Determination of double-and single-stranded DNA breaks in bovine sperm is predictive of their fertilizing capacity

Background:The analysis of chromatin integrity has become an important determinant of sperm quality.In frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood.The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics,and to assess if this parameter can predict pregnancy rates in cattle.Results:A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods(0-2 h and 2-4 h),analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays,chromatin deprotamination and decondensation,sperm motility,viability,acrosomal status,and intracellular levels of total ROS,superoxides and calcium.Insemination of 59,605 females was conducted using sperm from the same bulls,thus obtaining the non-return to estrus rates after 90 d(NRR).Results showed an increased rate of double-stranded breaks in the first period(0-2 h:1.29±1.01%/h vs.2-4 h:0.13±1.37%/h;P<0.01),whereas the rate of sperm with moderate+high single-stranded breaks was higher in the second period(0-2 h:3.52±7.77%/h vs.2-4h:21.06±11.69%/h;P<0.0001).Regarding sperm physiology,viability decrease rate was different between the two periods(0-2 h:-4.49±1.79%/h vs.2-4 h:-2.50±3.39%/h;P=0.032),but the progressive motility decrease rate was constant throughout post-thawing incubation(0-2 h:-4.70±3.42%/h vs.2-4 h:-1.89±2.97%/h;P>0.05).Finally,whereas no correlations between bull fertility and any dynamic parameter were found,there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet(Rs=-0.563,P=0.003),between NRR and basal progressive motility(Rs=0.511,P=0.009),and between NRR and sperm with high ROS at 4 h post-thaw(Rs=0.564,P=0.003).Conclusion:The statistically significant correlations found between intracellular ROS,sperm viability,sperm motility,DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress,where viability and motility would be affected first and sperm chromatin would be altered at a later stage,thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw.Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.

Non-genetic contributions of the sperm nucleus to embryonic development

Recent data from several laboratories have provided evidence that the newly fertilized oocyte inherits epigenetic signals from the sperm chromatin that are required for proper embryonic development. For the purposes of this review, the term epigenetic is used to describe all types of molecular information that are transmitted from the sperm cell to the embryo. There are at least six different forms of epigenetic information that have already been established as being required for proper embryogenesis in mammals or for which there is evidence that it may do so. These are （i） DNA methylation; （ii） sperm-specific histones, （iii） other chromatin-associated proteins; （iv） the perinuclear theca proteins; （v） sperm-born RNAs and, the focus of this review; and （vi） the DNA loop domain organization by the sperm nuclear matrix. These epigenetic signals should he considered when designing protocols for the manipulation and cryopreservation of spermatozoa for assisted reproductive technology as necessary components for effective fertilization and subsequent embryo development.

Pum 1 sequesters apoptosis during spermatogenesis

RNA binding proteins have long been known to play a role in spermatogenesis. The laboratory of Norman Hecht was one of the leaders in this field, having characterized three RNA binding proteins that regulate mRNAs during sperm differentiation. Polypyrimidine tract-binding pro- tein 2 （PTB2） has two functions--stabilizing mRNA transcripts in the cytoplasm and being involved in splicing,l Using in vivo cross- linking of protein to RNA, several mRNA and small RNA targets were identified in the testis.2 Translin is another protein that both stabilizes mRNAs from a specific transcription factor, and is associated with small RNAs.3 Perhaps the most interesting is the DNA/RNA binding protein MSY2. At low concentrations,

Hematopoietic stem cells are a critical sub-population of whole bone marrow in the treatment of myocardial infarction

Recent studies suggest that whole bone marrow (WBM) derived stem cells may facilitate recovery following myocardial infarction. However, the sub-population of WBM responsible for recovery remains uncertain. By adjusting the abundance of CD34+LinNeg cells in human bone marrow we examined the relative significance of hematopoietic stem cells (HSC) in the recovery of cardiac function in a murine model of induced myocardial infarction. Enrichment of HSC by ~100-fold in WBM transplanted into mice significantly increased recovery of heart function and reduced scar size compared to transplantation of WBM depleted in HSC by ~10-fold (P P < 0.01 respectively). Peri-infarct capillary density was significantly increased in recipients of HSC-enriched samples (P < 0.01) or WBM samples (P < 0.01) compared to controls. These results strongly suggest?a critical role for HSC in the effective treatment of myocardial infarction with human bone marrow, and imply that enrichment of HSC may markedly benefit the clinical application of WBM treatments.

A new Speedy/RINGO protein may help regulate male meiosis

Reproductive biology, although seen as a specialty study area, has many unique biology models that offer insight into the regulation of cellular processes that are shared by many different cell types. The most celebrated example of this was the discovery of the cyclins and their role in cell cycle regulation in Xenopus oocytes.

A model for the control of DNA integrity by the sperm nuclear matrix

The highly condensed chromatin of mammalian spermatozoa is usually considered to be biologically inert before fertilization. However, we have demonstrated that even in this compacted state, sperm chromatin is subject to degradation at open configurations associated with the nuclear matrix through a process we have termed sperm chromatin fragmentation （SCF）. ＂[his suggests that a mechanism exists to monitor the health of spermatozoa during transit through the male reproductive tract and to destroy the genome of defective sperm cells. The site of DNA damage in SCF, the matrix attachment sites, are the same that we hypothesize initiate DNA synthesis in the zygote. When sperm that have damaged DNA are injected into the oocyte, the newly created zygote responds by delaying DNA synthesis in the male pronudeus and, if the damage is severe enough, arresting the embryo＇s development. Here we present a model for paternal DNA regulation by the nuclear matrix that begins during sperm maturation and continues through early embryonic development.