Sex-sorted bovine spermatozoa and DNA damage: I. Static features

This study examined the static response of Spermatozoa DNA Fragmentation (SDF) after sex selection in bulls using a MoFlo^® SX (Beckman Coulter, Miami FL) spermatozoa sorter to produce three different subpopulations: 1) Spermatozoa bearing X- chromosomes with a purity of 95%, 2) Spermatozoa bearing Y-chromosomes with a purity of 95%, and 3) non-viable spermatozoa. The static response of SDF refers to the baseline values observed for DNA damage when analyzed pre- and post sex-sorting. Results showed that while the baseline level SDF in pre-sorted bull spermatozoa samples ranged from 5.3% to 11% with an average of 7.9% ± 2.1%, the level of SDF obtained in X- and Y-chromosome sorted samples was much lower (3.1% ± 1.9%) and statistical differences were obtained after comparing both groups (P < 0.01). Spermatozoa containing a fragmented DNA molecule tend to be accumulated in the non-viable subpopulation. The baseline SDF level in X- and Y-chromosome sorted subpopulations is reduced, by 63% on average when compared to the values obtained in the neat semen sample. Different bulls exhibit unique SDF reduction efficiencies via the X- and Y-chromosome sex selection process.     

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.     

Electroporation of bovine spermatozoa to carry foreign DNA in oocytes

In the present study, electroporation was used to test the ability of spermatozoa to carry foreign DNA into the bovine oocytes. Frozen-thawed bovine spermatozoa (10(7)/ml) were electroporated using six different combinations of voltage (500, 1,000, or 1,500 V) and capacitance (1 or 25 microFarads) in the presence of 1 mg/ml of plasmid pRGH527. The portions of plasmids retained by sperm cells after three washings (stable for ten washings) were 4.3, 5.5, 5.1, 6.0, 6.8, and 5.8% for 1 microFarad, 500, 1,000, and 1,500 V and 25 microFarads, 500, 1,000, and 1,500 V, respectively. Nonelectroporated cells have retained only 1% of plasmids. In the same experiment, electroporated spermatozoa were acrosome reacted by treatment with ionophore A23187 to evaluate the fraction of marked plasmids joined at the acrosomal membrane. The results show that 3.5, 5.0, 4.4, 5.0, 6.3, and 4.4% remain tied to the ionophore-treated sperm. Only 0.7% of plasmid was retained after removal of the acrosome of nonelectroporated cells. Acrosome reaction was not significantly induced by the electrical field (EF) (P less than 0.005). EF decrease motility significantly for greater than 100 V in 0.3 M mannitol (M) and mannitol-TALP (MT) (1/1) media and greater than or equal to 500 V (P less than 0.05) in TALP medium. The retained plasmid rate was compared between TALP medium M and MT media and resulted in a percentage of 1.0, 2.5, 6.5 at 1 microFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 microFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 microFarad, 500 or 1,000 V, 25 microFarad, 500 V or 1,000 in TALP medium hold plasmids in proportion of 5.2, 5.4, 7.4, and 6.0%. Electroporation above 100 V in M and MT killed the cells. In a part of this experiment, spermatozoa electroporated in the presence of radiolabeled plasmids have been treated with DNase I and results revealed that 35, 28, 54, 58, and 3% of marked DNA remains in sperm cells following digestion after electroporation in TALP (1,000 V, 1 and 25 microFarads), M medium (100 V, 1 and 25 microFarads), and control, respectively. Using in vitro matured bovine oocytes, the electroporation conditions were correlated with the fertilization rate (85% for control and 55% for electroporated spermatozoa). Autoradiography of embryos following fertilization indicated the presence of plasmids in the cytoplasm and in the zona pellucida.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association of exogenous DNA with cattle and insect spermatozoa in vitro

Spermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina), and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.     

Cryopreservation and Sperm DNA Integrity

Cryopreservation of sperm is an extremely important issue in the field of male infertility as freezing can have detrimental effects on a variety of sperm functions, some of them not accessible to the traditional semen quality analysis. In this study, chromatin structure variations in human spermatozoa in semen were studied with the sperm chromatin structure assay (SCSA), both before and after cryopreservation. Samples were divided into two aliquots: the first was analysed without further treatment, while the second was stored in liquid nitrogen at −196 °C using standard cryopreservation techniques. The fresh and thawed aliquots were also assessed by light and fluorescence microscopy (after Acridine Orange staining, AO), and computer-assisted semen analysis (CASA) of motility. Overall sperm quality was found to deteriorate after cryopreservation. When thawed spermatozoa were subjected to an extra swim-up round, a general improvement in nuclear maturity was seen in post-rise spermatozoa.     

Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P = 0.493). However, when thawed semen was subsequently incubated at 37 °C for periods of between 2 and 48 h, differences were recorded in the rate of sperm DNA fragmentation (r-SDF), that are indicative of seasonal changes in reproduction (Log Rank: Mantel–Cox; χ² = 158.6; DF = 3; P < 0.001). Goat semen collected and cryopreserved in the Spanish winter and spring resulted in a poor post-thaw sperm DNA longevity and recorded a r-SDF of 0.8 and 1.3 per hour, after 48 h of incubation respectively. The r-SDF subsequently decreased to 0.16 and 0.36 per hour during summer and autumn. It is therefore, recommended that semen samples from Murciano-Granadina goats housed above the 40°N latitude be collected and cryopreserved in the summer and early autumn. This would result in a more stable DNA molecule, which is likely to improve the reproductive success.     

DNA oxidative damage in mammalian spermatozoa: where and why is the male nucleus affected?

Gamete DNA integrity is one key parameter conditioning reproductive success as well as the quality of life for the offspring. In particular, damage to the male nucleus can have profound negative effects on the outcome of fertilization. Because of the absence of repair activity of the quiescent mature spermatozoa it is easily subjected to nuclear damage, of which oxidative damage is by far the most prominent. In relation to the organization of the mammalian sperm nucleus we show here that one can correlate the nuclear regions of lower compaction with areas preferentially showing oxidative damage. More precisely, we show that oxidative DNA damage targets primarily histone-rich and nuclear matrix-attached domains located in the peripheral and basal regions of the mouse sperm nucleus. These particular sperm DNA domains were recently shown to be enriched in genes of paramount importance in postfertilization DNA replication events and in the onset of the embryonic developmental program. We propose that monitoring of sperm DNA oxidation using the type of assay presented here should be considered in clinical practice when one wants to estimate the integrity of the paternal nucleus along with more classical assays that essentially analyze DNA fragmentation and nucleus compaction.     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay

This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R² value = 0.949; P < 0.01; n = 16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n = 6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 °C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P = 0.001) in DNA damage, as soon as 4 h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6 h of incubation and revealed individual animal variation. Freshly collected and incubated (37 °C) rhinoceros (n = 3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage.     

DNA damage to spermatozoa has impacts on fertilization and pregnancy

DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. The causes of this DNA damage are still uncertain but the major candidates are oxidative stress and aberrant apoptosis. The weight of evidence currently favours the former and, in keeping with this conclusion, positive results have been reported for antioxidant therapy both in vivo and in vitro. Resolving the causes of DNA damage in the male germ line will be essential if we are to prevent the generation of genetically damaged human embryos, particularly in the context of assisted conception therapy.     

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species

The dynamic onset of DNA fragmentation in mammalian sperm populations varies widely in different species when the spermatozoa are incubated in vitro at body temperature for several hours, and recent studies have shown that the dynamic rate of DNA fragmentation within a species has considerable predictive value in terms of fertility. The reasons for such variation are unclear, but here we show that differences in protamine sequence and identity could be partially responsible. Sets of 10 normal semen samples from 11 species (ram, goat, boar, white-tailed deer, rabbit, human, domestic and Spanish fighting bull, horse, donkey, rhinoceros, and koala) were cryopreserved, thawed, diluted in an appropriate extender for each species, and then incubated for 4 hr at 37 °C. Semen samples from human infertility patients were also included for comparison with the donors. DNA fragmentation analysis was undertaken immediately after thawing (t(0)) and after 4 hr (t(4)) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t(0) and t(4) were examined in the context of the respective protamine genomes. The expression of protamine 2 in a species significantly enhanced the likelihood of sperm DNA fragmentation; greater numbers of cysteine residues in protamine 1 tended to confer increased sperm DNA stability, and there were logical evolutionary relationships between species in terms of their sperm DNA stability. Human spermatozoa from infertility patients exhibited considerably higher DNA instability than the normal semen donors, a difference that could be indirectly attributed to unbalanced protamine 1-to-protamine 2 ratios.     

Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters,DNA integrity and fertilizing ability

Supplementation of the semen extender with antioxidants did not produce any significant effect on CASA and progressive motilities and sperm motility characteristics, in comparison to the control group (P > 0.05).     

Ultrastructure of spermatids and spermatozoa in three polychaetes with modified biology of reproduction: Autolytus sp., Chitinopoma serrula,and Capitella capitata

Unlike the primitive type of spermatozoon found in most polychaetes, the spermatozoon of Autolytus has a bilateral symmetry with elongated nucleus, and the mitochondria surround the posterior part of the nucleus. A rather large disk-shaped acrosome is situated along one side of the anterior part of the nucleus. From the anterior margin of the distal centriole emerge long striated rootlets, which run along the nuclear envelope to the anterior part of the nucleus. The spermatozoon of Chitinopoma serrula has an elongated, slightly bent nucleus, a thimble-like acrosome apically on the anterior surface of the nucleus, and an elongated middle piece containing 4 rod-like mitochondria developed from spherical mitochondria surrounding the basal part of the tail flagellum. In the spermatozoon of Capitella capitata, both nucleus and middle piece are elongated compared to the primitive type. The large and conical acrosome is placed asymmetrically at the nucleus and consists of an acrosomal vesicle and subacrosomal substance. The greater part of the middle piece forms a collar around the initial part of the tail flagellum. The cytoplasm of the collar contains granular material. One or two small mitochondria lie around the 2 centrioles at the base of the nucleus.

These types of spermatozoa represent early steps in the evolution of modified spermatozoa combined with changed biology of reproduction. The modified spermatozoa are larger than the primitive ones.     

DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes

Immunostaining is the preferred technique to assess differences in methylation and hydroxymethylation status of both pronuclei in single zygotes. DNA counterstaining is needed to delimitate the pronuclear area for quantification purposes. For a correct epitope retrieval of 5-methylcytosine and 5-hydroxymethylcytosine in bovine zygotes, 1 h of denaturation with 4 N HCl is needed. However, DNA stains are sensitive to denaturation. Therefore, four DNA stains were tested after 1 h of denaturation with 4 N HCl in this study. After this treatment, DAPI (4′,6-diamidino-2-phenylindole) and Hoechst failed to bind DNA, but both propidium iodide and ethidium homodimer-2 successfully bound it and both pronuclei were stained.     

Propolis protects human spermatozoa from DNA damage caused by benzo[a]pyrene and exogenous reactive oxygen species

Many environmental, physiological and genetic factors have been implicated in defective sperm function, the most common cause of infertility. In addition, sperm preparation techniques such as centrifugation, used prior to in vitro fertilization, are associated with the generation of reactive oxygen species (ROS) and an increase in the level of DNA damage. Factors that can offer spermatozoa protection are, therefore, of great importance. This study was designed to examine in vitro the effect of a Chilean propolis ethanolic extract on human spermatozoa treated with benzo[a]pyrene and exogenous reactive oxygen species. Our experimental evidence demonstrated that the natural drug under investigation is able to protect genomic DNA by damage induced by benzo[a]pyrene, hydrogen peroxide (H2O2) and hydrogen peroxide in combination with adenosine 5'-diphosphate (ADP) and ferrous sulfate (FeSO4), determining a significant reduction of the intracellular oxidants. An increase in membrane damage, measured by monitoring the formation of thiobarbituric acid-reactive substances (TBARS) and lactic dehydrogenase (LDH) release, was observed only in sperm treated with H2O2, ADP and FeSO4. The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack, reducing TBARS formation and LDH release. In summary, our results evidence that the protective effect exhibited by this natural compound in human spermatozoa is correlated, at least in part, to the antioxidant capacity of its active components, and suggest that propolis may have a role in protection against male infertility.     

Clinical implications of sperm DNA damage in IVF and ICSI: updated systematic review and meta-analysis

The clinical effect of sperm DNA damage in assisted reproduction has been a controversial topic during recent decades, leading to a variety of clinical practice recommendations. While the latest European Society of Human Reproduction and Embryology (ESHRE) position report concluded that DNA damage negatively affects assisted reproduction outcomes, the Practice Committee of the American Society for Reproductive Medicine (ASRM) does not recommend the routine testing of DNA damage for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Herein, our aim was to perform a systematic review and meta-analysis of studies investigating whether sperm DNA damage affects clinical outcomes in IVF and ICSI, in order to contribute objectively to a consistent clinical recommendation. A comprehensive systematic search was conducted according to PRISMA guidelines from the earliest available online indexing year until March 2020, using the MEDLINE-PubMed and EMBASE databases. We included studies analysing IVF and/or ICSI treatments performed in infertile couples in which sperm DNA damage was well defined and assessed. Studies also had to include information about pregnancy, implantation or live birth rates as primary outcomes. The NHLBI-NIH quality assessment tool was used to assess the quality of each study. Meta-analyses were conducted using the Mantel–Haenszel method with random-effects models to evaluate the Risk Ratio (RR) between high-DNA-damage and control groups, taking into account the 95% confidence intervals. Heterogeneity among studies was evaluated using the I² statistic. We also conducted sensitivity analyses and post-hoc subgroup analyses according to different DNA fragmentation assessment techniques. We identified 78 articles that met our inclusion and quality criteria and were included in the qualitative analysis, representing a total of 25639 IVF/ICSI cycles. Of these, 32 articles had sufficient data to be included in the meta-analysis, comprising 12380 IVF/ICSI cycles. Meta-analysis revealed that, considering IVF and ICSI results together, implantation rate (RR = 0.74; 95% CI = 0.61–0.91; I² = 69) and pregnancy rate (RR = 0.83; 0.73–0.94; I² = 58) are negatively influenced by sperm DNA damage, although after adjustment for publication bias the relationship for pregnancy rate was no longer significant. The results showed a non-significant but detrimental tendency (RR = 0.78; 0.58–1.06; I² = 72) on live birth rate. Meta-analysis also showed that IVF outcomes are negatively influenced by sperm DNA damage, with a statistically significant impact on implantation (RR = 0.68; 0.52–0.89; I² = 50) and pregnancy rates (RR = 0.72; 0.55–0.95; I² = 72), although the latter was no longer significant after correction for publication bias. While it did not quite meet our threshold for significance, a negative trend was also observed for live birth rate (RR = 0.48; 0.22–1.02; I² = 79). In the case of ICSI, non-significant trends were observed for implantation (RR = 0.79; 0.60–1.04; I² = 72) or pregnancy rates (RR = 0.89; 0.78–1.02; I² = 44), and live birth rate (RR = 0.92; 0.67–1.27; I² = 70). The current review provides the largest evidence to date supporting a negative association between sperm DNA damage and conventional IVF treatments, significantly reducing implantation and pregnancy rates. The routine use of sperm DNA testing is therefore justified, since it may help improve the outcomes of IVF treatments and/or allow a given couple to be advised on the most suitable treatment. Further well-designed controlled studies on a larger number of patients are required to allow us to reach more precise conclusions, especially in the case of ICSI treatments.     

Calculation of binding length of base-specific DNA dyes by comparison of sequence and flow cytometric data. Application to Oryza sativa and Arabidopsis thaliana

From biochemical experiments it has been found that AT- and GC-specific dyes need a certain number of consecutive bases of the same type for binding one dye molecule. From known base sequences the amount of bases included in dye binding can be calculated and compared with experimental data from flow cytometry. Oryza sativa and Arabidopsis thaliana are the first higher plants which are nearly completely (>90%) sequenced. From the published sequences the theoretical fluorescence intensity of base-specific dyes in relation to a base-unspecific dye is calculated for different binding lengths. These values are compared with the actual fluorescence intensities of nuclei analyzed by flow cytometry. For all investigated dyes (DAPI, Hoechst 33258, Hoechst 33342 (all AT specific) and Mithramycin A (GC specific)) a binding length of 1 bp results from the comparison of theoretical and experimental data. This is, however, in disagreement with former results on dye binding. The main reason for the discrepancy seems to be the remaining gap in the sequencing of the Arabidopsis genome.     

Induction of SCEs and DNA fragmentation in bovine peripheral lymphocytes by in vitro exposure to tolylfluanid-based fungicide

The potential for genotoxic and cytotoxic effects of tolylfluanid-based fungicide (50% active agent) was evaluated using sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. For the detection of possible genetic damage, DNA fragmentation assay was also applied. Bovine lymphocytes cultured for 72 h were treated with the fungicide at the final concentrations of 1.75, 3.5, 8.75, and 17.5 μg/mL for the last 24 and 48 h of culture without S9 metabolic activation, and during the last 2 h of culture with S9 metabolic activation. In the SCE assays no evidence for genotoxic activity of the fungicide was found in treatments of 24 h without and 2 h with S9. After the 24 h exposure to tolylfluanid, a weak decrease in the PI was observed. With the prolonged exposure time (48 h), dose dependence in the increase of SCE frequencies was observed. Moreover, after 48 h exposure slight fragmentation of DNA at the concentrations of 3.5 and 8.75 μg/mL was demonstrated. SCE quantification is the most widely used approach for the assessment of genotoxic/cytogenetic effects of chemical compounds. Positive results in the assay at 48 h exposure indicated a potential of the fungicide to increase frequency of chromosomal damage (replication injuries) that is the confirmation of early effect of exposure.     

Bovine oviduct-specific glycoprotein: A potent factor for maintenance of viability and motility of bovine spermatozoa in vitro

In the cow, a specific glycoprotein-bovine oviduct-specific glycoprotein (BOGP)-is secreted by the epithelial cells of the oviduct at the follicular stage of the estrous cycle. In this study, we examined the effects of purified BOGP on the viability and motility of bovine spermatozoa in culture in vitro. Frozen-thawed bovine spermatozoa were incubated in modified Tyrode's solution (TALP) that contained purified BOGP (TALP-BOGP). In TALP-BOGP, both the viability and motility of bovine spermatozoa were more effectively maintained than in the control medium without any added protein. The increases in both the viability and motility of spermatozoa were dose-dependent. Spermatozoa were also incubated in TALP medium supplemented with bovine serum albumin, egg albumin, lactalbumin, or gastric mucin, and their viability and motility in these media were compared with that in TALP-BOGP. Both the viability and motility of spermatozoa were more effectively maintained in TALP-BOGP throughout a 12-hr incubation than in other media tested. An immunolabeling study demonstrated that a monoclonal antibody specific for BOGP reacted with the posterior region of the head, the middle portion, and the tail of spermatozoa that had been incubated with TALP-BOGP, suggesting that BOGP becomes specifically associated with particular regions of the spermatozoon. These results suggest that BOGP is a potent factor for maintenance of the viability and motility of sperm. On the basis of the present results, we also propose that BOGP may play an important role in sperm functions during the reproductive process. © 1995 wiley-Liss, Inc.     

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Variability in DNA content to testis cells and sperm from F₁ hybrids between the laboratory mouse (M. muscullus) and the tobacco mouse (M. poschiavinus), has been determined by flow cytometry (FMC). The F₁ hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F₁ hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f₁ hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F₁ hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.