Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.

Reactive oxygen species have a great impact on spermatozoa function. Gametes from sole males born in captivity (F1) display lower quality than those from wild individuals. In this paper, the percentage of cells positive for dichlorofluorescein (DCF(+)) was determined by flow cytometry in wild and F1 animals, the effect of cryopreservation on DCF(+) cells was evaluated in both groups and the distribution of H2O2 within the cell was studied by confocal microscopy. Our results indicated that there are no differences in either viability or DCF(+) cells between wild and F1 animals when fresh samples were evaluated. However, when data were analyzed considering two different sperm populations in terms of motility, a significant decrease in viability and DCF(+) cells was reported in low-motile F1 spermatozoa. Cryopreservation did not alter the viability or the presence of DCF(+) cells in sperm samples from wild animals, but significantly decreased the viability in F1 samples. Distribution patterns of H2O2 have been established by confocal microscopy in Solea senegalensis spermatozoa: co-localization of H2O2 with active mitochondria (MitoTracker(+)) and co-localization with nuclear DNA (DAPI). Compared with H2O2 distribution in other marine species such as Scophthalmus maximus, Solea senegalensis spermatozoa showed widespread presence of H2O2 particularly in the nuclei, which could potentially compromise DNA integrity.

Effect of cryopreservation on human sperm messenger RNAs crucial for fertilization and early embryo development.

During recent years, several studies have pointed out the importance of key paternal transcripts in early embryo development. Sperm cryopreservation is commonly applied in assisted reproductive technologies (ARTs) and it is important to know if it produces any relevant effect at this level. In this study, using normozoospermic donors, we present a candidate transcript approach in which we quantify the presence of sperm mRNAs considered as markers for male fertility and pregnancy success. Analyses were done using quantitative PCR. Our results demonstrated that the used cryopreservation protocol, which is routinely employed in clinical practice, alter transcripts considered as spermatozoa quality markers and markers for pregnancy success. Most of the studied transcripts considered as male quality markers (PRM1, PRM2, and PEG1/MEST) and one of studied mRNAs reported as markers of pregnancy success (ADD1) were reduced after cryopreservation. In order to check if vitrification protocols could reduce this alteration, another assay was carried out analyzing those transcripts with higher differences in the first study (PRM1 and PRM2). The results showed the same tendency. Although maternal mRNAs can compensate these deficiencies, these results could make advisable the optimization of freezing/thawing procedures.

Probiotic administration improves sperm quality in asthenozoospermic human donors.

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H2O2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H2O2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

Selection of nonapoptotic sperm by magnetic-activated cell sorting in Senegalese sole (Solea senegalensis).

Senegalese sole (Solea senegalensis) is a promising species in aquaculture. However, owing to decreased sperm quality in F1 generations and the absence of courtship in those individuals born in captivity, artificial fertilization is being used to generate new progenies. The objective of this study was to implement a sperm selection method for nonapoptotic sperm subpopulation recovery before sperm cryopreservation. In particular, magnetic-activated cell sorting is used to eliminate apoptotic spermatozoa. This study represents the proof-of-concept for magnetic-activated cell sorting applicability in teleost species relevant in aquaculture. Apoptotic cell population was studied by flow cytometry using YO-PRO-1 and a caspase detection kit. Also, reactive oxygen species were measured in sperm samples. Our data demonstrated that caspase detection is more specific than YO-PRO-1 in the identification of apoptotic cells in S senegalensis seminal samples. The results showed that the percentage of apoptotic cells (caspase positive) was significantly higher (P = 0.04) in seminal samples from F1 than that from wild individuals. Magnetic-activated cell sorting removed a significant number of apoptotic cells from the samples (54% and 75% in wild and F1 individuals, respectively), decreasing the level of cells positive for reactive oxygen species (P = 0.17). In conclusion, this technique reduces the percentage of nonfunctional spermatozoa in a seminal sample before cryopreservation. This novel technique can be applied directly in the aquaculture industry.

Analysis of DNA damage after human sperm cryopreservation in genes crucial for fertilization and early embryo development.

Sperm cryopreservation is widely used in clinic for insemination, in vitro fertilization and other procedures such as intracytoplasmic sperm injection. The assessment after freezing/thawing of spermatozoa viability, motility and sometimes DNA integrity (mainly using fragmentation assays) has been considered enough to guarantee the safety and effectiveness of the technique. However, it is known that, even when fragmentation is absent, a significant DNA damage could be detected in some genome regions. This is particularly important considering that, during the last years, several studies have pointed out the importance of key paternal genes in early embryo development. In this study, using normozoospermic donors, we present a candidate gene approach in which we quantify the number of lesions produced by freezing/thawing over key genes (PRM1, BIK, FSHB, PEG1/MEST, ADD1, ARNT, UBE3A, SNORD116/PWSAS) using quantitative PCR. Our results demonstrated that the cryopreservation protocol used, which is routinely employed in clinic, produced DNA lesions. The genes studied are differentially affected by the process, and genome regions related to Prader-Willi and Angelman syndromes were among the most damaged: SNORD116/PWSAS (4.56 ± 1.84 lesions/10 kb) and UBE3A (2.22 ± 1.3 lesions/10 kb). To check if vitrification protocols could reduce these lesions, another experiment was carried out studying some of those genes with higher differences in the first study (FSHB, ADD1, ARNT and SNORD116/PWSAS). The number of lesions was not significantly reduced compared to cryopreservation. These results could be relevant for the selection of the most adequate available cryopreservation protocol in terms of the number of lesions that produced over key genes, when no differences with other traditional techniques for DNA assessment could be detected.