males exhibit severe sperm defects and sterility. XY(RIII)qdel males have a smaller interstitial Yq deletion, removing approximately two thirds of Ssty/Sly gene copies, and display an increased incidence of mild sperm head anomalies with impairment of fertility and an intriguing distortion in the sex ratio of offspring in favor of females. Here we used intracytoplasmic sperm injection (ICSI) to investigate the functional capacity of sperm from these Yq deletion males. Any selection related to the ability of sperm to fertilize in vitro is removed by ICSI, and we obtained two generations of live offspring from the infertile males. Genotyping of ICSI-derived offspring revealed that the Y(Tdym1)qdel deletion does not interfere with production of Y chromosome-bearing gametes, as judged from the frequency of Y chromosome transmission to the offspring. ICSI results for XY(RIII)qdel males also indicate that there is no deficiency of Y sperm production in this genotype, although the data show an excess of females following in vitro fertilization and natural mating. Our findings suggest that 1) Yq deletions in mice do not bias the primary sex ratio and 2) Y(RIII)qdel spermatozoa have poorer fertilizing ability than their X-bearing counterparts. Thus, a normal complement of the Ssty and/or Sly gene families on mouse Yq appears necessary for normal sperm function. Summary: ICSI was successfully used to reproduce infertile mice with Yq deletions, and the analysis of sperm function in obtained offspring demonstrated that gene families located within the deletion interval are necessary for normal sperm function.     

Abnormal genetic segregation associated with the presence of a Y. w+ chromosome in Drosophila melanogaster

We have observed an abnormal genetic segregation in the progeny of crosses between males of the F71 (y wa/Y.w+) strain and females of various strains carrying marker mutations on their chromosome 2. The Y.w+ chromosome, previously described as possibly being associated with a translocation of the 22D region of chromosome 2, was shown to carry the 21A1-22E4 tip of the 2L chromosome. One chromosome 2 of F71 had a deletion of this region. The abnormal genetic segregation observed in the progeny of different crosses can be explained both by the partial lethality (which becomes severe in some homogeneous genetic backgrounds) due to trisomy of the 21A1-22E4 chromosome 2 fragment and by the lethality associated with monosomy of this 21A1-22E4 segment.     

Disruption of genetic interaction between two autosomal regions and the X chromosome causes reproductive isolation between mouse strains derived from different subspecies

Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F1 hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX(MSM), which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX(MSM) males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX(MSM) background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX(MSM) spermatozoa have a defect in penetration through the zona pellucida of eggs.     

Low incidence of chromosome aberrations in spermatozoa of fertile boars

Chromosomal imbalance in gametes and embryos is one of the factors contributing to early embryonic mortality. Although the rate of chromosomally abnormal sperm cells is low and usually does not exceed 1%, there is no clear indication of fertilizing potential of such gametes. The aim of the experiment was to investigate the type and incidence of numerical chromosomal aberrations in spermatozoa produced by fertile boars used in artificial insemination (AI). We used the protocol of fluorescent in situ hybridization (FISH) on sperm interphase nuclei with molecular probes for porcine chromosome pairs 1 and 10. Altogether 12?348 sperm cells were examined. Disomy was observed in spermatozoa of all seven AI boars whereas only one diploid cell was identified in all screened sperm cells. The average rate of chromosomally unbalanced sperm was 0.105% (13/12 348) with an inter-individual variation from 0.048% to 0.194%. Among abnormal sperm cells, both disomy (0.097%) and diploidy (0.008%) were detected. Nullisomy was not included into calculations. The estimated aneuploidy rate calculated by doubling the number of disomic cells was 0.194%. Chromosome pair 10 was significantly more often involved in non-disjunction (75%, 9/12 aneuploid sperm cells) than chromosome pair 1 (25%, 3/12). We have shown for the pig that the rate of disomic cells falls into a range presented by other authors, whereas that of diploid spermatozoa appeared to be lower in the present study. In conclusion, numerical chromosome aberrations were present in spermatozoa of all AI boars analyzed in this study. Therefore, it can be assumed that the presence of unbalanced spermatozoa at the level observed in fertile males does not significantly affect their reproductive potential.     

Hybrid breakdown caused by substitution of the X chromosome between two mouse subspecies

Hybrid breakdown is a type of reproductive failure that appears after the F2 generation of crosses between different species or subspecies. It is caused by incompatibility between interacting genes. Genetic analysis of hybrid breakdown, particularly in higher animals, has been hampered by its complex nature (i.e., it involves more than two genes, and the phenotype is recessive). We studied hybrid breakdown using a new consomic strain, C57BL/6J-X(MSM), in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereas F1 hybrids between C57BL/6J and MSM/Ms are completely fertile. The C57BL/6J-X(MSM) males showed reduced testis weight with variable defects in spermatogenesis and abnormal sperm head morphology. We conducted quantitative trait locus (QTL) analysis for these traits to map the X-linked genetic factors responsible for the sterility. This analysis successfully detected at least three distinct loci for the sperm head morphology and one for the testis weight. This study revealed that incompatibility of interactions of X-linked gene(s) with autosomal and/or Y-linked gene(s) causes the hybrid breakdown between the genetically distant C57BL/6J and MSM/Ms strains.     

The contribution of the y chromosome to hybrid male sterility in house mice

Hybrid sterility in the heterogametic sex is a common feature of speciation in animals. In house mice, the contribution of the Mus musculus musculus X chromosome to hybrid male sterility is large. It is not known, however, whether F(1) male sterility is caused by X-Y or X-autosome incompatibilities or a combination of both. We investigated the contribution of the M. musculus domesticus Y chromosome to hybrid male sterility in a cross between wild-derived strains in which males with a M. m. musculus X chromosome and M. m. domesticus Y chromosome are partially sterile, while males from the reciprocal cross are reproductively normal. We used eight X introgression lines to combine different X chromosome genotypes with different Y chromosomes on an F(1) autosomal background, and we measured a suite of male reproductive traits. Reproductive deficits were observed in most F(1) males, regardless of Y chromosome genotype. Nonetheless, we found evidence for a negative interaction between the M. m. domesticus Y and an interval on the M. m. musculus X that resulted in abnormal sperm morphology. Therefore, although F(1) male sterility appears to be caused mainly by X-autosome incompatibilities, X-Y incompatibilities contribute to some aspects of sterility.     

X-Y chromosome dissociation in mouse strains difering in efficiency of spermatogenesis: Elevated frequency of univalents in pubertal males

Dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I spermatocytes of adult mice was significantly more frequent in the CBA strain (29%) than in C57, KP, or KE strains (7–11%). Autosome dissociatio (1–5%) involved only the smallest chromosome pairs. Eleyatedfrequency of X-Y dissociation in the CBA strain correlates with significantly lower testes weight and lower yield of spermatogenesis, which suggests that sex bivalent dissociation man be responsible for some loss of spermatogenic cells. However, sperm quality is not affected, the percentage of normal spermatozoa and their fertlizing capacity being higher in CBA thatn in the remaining strains. Two congenic strains, KE and KE. CBA (the latter with the Y chromosome introduced from CBA), had the same level of X-Y dissociatios, suggesting that the Y chromosome plays no rle in the determination of this character. In comparison with adult males pubertal (27–29 day-old) males had twice as hig a frequency of X-Y dissociation in KE an KP strains, and combined frequeicies of dissociated sex and autosome bivalents were significantly higher in pubertal males of all tested strains. Although te level of chromosome dissociation is not sufficient to explain increased mortality of germ cells observed in pubertal males, it could be one of the contributing factors.     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.     

Interstrain competition amongst mouse spermatozoa inseminated in various proportions, as affected by the genotype of the Y chromosome

Nonagouti (KP X C57BL)F1 hybrid females were artificially inseminated with a mixture of spermatozoa from males of the KE (nonagouti) and CBA (agouti) strains and the genotype of young was estimated by fur pigmentation. When KE and CBA spermatozoa mixed in the ratios of 1:1, 2:1 and 4:1 were inseminated after ovulation, 87%, 56% and 29% of progeny, respectively, were sired by CBA males, i.e. proportions of CBA progeny were significantly higher than ratios of CBA spermatozoa in the mixture. The surplus of CBA progeny was significantly less in females inseminated before ovulation, which may suggest that more rapid capacitation of CBA spermatozoa is partly responsible for their competitive advantage. In preparations from oviducal flushings of females killed 2-3 h after insemination, CBA spermatozoa (recognized by their shape) were found in similar proportions as in the inseminated mixture. There was therefore no evidence of their preferential selection at the uterotubal junction. No competitive advantage of CBA spermatozoa occurred when they were inseminated with spermatozoa from males of the KE.CBA strain, congenic with KE but with the Y chromosome derived from the CBA strain. This indicates that genetic factors linked with the Y chromosome may influence competitive ability of spermatozoa.     

Intracytoplasmic sperm injection effects in infertile azh mutant mice

Several reports in the literature describe men with infertility resulting from abnormal sperm head shape or decapitation defects of their spermatozoa. These defects are similar to those shown for the spermatozoa from azh (abnormal spermatozoon head shape) mice. The present study examines the efficiency and effects of intracytoplasmic sperm injection (ICSI) in successive generations of azh mice generated with this method. Three successive generations of azh mice were produced with ICSI. In all three ICSI series, more than 80% of 2-cell embryos were obtained, and more than 35% of embryos transferred gave rise to normal live offspring. In addition, ICSI was used to cross homozygous azh/azh males with homozygous azh/azh females, and live offspring were obtained. The ICSI-derived males were tested for their fecundity and abnormalities of sperm morphology. Spermatozoa from ICSI-derived azh/+ males did not show any impairment of fecundity in in vitro fertilization. These spermatozoa successfully fertilized oocytes from both C57BL/6 and B6D2F1 females, with fertilization rates ranging from 70%- 92%. The proportion of morphologically normal spermatozoa was similar in azh/+ males from three successive generations of ICSI (57.8%, 54.8%, and 49.0%, respectively), and no differences were noted when comparing ICSI-derived males with males derived by mating (57.6%) and with wild-type controls (61.6%). Detailed analysis differentiating between specific types of anomalies of sperm morphology did not reveal significant differences among the examined groups. The results of the present study demonstrate that ICSI does not enhance the azh mutation phenotype in the offspring and brings no risks when applied continuously. Moreover, serial (successive generations) ICSI is highly efficient in maintaining valuable mice with fertility problems.     

Comparison of immune responses to diphtheria and tetanus toxoids of various mouse strains

Immune responses of 11 mouse strains with known genetical characteristics and two outbred strains to diphtheria and to tetanus toxoids were compared. Both diphtheria and tetanus antitoxins were titrated by passive hemagglutination. From the pattern of the immune response, the mouse strains tested may be classified into four groups. [1] Strains ddY (SPF) and ddY (conv) and those with haplotype H-2b, such as C57BL/6 and C57BL/10, were high responders to both toxoids. [2] Strains with H-2d, such as BALB/c, B10.D2 and DBA/2Cr, were intermediate responders to both toxoids. [3] Strains with H-2k, H-2a or, H-2m, such as C3H/He, B10.BR, B10.BR/SgSn, B10.A/SgSnJ and B10.AKM/O1a, were high responders to diphtheria toxoid but low responders to tetanus toxoid. [4] The strain with H-2h4, B10.A (4R), was a poor responder to both toxoids.     

Fluorescence in situ hybridization with Y chromosome-specific probe in decondensed bovine spermatozoa

This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreitol (DTT) and glutathione (GSH) with or without heparin supplementation. Concentrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used with 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially that of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), however, could induce reliable, repeatable detection of the Y chromosome signal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa.     

Male-Sex-Ratio Trait in Drosophila Pseudoobscura: Frequency of Autosomal Aneuploid Sperm

Males with the SR X chromosome show the "sex-ratio" (sr) phenotype in which they produce almost entirely daughters. The few sons (about 1%) are invariably sterile X/O males and result entirely from nullo-XY sperm. The "male-sex-ratio" (msr) phenotype is a modified form of sr in which SR/Y males produce a higher frequency of sterile X/O sons. The msr trait is due to the presence of the SR X-chromosome in males which are also homozygous for one or more autosomes from the L116 strain. Here the frequency of nullo-3 and diplo-3 sperm from msr males was measured by crossing to a compound-3 strain and found to be 13.8% and 3.2%, respectively, of the total viable sperm. The sr males produced very low levels of nullo-3 sperm at a frequency not different from control X/Y males and a slightly elevated frequency of diplo-3 sperm over X/Y males. The msr males were found to have only 12% the fecundity of sr males and in matings to cause a high frequency of brown inviable eggs. These results indicate that high rates of autosomal aneuploidy are not restricted to chromosome 3 but also occur for chromosomes 2, 4 and 5. The overall frequency of autosomal aneuploid sperm is estimated to be approximately 50%. Microscopic studies of meiosis in testes from msr males indicates meiotic nondisjunction and meiotic chromosome loss are responsible for the msr phenotype. Last, microscopic studies of sperm cysts from msr males reveal high levels of spermiogenic failure.