An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis.

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology.

In vitro culture of ovine embryos up to early gastrulating stages.

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview.

MEK signalling pathway is required for hypoblast specification and migration in ovine.

In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.

Embryonic disc formation following post-hatching bovine embryo development in vitro.

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion.

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 Zrsr1mu and Zrsr2mu 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 Zrsr2mu/mu female with Zrsr1mu/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 Zrsr1/2mu 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.

D-Chiro-Inositol Treatment Affects Oocyte and Embryo Quality and Improves Glucose Intolerance in Both Aged Mice and Mouse Models of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) is the main cause of female infertility. It is a multifactorial disorder with varying clinical manifestations including metabolic/endocrine abnormalities, hyperandrogenism, and ovarian cysts, among other conditions. D-Chiro-inositol (DCI) is the main treatment available for PCOS in humans. To address some of the mechanisms of this complex disorder and its treatment, this study examines the effect of DCI on reproduction during the development of different PCOS-associated phenotypes in aged females and two mouse models of PCOS. Aged females (8 months old) were treated or not (control) with DCI for 2 months. PCOS models were generated by treatment with dihydrotestosterone (DHT) on Days 16, 17, and 18 of gestation, or by testosterone propionate (TP) treatment on the first day of life. At two months of age, PCOS mice were treated with DCI for 2 months and their reproductive parameters analyzed. No effects of DCI treatment were produced on body weight or ovary/body weight ratio. However, treatment reduced the number of follicles with an atretic cyst-like appearance and improved embryo development in the PCOS models, and also increased implantation rates in both aged and PCOS mice. DCI modified the expression of genes related to oocyte quality, oxidative stress, and luteal sufficiency in cumulus-oocyte complexes (COCs) obtained from the aged and PCOS models. Further, the phosphorylation of AKT, a main metabolic sensor activated by insulin in the liver, was enhanced only in the DHT group, which was the only PCOS model showing glucose intolerance and AKT dephosphorylation. The effect of DCI in the TP model seemed mediated by its influence on oxidative stress and follicle insufficiency. Our results indicate that DCI works in preclinical models of PCOS and offer insight into its mechanism of action when used to treat this infertility-associated syndrome.

Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality.

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 µM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 µM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

Longitudinal analysis of somatic and germ-cell telomere dynamics in outbred mice.

Although telomere length (TL) shortens with age in most tissues, an age-related increase in length has been described in sperm through a mechanism that is not yet fully understood. Changes in TL with age in the same individual have not been explored. This longitudinal study examines TL dynamics in somatic tissue and gametes during an entire lifespan in an outbred mouse population (from 8 to up to 114 weeks of age). Our findings indicate a reduced life expectancy in males compared to females (84.75 ± 9.23 vs. 113.16 ± 0.20 weeks) and significant variability in TL dynamics between individuals. While with aging, a clear reduction in TL was produced in somatic cells and oocytes, telomeres in sperm cells significantly lengthened. Finally, we found evidence indicating that telomere elongation in sperm during aging may be dependent on different mechanisms, such as the survival of spermatogonia with longer telomeres and the alternative lengthening of telomeres mechanism in meiotic and postmeiotic spermatogenic cells.

Embryo responses to stress induced by assisted reproductive technologies.

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.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

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 (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu 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, Zrsr1mu 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.

The oviduct: from sperm selection to the epigenetic landscape of the embryo.

The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed.

Early sex-dependent differences in response to environmental stress.

Developmental plasticity enables the appearance of long-term effects in offspring caused by exposure to environmental stressors during embryonic and foetal life. These long-term effects can be traced to pre- and post-implantation development, and in both cases, the effects are usually sex specific. During preimplantation development, male and female embryos exhibit an extensive transcriptional dimorphism mainly driven by incomplete X chromosome inactivation. These early developmental stages are crucial for the establishment of epigenetic marks that will be conserved throughout development, making it a particularly susceptible period for the appearance of long-term epigenetic-based phenotypes. Later in development, gonadal formation generates hormonal differences between the sexes, and male and female placentae exhibit different responses to environmental stressors. The maternal environment, including hormones and environmental insults during pregnancy, contributes to sex-specific placental development that controls genetic and epigenetic programming during foetal development, regulating sex-specific differences, including sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review summarizes several human and animal studies examining sex-specific responses to environmental stressors during both the periconception period (caused by differences in sex chromosome dosage) and placental development (caused by both sex chromosomes and hormones). The identification of relevant sex-dependent trajectories caused by sex chromosomes and/or sex hormones is essential to define diagnostic markers and prevention/intervention protocols.

Pyruvate antioxidant roles in human fibroblasts and embryonic stem cells.

Oxidative stress has been related to multiple diseases, especially during early embryonic development, when environmental alterations can lead to long-term deleterious effects. In vitro studies of oxidative stress have been mainly focused on somatic cells, but embryonic stem cells (ESCs) represent a promising model of early embryonic development as they are the in vitro equivalent to pluripotent cells in the embryo. Human fibroblasts and ESCs were exposed to different pro-oxidant agents (hydrogen peroxide, tert-butyl hydroperoxide (TBHP), and rotenone) and antioxidants (sodium pyruvate, N-acetylcysteine, Trolox, and sodium selenite) during a 72 h oxidative stress treatment. Then, cell viability, oxidative stress, mitochondrial activity, and gene expression were analyzed, focusing on the antioxidant effect of pyruvate. Pyruvate protected both somatic and pluripotent cells against different pro-oxidant agents, showing strong ROS scavenging capacity, protecting mitochondrial membrane potential, and regulating gene expression and cell metabolism through different mechanisms in fibroblasts and ESCs. In fibroblasts, pyruvate avoided NFKß nuclear translocation and the upregulation of genes related to the oxidative stress response, while in ESCs pyruvate stimulated the expression of genes involved in anaerobic glycolysis. Fibroblasts and ESCs reacted in different ways to oxidative stress and antioxidant treatment, and pyruvate was the most complete antioxidant, protecting both cell types at different levels.

Elimination of methylation marks at lysines 4 and 9 of histone 3 (H3K4 and H3K9) of spermatozoa alters offspring phenotype.

The contribution of the contents of spermatozoa to the development of the embryo is currently being considered wider than was previously thought. Recent findings point to the participation of epigenetic marks present in the retained histones of mature spermatozoa on embryo and fetal development. Here we created a novel conditional transgenic mouse that expresses lysine (K) demethylase 1a (Kdm1a) during spermatogenesis when the testicles are subjected to heat stress. Using these animals under these conditions we were able to reduce the methylation level of histone 3 at lysines 4 and 9 (H3K4 and H3K9, respectively) in mature spermatozoa. The offspring of these transgenic mice were followed for correct development and growth after birth. We found that the offspring of males expressing Kdm1a suffered 20% of reabsorptions at Day 15 after implantation (vs 0.3% in the control). In addition, 35% of the offspring sired by these males showed some kind of abnormality (suckling defects, lack of movement coordination, dropping forelimbs, abnormal body curvature, absence of eyes, gigantisms and neuromuscular defects) and 25% died before postnatal Day 21. Some abnormalities were maintained to adulthood. These results show that alteration of epigenetic marks present in the retained histones of mature spermatozoa affect fetal development and have phenotypic consequences in the newborn.

The effect of human follicular fluid on bovine oocyte developmental competence and embryo quality.

In this study, the hypothesis that embryo development during routine IVF procedures is determined by the pre-ovulatory follicular fluid composition was tested. Follicular fluid from women with obesity ('obese') and a 'positive' or 'negative' IVF outcome was added during the in-vitro maturation of bovine oocytes. 'Negative' and 'obese' follicular fluid reduced bovine embryo development, compared with laboratory control embryo development (P < 0.05 or P < 0.1). The addition of follicular fluid also altered bovine blastocyst gene expression. Furthermore, LDHA and PPARGC1B gene expression differed between follicular fluid groups. Data suggest that pre-ovulatory follicular fluid can potentially affect oocyte developmental competence and embryo quality. Furthermore, the bovine model may be used as a screening tool.

Characterisation of the deleted in azoospermia like (Dazl)-green fluorescent protein mouse model generated by a two-step embryonic stem cell-based strategy to identify pluripotent and germ cells.

The deleted in azoospermia like (Dazl) gene is preferentially expressed in germ cells; however, recent studies indicate that it may have pluripotency-related functions. We generated Dazl-green fluorescent protein (GFP) transgenic mice and assayed the ability of Dazl-driven GFP to mark preimplantation embryo development, fetal, neonatal and adult tissues, and in vitro differentiation from embryonic stem cells (ESCs) to embryoid bodies (EBs) and to primordial germ cell (PGC)-like cells. The Dazl-GFP mice were generated by a two-step ESC-based strategy, which enabled primary and secondary screening of stably transfected clones before embryo injection. During preimplantation embryo stages, GFP was detected from the zygote to blastocyst stage. At Embryonic Day (E) 12.5, GFP was expressed in gonadal ridges and in neonatal gonads of both sexes. In adult mice, GFP expression was found during spermatogenesis from spermatogonia to elongating spermatids and in the cytoplasm of oocytes. However, GFP mRNA was also detected in other tissues harbouring multipotent cells, such as the intestine and bone marrow. Fluorescence was maintained along in vitro Dazl-GFP ESC differentiation to EBs, and in PGC-like cells. In addition to its largely known function in germ cell development, Dazl could have an additional role in pluripotency, supporting these transgenic mice as a valuable tool for the prospective identification of stem cells from several tissues.

An efficient system to establish biopsy-derived trophoblastic cell lines from bovine embryos.

Trophoblastic cells play a crucial role in implantation and placentogenesis and can be used as a model to provide substantial information on the peri-implantation period. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stocks in the long-term. Our results show that the combination of a monolayer culture system in microdrops on a surface treated with gelatin and the employment of conditioned media from mouse embryonic fibroblasts support the growth of bovine trophoblastic cells lines from an embryo biopsy. Expression profiles of mononucleate- and binucleate-specific genes in established trophoblastic cells lines represented various stages of gestation. Moreover, the ability to expand trophoblastic cell lines for more than 2 yr together with pluripotency-related gene expression patterns revealed certain self-renewal capacity. In summary, we have developed a system to expand in vitro trophoblastic cells from an embryo biopsy that solves the limitations of using amplified DNA from a small number of cells for bovine embryo genotyping and epigenotyping and, on the other hand, facilitates the establishment of trophoblastic cell lines that can be useful as peri-implantation in vitro models.

Germ-cell culture conditions facilitate the production of mouse embryonic stem cells.

The derivation of embryonic stem-cell (ESC) lines from blastocysts is a very inefficient process. Murine ESCs are thought to arise from epiblast cells that are already predisposed to a primordial-germ-cell fate. During the process of ESC derivation from B6D2 F1 hybrid mice, if we first culture the embryo from the two-cell stage in medium supplemented with LIF, we improve the quality of the blastocyst. When the blastocyst is then cultured in a germ-line stem-cell culture medium (GSCm), we are able to more efficiently (28.3%) obtain quality ESC lines that have a normal karyotype, proper degree of chimerism, and exhibit germ-line transmission when microinjected into blastocysts. Although germ-cell-specific genes were expressed in all culture medium conditions, GSCm did not shift the transcriptome towards germ-cell specification. A correlation was further observed between ESC derivation efficiency and the expression of some imprinted genes and retrotransposable elements. In conclusion, the combination of LIF supplementation followed by culture in GSCm establishes a higher efficiency method for ESC derivation.

Transforming growth factor beta (TGFß) pathway is essential for hypoblast and epiblast development in ovine post-hatching embryos.

The developmental failures occurring between blastocyst hatching and implantation in farm ungulates are a major cause of pregnancy losses. At the expanded blastocyst stage, three cell lineages emerge in the embryo: trophoblast, hypoblast and epiblast, the latter being the most vulnerable during post-hatching development. Transforming growth factor beta (TGFß) signaling pathway is involved in hypoblast and epiblast development; however, previous in vitro functional studies are limited to the expanded blastocyst stage. In this study, we have analyzed the effect of TGFß inhibition with 10, 20 or 40 µM SB431542 during ovine post-hatching developmental period using a recently developed culture system able to recapitulate major developmental landmarks. We have found a negative effect of TGFß inhibition on hypoblast and epiblast development that could be partially reverted by Rho-associated protein kinase (ROCK) inhibitor Y-27632. Our findings provide new insights into the molecular networks regulating embryo development beyond the expanded blastocyst and could help to elucidate the causes of early pregnancy losses in farm ungulates.

Loss of the importin Kpna2 causes infertility in male mice by disrupting the translocation of testis-specific transcription factors.

Karyopherins mediate the movement between the nucleus and cytoplasm of specific proteins in diverse cellular processes. Through a loss-of-function approach, we here examine the role of Karyopherin Subunit Alpha 2 (Kpna2) in spermatogenesis. Knockout male mice exhibited reduced body size and sperm motility, increased sperm abnormalities, and led to the dysregulation of testis gene expression and ultimately to infertility. Impaired mRNA expression mainly affected clusters of genes expressed in spermatids and spermatocytes. Downregulated genes included a set of genes that participate in cell adhesion and extracellular matrix (ECM) organization. We detected both the enrichment of some transcription factors that bind to regions around transcription start sites of downregulated genes and the impaired transport of specific factors to the nucleus of spermatid cells. We propose that Kpna2 is essential in the seminiferous tubules for promoting the translocation of testis-specific transcription factors that control the expression of genes related to ECM organization.