Pablo Bermejo-Alvarez

• Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria

Factor V deficiency is a congenital coagulation disorder characterized by the absence or malfunction of factor V (FV). The purpose of this study was to develop a viable FV-deficient mouse model using CRISPR/Cas9 technology. A viable pathological model of the disease was not available to develop new therapies. A previous in silico study was performe...

In brief GATA3 is a transcription factor exclusively expressed by the trophectoderm. In this paper, we demonstrate that although GATA3 ablation is dispensable for trophectoderm specification and blastocyst formation in sheep, its ablation negatively affects epiblast survival during post-hatching development, making the first such observation in non...

This work represents a retrospective and updated review in a scientific context of itself, the Advanced Therapies group of the Complutense University of Madrid. The project, which began almost 7 years ago, has pursued the establishment of gene and cell therapy protocols for the treatment of coagulation factor V deficiency. This “trip” has been poss...

In brief Bovine embryos lacking SMC2 (a core component of condensins I and II) are unable to survive maternal recognition of pregnancy. SMC2 KO embryos are able to form blastocysts, exhibiting a reduced cell proliferation ability, and arrest their development shortly after hatching. Abstract Condensins are large protein complexes required for chro...

The first lineage differentiation in mammals gives rise to the inner cell mass (ICM) and the trophectoderm (TE). In mice, TEAD4 is a master regulation of TE commitment, as it regulates the expression of other TE-specific genes and its ablation prevents blastocyst formation, but its role in other mammals remains unclear. Herein, we have observed tha...

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the...

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 blasto...

Assisted reproductive technologies are key to maintain genetic stocks of endangered wild species, such as the Iberian ibex (Capra pyrenaica). Due to the low availability of ibex ovaries, heterologous in vitro fertilization (IVF) with domestic goat (Capra hircus) oocytes constitutes an excellent alternative to determine the fertilization capacity of...

Study question: Is the abundance of certain biochemical compounds in human cumulus cells (CCs) related to oocyte quality? Summary answer: Malonate, 5-oxyproline, and erythronate were positively associated with pregnancy potential. What is known already: CCs are removed and discarded prior to ICSI, thereby constituting an interesting biological...

Context Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. Aims To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro–produced bovine embryos. Methods Pre-hatching effects of AA were tested by culturing bovine zygotes in synthe...

Ungulate embryos undergo critical cell differentiation and proliferation events around and after blastocyst hatching. Failures in these processes lead to early pregnancy losses, which generate an important economic impact on farming. Conventional embryo culture media, such as SOF, are unable to support embryo development beyond hatching. In contras...

Research question: Is the transcriptome of cumulus cells a good predictor of the embryo's developmental competence? Design: Cumulus cells were collected from donor oocytes and their transcriptome was analysed by RNA sequencing analysis at >30 × 106 reads in samples grouped according to the developmental potential of their enclosed oocyte: not ab...

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...

STUDY QUESTION Is relative mitochondrial DNA (mtDNA) content in cumulus cells related to embryo developmental competence in humans and/or the bovine model? SUMMARY ANSWER mtDNA content in cumulus cells provides poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer. WHAT IS KNOWN ALREADY Cumulus cells...

Cumulus cells provide an interesting biological material to perform analyses to understand the molecular clues determining oocyte competence. The objective of this study was to analyze the transcriptional differences between cumulus cells from oocytes exhibiting different developmental potentials following individual in vitro embryo production by R...

Study question What is the maximum particle size that can traverse through human zona pellucida (ZP) and that of other mammals? Summary answer Human oocytes ZP is impermeable to 40-100 nm nanospheres. ZP of other mammals is impermeable to 100 nm particles, and permeability changes along preimplantation development. What is known already ZP is a p...

Fertilization involves the fusion of two gametes by means of yet unknown membrane binding and fusion events. Over the last years, many sperm proteins have been uncovered to play essential roles in sperm-egg fusion in mammals, but their precise role in fertilization remains unknown, being unclear how these proteins interact with each other or with o...

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 form...

Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzona...

Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term...

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike...

Cumulus cells play fundamental metabolic roles during folliculogenesis, being closely connected to the oocyte through transzonal projections. These oocyte-supporting cells are removed and discarded before intracytoplasmic sperm injection (ICSI), thereby constituting an interesting biological material on which to perform molecular analyses aimed to...

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo...

Prostaglandins (PGs) are lipid signalling molecules that play critical roles in gestation by promoting corpus luteus maintenance or luteolysis, and have been suggested to play other roles in early pregnancy, including embryo–maternal crosstalk. The signalling roles of PGs and other lipids are often mediated by peroxisome proliferator-activated rece...

Current advances on in vitro fertilization (IVF) techniques in pig, allow the generation of embryos with improved quality, enabling the use of IVF as a means to produce genetically modified pigs. In contrast to in vivo production, IVF allows precise control of microinjection timing of gene editing tools with respect to fertilization and the beginni...

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 (P...

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-e...

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-e...

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-e...

Considerable variation in fertility exists between bulls in AI centres, despite passing minimum post-thaw quality control checks. The development of a reliable in vitro test to predict bull fertility could enable the identification and selection of high fertility bulls, without the need to resort to test inseminations. An in-depth knowledge of the...

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved...

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In t...

Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that...

Physical exercise has positive effects on cognition, but very little is known about the inheritance of these effects to sedentary offspring and the mechanisms involved. Here, we use a patrilineal design in mice to test the transmission of effects from the same father (before or after training) and from different fathers to compare sedentary- and ru...

The ablation (KO) or targeted insertion (KI) of specific genes or sequences has been essential to test their roles on a particular biological process. Unfortunately, such genome modifications have been largely limited to the mouse model, as the only way to achieve targeted mutagenesis in other mammals required from somatic cell nuclear transfer, a...

In vitro maturation (IVM) leads to reduced developmental rates compared to the use of in vivo matured oocytes. This reduction can be attributed to the suboptimal environment experienced during IVM, but the use of incompetent oocytes also plays a significant role. The objective of this study has been to characterize the mitochondrial and metabolic d...

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim...

Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, t...

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Heterogametic sex determination systems, where one sex is homogametic –XX or ZZ– and the other is heterogametic –XY or ZW–, entail a transcriptional dosage problem: in the absence of a compensatory mechanism, the homogametic sex expresses a double dose of those genes allocated on the duplicated sex chromosome. Eutherian mammals have evolved an epig...

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 105 EV/mL from a...

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts...

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mu...

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C–) supplemented with OF or 5% fetal calf serum (positive control; C⁺). Embryo development was recorded on Days...

Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, sperm...

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C-) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days...

Artificial reproductive techniques are currently responsible for 1.7–4% of the births in developed countries and intracytoplasmatic sperm injection (ICSI) is the most commonly used, accounting for 70–80% of the cycles performed. Despite being an invaluable tool for infertile couples, the technique bypasses several biological barriers that naturally...

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial...

A high-glucose concentration in the reproductive tract during early development may result in aberrant embryo or fetal development, with effects that could have a greater impact on one sex than the other. Here, we determine if a high-glucose concentration impacts embryo development and pregnancy outcomes in a sex-specific manner in the mouse. Zygot...

Preimplantation developmental plasticity has evolved in order to offer the best chances of survival under changing environments. Conversely, environmental conditions experienced in early life can dramatically influence neonatal and adult biology, which may result in detrimental long-term effects. Several studies have shown that small size at birth,...

A critical issue for species integrity is the existence of a telomere elongation program during embryogenesis that ensures sufficient telomere reserves in mammalian newborns. Two different mechanisms have been reported to act on telomere elongation during early embryogenesis: first, the telomerase, the ribonucleoprotein that adds telomeric repeats...

Study question: Does maternal obesity affect estrous cyclicity, embryo development and blastocyst gene expression in mice? Summary answer: Maternal obesity alters estrous cyclicity and causes the down-regulation of two key metabolite receptors (Slc2a1 and Ldlr) in blastocysts recovered from diet-induced obese females, but embryo development is n...

Lentiviral mediated transgenesis of trophectoderm in mouse blastocysts is a powerful means for exploring the roles of a candidate gene in placenta. When zona-denuded blastocysts are transduced with lentiviral vectors carrying a transgene, the tight junctions of the epithelial trophectoderm impede access to the inner cell mass and restrict transgene...

A high-glucose concentration in the reproductive tract during early development may result in aberrant embryo or fetal development, with effects that could have a greater impact on one sex than the other. Here, we determine if a high-glucose concentration impacts embryo development and pregnancy outcomes in a sex-specific manner in the mouse. Zygot...

X-chromosome inactivation (XCI) is a complex epigenetic process that ensures that most X-linked genes are expressed equally for both sexes. Female eutherian mammals inactivate randomly the maternal or paternal inherited X-chromosome early in embryogenesis, whereas the extra-embryonic tissues experience an imprinting XCI that results in the inactiva...

Using the mouse model, intracytoplasmic sperm injection (ICSI)-mediated transgenesis has been shown to be a valuable tool for the production of transgenic animals, an essential instrument for basic and applied research in bioscience. This method of transgenesis consists of the microinjection of spermatozoa preincubated with foreign DNA. ICSI of DNA...

The establishment of telomere length (TL) during embryogenesis determines telomere reserves in newborn mammals. However, limited information is available on TL dynamics during preimplantation in contrast to the extensive existing data on telomerase activity in germ cells and embryogenesis. Telomerase activity is high in the male germ line, low or a...

Obesity is frequently associated with infertility in humans and domestic animals. In this study, we aimed to study the effect of diet-induced obesity on oestrus cyclicity, ovulation rate, pre-implantation embryo development and blastocysts gene expression. Twenty-one NIH Swiss female mice were divided in 2 groups: one fed with a control diet (C, n=...

Contents In the last years, enormous progress has been made in the analysis of gene transcription at the blastocyst stage. The study of gene expression at this early stage of development is challenging because of the very small amount of starting material, which limits the use of traditional mRNA analysis approaches such as Northern blot. Another p...

In Vitro Embryo Production. (DOC)

mRNA Expression Analysis. (DOC)

Details of primers used for qRT-PCR. (DOCX)

Amino Acid Profiling. (DOC)

During the in vitro maturation of adult bovine oocytes, leptin has beneficial effects on blastocyst development, apoptosis and transcription levels of developmentally important genes. The present study analyzes the differential effects of leptin on prepubertal bovine oocytes and cumulus cells. Effects were determined of leptin treatment during oocy...

The mouse is a convenient model to analyze the impact of in vitro culture (IVC) on the long-term health and physiology of the offspring, and the possible inheritance of these altered phenotypes. The preimplantation period of mammalian development has been identified as an early 'developmental window' during which environmental conditions may influe...

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte...

Studies concerning oocyte quality markers, oocyte/embryo metabolism or commercial OPU settings treating donors with low oocyte yields, indicate a need for optimization of IVP protocols to culture single oocytes to the blastocyst stage. However, culture conditions for single oocyte usually impair development, although previous research showed that s...

Preimplantation development is a key period for the establishment of epigenetic marks, as complete epigenetic reprogramming involving DNA methylation and histone modifications occurs from the gametes to the blastocyst stage. The changing epigenetic landscape makes preimplantation embryos especially vulnerable to modifications of the epigenome initi...

Food deprivation affects female reproduction. The goal of the present study was to elucidate in the rabbit model the effects of acute energy restriction on ovarian function (follicle development, atresia rate and in vitro oocyte maturation) and embryonic development and gene expression of some candidate genes. Serum metabolic parameters (non-esteri...

Sex chromosome transcripts can lead to a broad transcriptional sexual dimorphism in the absence of concomitant or previous exposure to sex hormones, especially when X-chromosome inactivation (XCI) is not complete. XCI timing has been suggested to differ greatly among species, and in bovine, most of the X-linked transcripts are upregulated in female...

In adult tissues, sexual dimorphism is largely attributed to sex hormone effects, although there is increasing evidence for a major role of sex chromosome dosage. During preimplantation development, male and female embryos can display phenotypic differences that can only be attributed to the transcriptional differences resulting from their differen...

Telomeres are repeated DNA regions that provide protection from enzymatic end-degradation and maintain chromosome stability during DNA replication. In most mammalian somatic cell types, telomeres shorten with each cell cycle. Telomerase, a reverse transcriptase that can elongate telomeres, adds telomeric repeats into chromosome ends, and is involve...

In bovine blastocysts, a large transcriptional sexual dimorphism affecting almost one-third of the genes is observed. At this early stage, sex chromosomes impose extensive transcriptional regulation on autosomal genes. However, little is known about the situation in further stages of embryo development. The aim of the present study was to determine...

In recent years, high nonesterified fatty acid (NEFA) concentrations have been intensively discussed as an important metabolic cue linking negative energy balance in high yielding dairy cows early postpartum to disappointing fertility. Previous research has shown that high NEFA concentrations during in vitro oocyte maturation not only induce signif...

Leptin is a metabolic hormone related to body condition and nutritional status that influences fertility in assisted reproductive technologies modulating oocyte and embryo quality. The aim of the present study was to establish the effect of various leptin concentrations (0, 10, 100 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo...

In somatic cell nuclear transfer, an overall epigenetic reprogramming must occur to abolish the somatic cell expression profile and to establish a new embryo-specific expression profile. The epigenetic reprogramming involves processes such as DNA methylation, histone modifications, X-chromosome inactivation and adjustment of telomere length. Telome...

In the recently published article “Amino acid metabolism of bovine blastocysts: A biomarker of sex and viability” by R. Sturmey et al. (Mol Reprod Dev, volume 77 (3): 285–296, 2010 [DOI: 10.1002/mrd.21145]), the Acknowledgment of grant support was inadvertently omitted. The omitted text is herein printed to address this error: RGS wishes to acknowl...

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocy...

This study evaluated the effect of two oxygen concentrations (20 and 5%) on bovine embryo development (kinetics of first cleavage and blastocyst development) during maturation (M) and fertilization (F) and analysed differences in gene expression between cumulus-oocyte complexes (COC) matured at 5 or 20% oxygen and the resulting blastocysts. A total...

Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-p...