C. López-Fernández

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 (t0) and after 4 hr (t4) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t0 and t4 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. Mol. Reprod. Dev. 78:951–961, 2011.

Dynamics of sperm DNA fragmentation in domestic animals III. Ram.

From a biological viewpoint spermatozoa are ejaculated by the male and received into the female while maintaining roughly constant temperature, which in most mammals is below the temperature of the soma. When ejaculated spermatozoa are used for artificial reproductive purposes a temperature excursion episode is produced, because the spermatozoa are often stored as frozen or chilled samples and the biological temperature is only recovered after insemination. In this study we have analyzed the effects of cooling (to 15 degrees C) and freezing ram spermatozoa on the subsequent sperm DNA fragmentation index (sDFI) during a varying period of storage at 37 degrees C. The aim was to emulate in vivo processes that cooled or frozen-thawed spermatozoa experience after insemination. The study was performed using commercial semen samples derived from rams regularly used for reproductive purposes. Semen samples were studied after a cooling or cryopreservation episode followed by biological temperature recovery and incubation up to 48 h. The results indicated that when spermatozoa experience a severe (frozen) or mild (cooled) temperature excursion episode, major effects on sperm viability and DNA fragmentation are induced and cause the subsequent rapid decline of ram sperm quality. This effect could be detected just at the onset of the biological temperature recovery. Sperm DNA damage in cooled samples was observed after 5 h of incubation at 37 degrees C, while this time was reduced to less than 60 min in frozen-thaw samples. The dynamics of sDFI in different animals, analyzed under the same experimental conditions, was different from one sample to another, regardless of the method used for storage. Sperm viability was better preserved in cooled rather than in frozen samples. While for the frozen-thawed samples sperm viability was almost abolished after 5 h of incubation, a stable proportion of viable spermatozoa (ranging from 20% to 60%) was observed in the cooled samples at the corresponding time points. Finally, with respect to the prevalence of sDFI in ram, the level commonly found was lower than 5% at the onset of the experiment. However, sDFI was higher than 5% in 25% of the samples and in 15% of rams this index exceeded 10%.

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.

DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 degrees C, 25 degrees C, and 4 degrees C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20+/-14.77% and did not differ significantly from the results of a neutral comet assay (22.0+/-19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 degrees C than when incubated at 25 degrees C or 4 degrees C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing.

A dynamic assessment of sperm DNA fragmentation versus sperm viability in proven fertile human donors

OBJECTIVE To analyze any possible dynamic correlation between sperm DNA fragmentation and sperm viability. DESIGN The rate of viability loss and the rate of increase of the frequency of sperm cells with fragmented DNA were determined at 0, 1.5, 4.5, and 24.0 hours after thawing samples from donors with proven fertility. SETTING Academic biology and reproductive medicine centers. PATIENT(S) Fifteen male donors with proven fertility for a maximum of six births at the reproductive medicine center. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Sperm DNA fragmentation and viability dynamics expressed as logarithmic coefficients of change. RESULT(S) The dynamics of sperm DNA fragmentation and sperm viability adjusted to a logarithmic function with an initial highest velocity that progressively decreases. Nevertheless, the rates were not statistically significantly correlated. CONCLUSION(S) In the short term, dynamic dysfunction of membrane permeability does not result in DNA fragmentation and thus must be considered as independent parameters of sperm quality.

Sperm deoxyribonucleic acid fragmentation dynamics in fertile donors

OBJECTIVE To study the velocity of sperm DNA fragmentation in frozen-thawed sperm samples from male sperm donors of proved fertility. DESIGN Sperm DNA fragmentation assessment with use of sperm chromatin dispersion methodology after 0, 4, 8, and 24 hours of incubation in IVF medium. SETTING Academic biology and reproductive medicine centers. PATIENT(S) Twenty male fertility donors with proved fertility for a maximum of six births at the reproductive medicine center. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The velocity of sperm DNA fragmentation between two consecutive incubations was scored. Best adjustment of sperm DNA fragmentation index versus incubation time for linear, logarithmic, or exponential function was tested. RESULT(S) Increase of sperm DNA fragmentation through time accounted for a substantial percentage of the overall variation. The highest velocity of sperm DNA fragmentation was observed in the first 4 hours of incubation, decreasing by 50% during the second incubation period and being of the order of 1% in the final experimental period. The tendency to increase in sperm DNA fragmentation is not homogeneous among donors; they may adjust to a logarithmic, linear, or exponential function rendering high values for R(2). CONCLUSION(S) Sperm DNA fragmentation occurs rapidly after thawing, and it is an important cause of the rapid decline of sperm quality. Thus, the use of sperm samples as quickly as possible after thawing is highly recommended in clinical practice. Different sperm DNA fragmentation dynamics among individuals were observed.

DNA base sequence is not the only factor for restriction endonuclease activity on metaphase chromosomes: evidence using isoschizomers

Human and mouse fixed metaphase chromosomes were treated with the isoschizomer sets MboI/Sau3A and EcoRII/BstNI. In both cases we found that each member of the isoschizomer pairs produced different results, indicating that factors other than DNA base composition may affect in situ digestion by restriction endonucleases and that the structure of the enzymes is one factor. We also found that MboI and Sau3A isoschizomers produced the same effect on the chromosomes of the grasshopper Oedipoda germanica. This indicated that differences in the chromatin structure of different species may be important in determining restriction endonuclease activity on eukaryotic chromosomes.

Restriction endonucleases in the study of eukaryotic chromosomes

C. Lopez-Fernandezl, J. Gosalvezi, L. Ferrucci2 & R. Mezzannottej ’ Departamento de Biologia (Genktica), Facultad de Ciencias (C-XV), Universidad Autbnoma de Madrid, 28049 Madrid, Spain 2 Dipartimento di Biologia, Facoltci di Scienza M.F.W., II Universitci degli Studi di Roma, 00173 La Romanina, Roma, Italy 3 Dipartamento di Biologia Generale, Facolt6 di Medic& e Chirurgia, 09124 Via Ospedale, Cagliari, Italy

Correlation between constitutive heterochromatin and restriction enzyme resistant chromatin in Arcyptera tornosi (Orthoptera)

Fixed mitotic chromosomes of A. tornosi have been analysed by means of C-banding, DA-DAPI and Chromomicin A3 fluorescence, as well as by digestion in situ with Alu I, Hae III, Hinf I and Hind III restriction endonucleases. From the results obtained at least nine types of chromatin can be distinguished in A. tornosi, Some C-band positive areas (constitutive heterochromatin) which show a characteristic fluorescence pattern are digested by specific endonucleases, whilst others are undigested. C-band negative areas (euchromatin) are digested by some restriction endonucleases but not by others. Regions digested are supposed to contain highly repetitive DNAs. It is noteworthy, however, that the heterochromatin associated with NORs is not attacked by any of the enzymes we used, while regions believed to contain AT-rich DNA (DA-DAPI positive) are digested by Hae III that cleaves the GG↓CC base sequence target.

Dynamics of sperm DNA damage in fresh versus frozen–thawed and gradient processed ejaculates in human donors

The rate of increase of sperm DNA fragmentation (rsDF) in fresh and frozen–thawed and processed sperm samples after a density gradient for sperm selection was analysed after 0, 0.5, 1.5, 4.5, 6, 24, 48 and 72 h of incubation at 37 °C, in five donors with proven fertility. The results showed that: (i) sperm DNA fragmentation (sDF) at baseline in fresh samples (14.3 ± 3.3) was lower than that obtained after freeze‐thawing and selection (19.4 ± 4.1), significant differences; (ii) After 6 h of incubation the mean sDF in fresh samples (24.2 ± 10.2) was significantly lower than that in frozen–thawed samples (45.3 ± 7.1); (iii) Subsequently, the rsDF in fresh semen samples was 1.6% per h after 6 h of incubation, while after thawing and selection the rsDF was 4.3% per h; The tendency to increase in sDF showed high R2 values (R2 = 0.90) for exponential functions in case of fresh samples, whereas R2 values for linear functions were higher after sperm selection (R2 = 0.97). These results indicate that differences in sperm DNA fragmentation dynamics before and after storage are an important issue that must be considered for storage of sperm to be used for artificial reproduction techniques.