Background: Before ovulation, sperm-oviduct interaction mechanisms may act as checkpoint for the selection of fertilizing spermatozoa in mammals. Postovulatory mating does not allow the sperm to attach to the oviduct, and spermatozoa may only undergo some selection processes during the transport through the female reproductive tract and/or during the zona pellucida (ZP) binding/penetration. Methods: We have induced DNA damage in spermatozoa by two treatments, (a) a scrotal heat treatment (42 degrees C, 30 min) and (b) irradiation with 137Cs gamma-rays (4 Gy, 1.25 Gy/min). The effects of the treatments were analyzed 21-25 days post heat stress or gamma-radiation. Postovulatory females mated either with treated or control males were sacrificed at Day 14 of pregnancy, and numbers of fetuses and resorptions were recorded. Results: Both treatments decreased significantly implantation rates however, the proportion of fetuses/resorptions was only reduced in those females mated to males exposed to radiation, indicating a selection favoring fertilization of sperm with unfragmented DNA on the heat treatment group. To determine if DNA integrity is one of the keys of spermatozoa selection after postovulatory mating, we analyzed sperm DNA fragmentation by COMET assay in: a) sperm recovered from mouse epididymides; b) sperm recovered from three different regions of female uterine horns after mating; and c) sperm attached to the ZP after in vitro fertilization (IVF). Similar results were found for control and both treatments, COMET values decreased significantly during the transit from the uterine section close to the uterotubal junction to the oviduct, and in the spermatozoa attached to ZP. However, fertilization by IVF and intracytoplasmatic sperm injection (ICSI) showed that during sperm ZP-penetration, a stringent selection against fragmented-DNA sperm is carried out when the damage was induced by heat stress, but not when DNA fragmentation was induced by radiation. Conclusion: Our results indicate that in postovulatory mating there is a preliminary general selection mechanism against spermatozoa with low motility and fragmented-DNA during the transport through the female reproductive tract and in the ZP binding, but the ability of the ZP to prevent fertilization by fragmented-DNA spermatozoa is achieved during sperm-ZP penetration, and depends on the source of damage.

Background In the mouse, culture of embryonic stem (ES) cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs). In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it. Methods In order to investigate whether this phenotype was associated with chromosome alteration, inadvertent differentiation, or epigenetic modification, we characterized and compared this R1 ES cell line at passage 27 with an early passage and with a second ES cell line C57/CBAF1 generated in our laboratory. We assessed: i) karyotype; ii) expression of pluripotent and differentiation markers, iii) mRNA transcription by qRT-PCR of two REs, intracisternal-A particle (IAP) and murine endogenous-retrovirus-L (MuERV-L), and iv) methylation of IAP and MuERV-L. Results The R1 ES cell at passage 27, presented normal morphology, karyotype, and expression of genetic markers characteristic of pluripotent; however, it was detected an altered mRNA transcription of sense and antisense RNA strands of both REs, concomitantly with an altered methylation pattern for the IAP element but not for MuERV-L. These results indicate that besides methylation, other post-transcriptional processes are involved in gene silencing of some REs; and that culture of ES cells may decrease their pluripotency by producing inadvertent alterations in the expression of REs without significantly affecting the morphology, chromosome structure, and expression of pluripotent or differentiation markers. Conclusion Inadvertent REs instability may have important consequences for the use of ES cells in transgenesis (chimera formation) or in cell therapy.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1^mu and Zrsr2^mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2^mu/mu female with Zrsr1^mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2^mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

Spermatogenesis is a complex and highly regulated process involving the proliferation, differ-entiation, and apoptosis of germ cells. This process is controlled by various hormonal, genetic, and environmental factors, including temperature. In Hormonal regulation, Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH) and testosterone (T), are essential for a correct spermatogenesis development from early stages and spermatogonia proliferation to germ cells maturation. Other hormones, like inhibin and activin, finely participate tuning the process of spermatogenesis. Genetic regulation involves various transcription factors such as SOX9, SRY, and DMRT1, which are crucial for the development and maintenance of the testis and germ cells. MicroRNAs (miRNAs) play a significant role by regulating gene expression post-transcriptionally. Epigenetic modifications, including DNA methylation, histone modifica-tions, and chromatin remodelling, are also vital. Temperature regulation is another critical as-pect, with the testicular temperature maintained around 2-4°C below body temperature, essen-tial for efficient spermatogenesis. Heat shock proteins (HSPs) protect germ cells from heat-induced damage by acting as molecular chaperones, ensuring proper protein folding and preventing the aggregation of misfolded proteins during thermal stress. Elevated testicular temperature can impair spermatogenesis, increasing germ cell apoptosis, and inducing oxidative stress, DNA damage, and disruption of the blood-testis barrier, leading to germ cell death and impaired differentiation. Cellular mechanisms of germ cell proliferation, differentiation, and death include the mitotic divisions of spermatogonia to maintain the germ cell pool and pro-duce spermatocytes. Spermatocytes undergo meiosis to produce haploid spermatids, which then differentiate into mature spermatozoa. Apoptosis, or programmed cell death, ensures the re-moval of defective germ cells and regulates the germ cell population. Hormonal imbalance, ge-netic defects, and environmental stress can trigger apoptosis during spermatogenesis. Under-standing these mechanisms is crucial for addressing male infertility and developing therapeutic interventions. Advances in molecular biology and genetics continue to uncover the intricate de-tails of how spermatogenesis is regulated at multiple levels, providing new insights and poten-tial targets for treatment.

Spermatozoa undergo their last phase of spermiogenesis, known as maturation, as they pass through the epididymis. A recent report indicates that mouse immature spermatozoa retrieved from the caput epididymis for intracytoplasmic sperm injection (ICSI) give rise to embryos with multiple developmental defects. Further, these embryos were unable to develop to term after their transfer to surrogate mothers. Herein, we examined the potential of mouse caput spermatozoa to produce normal embryos by comparing the use of caput vs. cauda epididymal spermatozoa for in vitro fertilization (IVF) or ICSI. Two methods for the separation of sperm heads prior to ICSI were also compared: freezing/thawing or drawing through a syringe. We found that in contrast to caudal spermatozoa, caput spermatozoa failed to produce embryos via IVF, confirming their immature state. However, regardless of the method employed for the separation of sperm heads, similar efficiencies of blastocyst production in vitro and development to term after transfer to surrogate mothers were observed following ICSI using both caput and cauda epididymal spermatozoa. It therefore seems that mice spermatozoa recovered from the caput epididymis are as valid as caudal spermatozoa for the production of embryos and offspring by ICSI.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1^mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1^mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1^mu hypothalamus. The Zrsr1^mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1^mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.