Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution

The purpose of the present study was to investigate the impact of carcinogenic polycyclic aromatic hydrocarbons and volatile organic compounds on sperm quality in a group of city policemen in Prague during a period of increased concentrations of ambient air-pollutants (winter season) compared to a period of low exposure (spring). Polymorphisms in metabolic genes (CYP1A1, EPHX1, GSTM1, GSTP1, GSTT1), folic acid metabolism genes (MTR, MTHFR) and DNA repair genes (XRCC1, XPD6, XPD23, hOGG1) were evaluated in these men as potential modifiers of associations between air pollution exposure and changes in sperm quality. The study population was a group of 47 policemen working in the center of the city. Seasonal differences in exposure were verified by ambient and personal monitoring. Markers of sperm injury included semen volume, sperm concentration, sperm morphology, sperm motility, and sperm DNA damage measured with the sperm chromatin structure assay The sperm chromatin structure assay (SCSA) includes a measure of DNA damage called DNA Fragmentation Index (DFI). The % of cells with detectable DFI (detDFI) by this assay includes sperm with either medium or high DNA damage; the term hDFI is used to define the % of sperm with only high DNA damage. The assay also detects immature sperm defined by high density staining (HDS).No significant differences were found in any of the standard semen parameters between the sampling periods except for vitality of sperms. Both DFI and HDS were significantly higher in winter than in spring samples for all men and for non-smokers. At the bivariate level, significant associations between hDFI or detDFI and polymorphisms of the repair genes XRCC1, XPD6 and XPD23 were observed. In multivariate models, polymorphisms of the genes XPD6, XPD23 and CYP1A1MspI were associated with hDFI and HDS. Moreover, HDS was significantly associated with polymorphisms in GSTM1 gene.     

Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen

An association between sperm DNA integrity and fertility was recently shown for frozen–thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n = 20) in SMEY and Triladyl^® (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen–thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n = 20) and Holstein (n = 20) semen diluted in Triladyl^® during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 °C) and after freezing–thawing of NRF semen in SMEY, but only after freezing–thawing for NRF semen diluted in Triladyl^®. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl^®, while the degree of damage was higher in Triladyl^®, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl^® after 9 h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl^® maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.     

Major morphological sperm abnormalities in the bull are related to sperm DNA damage

The influence of sperm morphology and chromatin integrity on bull fertility suggests a strong but undefined biological relationship between these two parameters. In this study we explore this relationship, making use of the Sperm Chromatin Dispersion test, which allows simultaneous observation of sperm abnormalities and DNA fragmentation. Based on spermatozoa from 17 Holstein-Friesian bulls, we determined a relationship between DNA fragmentation and the presence of the “so called” major-type sperm defects. Values for DNA fragmentation index (mean ± SEM) calculated from cells with major-type abnormalities were significantly (P < 0.05) higher (85.05 ± 5.00%) than those from abnormal forms classified as minor-type (17.89 ± 5.55%). Some of the sperm abnormalities, such as double forms, narrow base heads, small heads, shortened tails and proximal cytoplasmic droplets, were only associated with sperm showing fragmented DNA. The simultaneous assessment of sperm morphology and DNA fragmentation has the potential to improve the efficacy of sperm quality assessment in this species.     

Dosimetry studies on the ethylation of mouse sperm DNA after in vivo exposure to (3H)ethyl metanesulfonate

Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [¹⁴C]dT/[³H]dC ratios for whole sperm and sperm DNA were the same for each animal tested.The spermatozoa of males used in the dosimetry studies were labeled with [¹⁴C]thymidine, and then the animals were given various [³H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of ³H and ¹⁴C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide (E/N) ranged from ～ 10^?7 at 3.3 mg/kg up to ～ 10^?4 at 400 mg/kg, and the data indicated that E/N increased with the 1.5 power of the dose. E/N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA.The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing–thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

Fresh and frozen-thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765 mg/kg C57BL6/J or 600 mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P < 0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P < 0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P < 0.05).     

Construction of a Catsper1 DNA Vaccine and Its Antifertility Effect on Male Mice

Cation channel of sperm 1 (CATSPER1) is a unique sperm cation channel protein, and essential for sperm function and male fertility. CATSPER1 exclusively expresses in meiotic and postmeiotic spermatogenic cells, thus belongs to the spermatogenesis-specific antigen that escape central tolerance. We have previously demonstrated the immunocontraceptive potential of its transmembrane domains and pore region, and reported the antifertility effects of its B-cell epitopes on male mice. Aiming to develop DNA vaccine targeting CATSPER1 for male contraception, here the whole open reading frame of mouse Catsper1 was cloned into the plasmid pEGFP-N1 to obtain a DNA vaccine pEGFP-N1-Catsper1. The vaccine was confirmed to be transcribed and translated in mouse N2a cell in vitro and mouse muscle tissue in vivo. Intramuscular injection with the vaccine on male mice induced specific immune reaction and caused significant inhibition on sperm hyperactivated motility and progressive motility (P<0.001 for both), and consequently reduced male fertility. The fertility rate of experimental group was 40.9%, which was significant lower (P=0.012) than control group (81.8%). No significant change in mating behavior, sperm production and histology of testis/epididymis was observed. Given that Catsper1 exhibits a high degree of homology among different species, Catsper1 DNA vaccine might be a good strategy for developing an immunocontraceptive vaccine for human and animal use.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Evaluation of the Relationship Between Height and Zinc,Copper, Iron,Calcium, and Magnesium Levels in Healthy Young Children in Beijing,China

Studies suggest a relationship between semen quality and the concentration of trace elements in serum or seminal plasma. However, trace elements may be linked to DNA and capable of altering the gene expression patterns. Thus, trace element interactions with DNA may contribute to the mechanisms for a trans-generational reproductive effect. We developed an analytical method to determine the amount of trace elements bound to the sperm DNA, and to estimate their affinity for the sperm DNA by the ratio: R = Log [metal concentration in the sperm DNA/metal concentration in seminal plasma]. We then analyzed the concentrations of 15 trace elements (Al, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Ti, V, Zn, As, Sb, and Se) in the seminal plasma and the sperm DNA in 64 normal and 30 abnormal semen specimens with Inductively Coupled Plasma/Mass Spectrometry (ICP-MS). This study showed all trace elements were detected in the seminal plasma and only metals were detected in the sperm DNA. There was no correlation between the metals’ concentrations in the seminal plasma and the sperm DNA. Al had the highest affinity for DNA followed by Pb and Cd. This strong affinity is consistent with the known mutagenic effects of these metals. The lowest affinity was observed for Zn and Ti. We observed a significant increase of Al linked to the sperm DNA of patients with oligozoospermia and teratozoospermia. Al’s reproductive toxicity might be due to Al linked to DNA, by altering spermatogenesis and expression patterns of genes involved in the function of reproduction.     

DNA ORGANIZATION IN THE MATURE SPERM OF SEVERAL ORTHOPTERA BY THE METHOD OF POLARIZED FLUORESCENCE MICROSCOPY

Mature sperm of Acheta domesticus, Acheta assimilis, Nemobius fasciatus, Nemobius confusus, Melanoplus femur-rubrum, Romalea microptera, Scudderia curvicauda, and Ceuthophilus nigricans were examined for DNA configuration by means of polarized fluorescence microscopy. In all cases, the results suggest that the DNA lies in unsupercoiled array, predominantly parallel to the elongate sperm head axes. Detailed calculations of the factors influencing the degree of polarization of the fluorescent emission from supercoiled DNA are contained in an appendix.     

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl–Lav-8, Cocl–Lav-18, Cocl–Lav-28, Cocl–Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5 × 10⁶ which is less than 0.1% of spermatozoa in standard 250 μL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.     

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl–Lav-8, Cocl–Lav-18, Cocl–Lav-28, Cocl–Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5 × 10⁶ which is less than 0.1% of spermatozoa in standard 250 μL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

Dynamics of sperm DNA fragmentation in domestic animals: II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 °C; n = 10) or frozen-thawed (n = 13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 °C and stored for 1 h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 °C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1 h of incubation at 37 °C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6 h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6 h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.     

Relationships between bacteriospermia,DNA integrity,nuclear protamine alteration,sperm quality and ICSI outcome

The Aim of this study was to evaluate the effects of bacteriospermia on human sperm parameters, nuclear protamines, DNA integrity and ICSI outcome in patients enrolled for ICSI treatment. 84 unselected couples consulting in infertility and obstetrics clinic and enrolled for ICSI treatment were included in this study. The semen specimens were screened bacteriologically; semen and sperm parameters were also evaluated according to WHO guidelines. DNA integrity, protamines concentration and protamine deficiency were estimated by TUNEL assay, AU-PAGE and Chromomycin (CMA3) respectively. The results of this study revealed that 34.52% of studied semen samples were infected with bacteria. The isolated bacteria were identified as Staphylococcus aureus, Staph. epidermidis, Staph. haemolyticus, Escherichia coli, Enterococcus faecalis and Streptococcus agalactiae. Bacteriospermia had a significant (p?<?.010) negative effect on sperm parameters; concentration, motility, progressive motility and chromatin condensation. Moreover, high DNA fragmentation with low P1 and P2 concentrations were noticed in infected patients in comparison to non-infected patients but non-significant. Also, the fertilization rate decreased significantly (p?<?.05) with infected patients. In conclusion: bacteriospermia has significant negative effect on sperm quality and fertilization rate in patients who underwent ICSI treatment.     

Genetic Variants in Nitric Oxide Synthase Genes and the Risk of Male Infertility in a Chinese Population: A Case-Control Study

Background

In recent years, oxidative stress has been studied extensively as a main contributing factor to male infertility. Nitric Oxide, a highly reactive free radical gas, is potentially detrimental to sperm function and sperm DNA integrity at high levels. Thus, the aim of this study was to investigate the associations between five polymorphisms in nitric oxide synthase genes (NOSs) and the risk of male infertility and sperm DNA damage as well.

Methods

Genotypes were determined by the OpenArray platform. Sperm DNA fragmentation was detected using the Tdt-mediated dUTP nick-end labeling assay, and the level of 8-hydroxydeoxyguanosine (8-OHdG) in sperm DNA was measured using immunofluorescence. The adjusted odds ratio (OR) and 95% confidence interval (CI) were estimated using unconditional logistic regression.

Results

Our results revealed a statistically significant difference between the cases and controls in both genotypic distribution (P<0.001) and allelic frequency (P = 0.021) only for the NOS3 rs1799983 SNP. Multivariate logistic regression analyses revealed that rs1799983 was associated with a borderline significantly increased risk of male infertility (GT vs. GG: adjusted OR = 1.30, 95% CI: 1.00–1.70; GT+TT vs. GG: adjusted OR = 1.34, 95% CI: 1.03–1.74; P _trend = 0.020). Moreover, NOS3 rs1799983 was positively associated with higher levels of sperm DNA fragmentation (β = 0.223, P = 0.044). However, the other 4 polymorphisms (NOS1 rs2682826, NOS1 rs1047735, NOS2 rs2297518, and NOS2 rs10459953) were not found to have any apparent relationships with male infertility risk.

Conclusions

Of five NOS gene polymorphisms investigated in the present study, we found NOS3 rs1799983 might cause oxidative sperm DNA damage, thereby contributing to male infertility.     

Transmission ratio distortion of mouse t haplotypes is not a consequence of wild-type sperm degeneration

A mouse t-complex-specific DNA probe was used to determine the ratio of t-carrying and (+)-carrying sperm in epididymal, vas deferens, and postejaculatory sperm cell populations from heterozygous ($\text{+}\text{t}$) mice with transmission ratios of greater than 95%. No detectable degeneration of (+)-carrying sperm was observed. In this respect, mouse t haplotypes differ from Drosophila melanogaster SD chromosomes. High transmission of t haplotypes must be a consequence of differential transport and/or differential sperm function during the fertilization process itself.