Welcome to the GAMETOGENESIS and EARLY EMBRYONIC DEVELOPMENT lab
Molecular mechanisms regulating gametogenesis, fertilization, and early embryonic development
Group leader
Prof. A. Gutierrez-Adan
RESEARCH LINES
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Understand the genetic and epigenetic mechanisms that controls the early embryo development and their impact on fetal development and adult health.
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​Analysis of in vivo/in vitro sperm selection and techniques for assisted reproduction.
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​Determine the role of mRNA splicing and ncRNAs on regulation of embryo development, sex determination, gametogenesis, and fertility.
Recent publications
Genome‑wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline
I Gómez-Redondo; B Planells; S Canovas; E Ivanova; G Kelsey; A Gutiérrez-Adán (2021)
Clinical Epigenetics 13:27. DOI: 10.1186/s13148-021-01003-x
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Background: Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming.
Results: Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magneticactivated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage.
Conclusions: Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.
I Gómez-Redondo, B Planells; P. Navarrete, A Gutiérrez-Adán (2021)
During the process of sex determination, a germ-cell-containing undifferentiated gonad is converted into either a male or a female reproductive organ. Both the composition of sex chromosomes and the environment determine sex in vertebrates. It is assumed that transcription level regulation drives this cascade of mechanisms; however, transcription factors can alter gene expression beyond transcription initiation by controlling pre-mRNA splicing and thereby mRNA isoform production. Using the key time window in sex determination and gonad development in mice, it has been reported that new non-transcriptional events, such as alternative splicing, could play a key role in sex determination in mammals. We know the role of key regulatory factors, like WT1(+/–KTS) or FGFR2(b/c) in pre-mRNA splicing and sex determination, indicating that important steps in the vertebrate sex determination process probably operate at a post-transcriptional level. Here, we discuss the role of pre-mRNA splicing regulators in sex determination in vertebrates, focusing on the new RNA-seq data reported from mice fetal gonadal transcriptome.
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The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.
Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion.
I Gómez-Redondo , P Ramos-Ibeas , E Pericuesta, R Fernández-González,
R Laguna-Barraza and A Gutiérrez-Adán (2020). IJMS 21(11), 4115
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.
Gene expression profiles of bovine genital ridges during sex determination and early differentiation of the gonads
Benjamín Planells, Isabel Gómez-Redondo, José María Sánchez, Michael McDonald, Ángela Cánovas, Patrick Lonergan, Alfonso Gutiérrez-Adán (2019) Biology of Reproduction 2019
Differential isoform expression and alternative splicing in sex determination in mice
B Planells, I Gomez-Redondo, E Pericuesta, P Lonergan and A Gutiérrez-Adán (2019)
BMC Genomics 20:202.
Most current knowledge of sex determination in mammals has emerged from mouse and human studies. To investigate the molecular regulation of the sex determination process in cattle, we used an RNA sequencing strategy to analyze the transcriptome landscape of male and female bovine fetal gonads collected in vivo at key developmental stages: before, during, and after SRY gene activation on fetal days D35 (bipotential gonad formation), D39 (peak SRY expression), and D43 (early gonad differentiation). Differentially expressed genes (DEGs) were identified in male vs. female germinal ridges and among group genes showing similar expression profiles during the three periods. There were 143, 96, and 658 DEG between males and female fetuses at D35, D39, and D43, respectively. On D35, genes upregulated in females were enriched in translation, nuclear export, RNA localization, and mRNA splicing events, whereas those upregulated in males were enriched in cell proliferation regulation and male sex determination terms. In time-course experiments, 767 DEGs in males and 545 DEGs in females were identified between D35 vs. D39, and 3157 DEGs in males and 2008 in females were identified between D39 vs. D43. Results highlight unique aspects of sex determination in cattle, such as the expression of several Y chromosome genes (absent in mice and humans) before SRY expression and an abrupt increase in the nuclear expression of SOX10 (instead of SOX9 expression in the Sertoli cell cytoplasm as observed in mice) during male determination and early differentiation.
The oviduct: from sperm selection to the epigenetic landscape of the embryo.
S Pérez-Cerezales, P Ramos-Ibeas, O Salvador Acuña, M Avilés, P Coy, D Rizos and A Gutiérrez-Adán (2018) Biol Reprod 98(3) 262-276.
Mammalian oviducts host fertilization and preimplantation development of the embryo. Recent research signifies the importance of the oviduct on sperm selection in naturally conceived fertilization, and in genetic and epigenetic reprograming of pre-implantation embryo development. This review highlights oviductal fluid composition taken special emphasis in the exosomes, the role of oviduct on sperm selection, on early embryo development, and on modeling epigenetic landscape of the embryo, giving insight into potential areas for the improvement of assisted reproductive technologies.
Sperm selection by thermotaxis improves ICSI outcome in mice. Pérez-Cerezales, S., Laguna-Barraza, R., Chacón de Castro, A., Sánchez-Calabuig, MJ., Cano-Oliva, E., de Castro-Pita, FJ., Montoro-Buils, L., Pericuesta, E., Fernández-González, R., Gutiérrez-Adán, A. Sci. Reports. 2018.(8):2902.
Alternative splicing (AS) may play an important role in gonadal sex determination (GSD) in mammals. The present study was designed to identify differentially expressed isoforms and AS modifications accompanying GSD in mice. By deep RNA-seq, we performed a transcriptional analysis of XX and XY gonads during sex determination on embryonic days 11 (E11) and 12 (E12). Through differentially expressed gene (DEG) analysis we identified hundreds of genes related to GSD and early sex differentiation that could be good candidates for sex reversal. Expression at time point E11 in males was significantly enriched in RNA splicing and mRNA processing gene ontology terms. Differentially expressed isoform analysis identified hundreds of specific isoforms related to GSD, many of which showed no differences in the DEG analysis. Hundreds of AS events were identified as modified at E11 and E12. Female E11 gonads featured sex-biased upregulation of intron retention (in genes related to regulation of transcription, protein phosphorylation, protein transport and mRNA splicing) and exon skipping (in genes related to chromatin repression) suggesting AS as a post-transcription mechanism that controls sex determination of the bipotential fetal gonad. Our data indicate an important role of splicing regulatory mechanisms for sex determination in mice.
Sex-dimorphic behavioral alterations and altered neurogenesis in U12 intron splicing-defective Zrsr1 mutant mice. F Alén, I Gómez-Redondo, P Rivera, J Suárez, P Ramos-Ibeas, E Pericuesta, R Fernández-González, S Perez-Cerezales, K Horiuchi, L Orio, F Rodriguez de Fonseca and A Gutiérrez-Adán (2019). IJMS 20, 3543
Mutant mice 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 spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to glutathione metabolic process, synaptotemal complex assembly, mRNA transport, and altered splicing events involving enrichment of U12-type intron retention (IR). Furthermore, increased IR in U-12 containing genes related with prolactin, progesterone and 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 is relevant for organizing the hypothalamic cell network controlling behavior.
Impaired spermatogenesis, muscle and erythrocyte function in U12 intron splicingdefective
Zrsr1 mutant mice. Horiuchi, K., Perez-Cerezales, S., Papasaikas, P., Ramos-Ibeas, P., López-Cardona, AP., Laguna-Barraza, R., Fonseca-Balvís, N., Pericuesta, E., Fernández-González, R., Planells, B., Viera, A., Suja, JA., Ross, PJ., Alén, F., Orio, L., Rodriguez de Fonseca, F., Pintado, B., Valcárcel, J., Gutiérrez-Adán, A., Cell Reports. 2018. 2, 1-13.
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.
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-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.
