Genetic and environmental factors produce different levels of DNA damage in spermatozoa. Usually, DNA-fragmented spermatozoa (DFS) are used with intracytoplasmic sperm injection (ICSI) treatments in human reproduction, and use of DFS is still a matter of concern. The purpose of the present study was to investigate the long-term consequences on development and behavior of mice generated by ICSI with DFS. Using CD1 and B6D2F1 mouse strains, oocytes were injected with fresh spermatozoa or with frozen-thawed spermatozoa without cryoprotector. This treatment increased the percentage of TUNEL-positive spermatozoa, tail length as measured by comet assay, and loss of telomeres as measured by quantitative PCR. The ICSI-generated embryos were cultured for 24 h in KSOM, and 2-cell embryos were transferred into CD1 females. The DFS reduced both the rate of preimplantation embryo development and number of offspring. Immunofluorescence staining with an antibody against 5-methylcytosine showed a delay of 2 h on the active demethylation of male pronucleus in the embryos produced by ICSI. Moreover, ICSI affected gene transcription and methylation of some epigenetically regulated genes like imprinting, X-linked genes, and retrotransposon genes. At 3 and 12 mo of age, ICSI with DFS-produced animals and in vivo-fertilized controls were submitted to behavioral tests: locomotor activity (open field), exploratory/anxiety behavior (elevated plus maze, open field), and spatial memory (free-choice exploration paradigm in Y maze). Females produced by ICSI showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, and age-dependent hypolocomotion in the open-field test (P<0.05). Postnatal weight gain of mice produced by ICSI with fresh or frozen sperm was higher than that of their control counterparts from 16 wk on (P<0.01). Anatomopathological analysis of animals at 16 mo of age showed some large organs and an increase in pathologies (33% of CD1 females produced with DFS presented some solid tumors in lungs and dermis of back or neck). Moreover, 20% of the B6D2F1 mice generated with DFS died during the first 5 mo of life, with 25% of the surviving animals showing premature aging symptoms, and 70% of the B6D2F1 mice generated with DFS died earlier than controls with different kind of tumors. We propose that depending on the level of DFS, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. The incomplete repair, however, may lead to long-term pathologies. Our data indicate that use of DFS in ICSI can generate effects that only emerge during later life, such as aberrant growth, premature aging, abnormal behavior, and mesenchymal tumors.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.

The ejaculate is a heterogeneous pool of spermatozoa containing only a small physiologically adequate subpopulation for fertilization. As there is no method to isolate this subpopulation, its specific characteristics are unknown. This is one of the main reasons why we lack effective tools to identify male infertility and for the low efficiency of assisted reproductive technologies. The aim of this study was to improve ICSI outcome by sperm selection through thermotaxis. Here we show that a specific subpopulation of mouse and human spermatozoa can be selected in vitro by thermotaxis and that this subpopulation is the one that enters the fallopian tube in mice. Further, we confirm that these selected spermatozoa in mice and humans show a much higher DNA integrity and lower chromatin compaction than unselected sperm, and in mice, they give rise to more and better embryos through intracytoplasmic sperm injection, doubling the number of successful pregnancies. Collectively, our results indicate that a high quality sperm subpopulation is selected in vitro by thermotaxis and that this subpopulation is also selected in vivo within the fallopian tube possibly by thermotaxis.

Endocannabinoids have been recognized as mediators of practically all reproductive events in mammals. However, little is known about the role of this system in oocyte maturation. In a mouse model, we observed that activation of cannabinoid receptor 1 (CB) during oocyte maturation modulated the phosphorylation status of Akt and ERK1/2 and enhanced the subsequent embryo production. In the absence of CB, oocyte maturation was impaired and embryo development delayed. Cannabinoid receptor 2 (CB) was unable to rescue these effects. Finally, we confirmed abnormal oocyte maturation rather than impaired embryonic transport through the oviduct in CB knockouts. Our data suggest that cannabinoid agonists may be useful maturation supplements. For fertilization patients intolerant to gonadotropins, this could be a promising and only option.-López-Cardona, A. P., Pérez-Cerezales, S., Fernández-González, R., Laguna-Barraza, R., Pericuesta, E., Agirregoitia, N., Gutiérrez-Adán, A., Agirregoitia, E. CB cannabinoid receptor drives oocyte maturation and embryo development PI3K/Akt and MAPK pathways.

Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health. K E Y W O R D S assisted reproductive technologies, bovine, embryo, human, mouse, stress

The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.

These results suggest that the intermittent hypoxia associated with obstructive sleep apnea (OSA) could induce fertility reduction in male patients with this sleep breathing disorder.

For epigenetic phenotypes to be passed on from one generation to the next, it is required that epigenetic marks between generations are not cleared during the two stages of epigenetic reprogramming: mammalian gametogenesis and preimplantation development. The molecular nature of the chromatin marks involved in these events is unknown. Using the epigenetically inherited allele Axin1(Fu) (the result of a retrotransposon insertion upstream of the Axin1 gene) we sought to establish the heritable mark during early embryonic development that determines transgenerational epigenetic inheritance and to examine a possible shift in the expression of this epiallele in future progeny induced by in vitro culture (IVC). To identify the heritable mark we analyzed 1) the level of DNA methylation shown by the Axin1(Fu) allele in sperm and embryos at blastocysts stage and 2) the histone marks (H3K4 me2, H3K9 me3, H3K9 ac, and H4K20 me3) present at the blastocyst stage at the specific Axin1(Fu) locus. According to our data, histone H3K4 me2 and H3K9 ac mark the differences between the Axin1(Fu) penetrant and the silent locus during the first period of demethylation of the preimplantation development. Moreover, suboptimal IVC (reported to produce epigenetic alterations in embryos) and the histone deacetylase inhibitor trichostatin A affect the postnatal expression of this epigenetically sensitive allele through histone modifications during early development. This finding indicates that altered histone modifications during preimplantation can drive altered gene expression later on in development.