Eline Wydooghe

Differential apoptotic staining of mammalian blastocysts based on double immunofluorescent CDX2 and active caspase-3 staining

Several approaches have been described for differential staining of blastocysts, but these methods are often time-consuming and unreliable. Here we describe a method for simultaneous differential staining and detection of apoptosis. The differential staining is based on the transcription factor CDX2 which is localized in the nucleus of trophectoderm (TE) cells but absent in the inner cell mass (ICM). Apoptosis is detected by staining of active caspase-3, a key player in several apoptotic pathways. This new approach represents a robust method for quantifying simultaneously ICM/TE ratio and apoptotic cell ratio in bovine, murine, porcine, and human blastocysts.

Replacing serum in culture medium with albumin and insulin, transferrin and selenium is the key to successful bovine embryo development in individual culture

Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality.

Influence of the uterine environment on the development of in vitro produced equine embryos

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.

Autocrine embryotropins revisited: how do embryos communicate with each other in vitro when cultured in groups?

In the absence of the maternal genital tract, preimplantation embryos can develop in vitro in culture medium where all communication with the oviduct or uterus is absent. In several mammalian species, it has been observed that embryos cultured in groups thrive better than those cultured singly. Here we argue that group‐cultured embryos are able to promote their own development in vitro by the production of autocrine embryotropins that putatively serve as a communication tool. The concept of effective communication implies an origin, a signalling agent, and finally a recipient that is able to decode the message. We illustrate this concept by demonstrating that preimplantation embryos are able to secrete autocrine factors in several ways, including active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles. Likewise, we broaden the traditional view that inter‐embryo communication is dictated mainly by growth factors, by discussing a wide range of other biochemical messengers including proteins, lipids, neurotransmitters, saccharides, and microRNAs, all of which can be exchanged among embryos cultured in a group. Finally, we describe how different classes of messenger molecules are decoded by the embryo and influence embryo development by triggering different pathways. When autocrine embryotropins such as insulin‐like growth factor‐I (IGF‐I) or platelet activating factor (PAF) bind to their appropriate receptor, the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) pathway will be activated which is important for embryo survival. On the other hand, the mitogen‐activated protein kinase (MAPK) pathway is activated when compounds such as hyaluronic acid and serotonin bind to their respective receptors, thereby acting as growth factors. By activating the peroxisome‐proliferator‐activated receptor family (PPAR) pathway, lipophilic autocrine factors such as prostaglandins or fatty acids have both survival and anti‐apoptotic functions. In conclusion, considering different types of messenger molecules simultaneously will be crucial to understanding more comprehensively how embryos communicate with each other in group‐culture systems. This approach will assist in the development of novel media for single‐embryo culture.

Autocrine embryotropins revisited: how do embryos communicate with each other in vitro when cultured in groups?: Autocrine embryotropins revisited

In the absence of the maternal genital tract, preimplantation embryos can develop in vitro in culture medium where all communication with the oviduct or uterus is absent. In several mammalian species, it has been observed that embryos cultured in groups thrive better than those cultured singly. Here we argue that group‐cultured embryos are able to promote their own development in vitro by the production of autocrine embryotropins that putatively serve as a communication tool. The concept of effective communication implies an origin, a signalling agent, and finally a recipient that is able to decode the message. We illustrate this concept by demonstrating that preimplantation embryos are able to secrete autocrine factors in several ways, including active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles. Likewise, we broaden the traditional view that inter‐embryo communication is dictated mainly by growth factors, by discussing a wide range of other biochemical messengers including proteins, lipids, neurotransmitters, saccharides, and microRNAs, all of which can be exchanged among embryos cultured in a group. Finally, we describe how different classes of messenger molecules are decoded by the embryo and influence embryo development by triggering different pathways. When autocrine embryotropins such as insulin‐like growth factor‐I (IGF‐I) or platelet activating factor (PAF) bind to their appropriate receptor, the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) pathway will be activated which is important for embryo survival. On the other hand, the mitogen‐activated protein kinase (MAPK) pathway is activated when compounds such as hyaluronic acid and serotonin bind to their respective receptors, thereby acting as growth factors. By activating the peroxisome‐proliferator‐activated receptor family (PPAR) pathway, lipophilic autocrine factors such as prostaglandins or fatty acids have both survival and anti‐apoptotic functions. In conclusion, considering different types of messenger molecules simultaneously will be crucial to understanding more comprehensively how embryos communicate with each other in group‐culture systems. This approach will assist in the development of novel media for single‐embryo culture.

Proteome of equine oviducal fluid: effects of ovulation and pregnancy

The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC-tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways.

Emerging role of extracellular vesicles in communication of preimplantation embryos in vitro

In vitro, efficient communication between mammalian embryos in groups or between embryos and cocultured somatic cells implies that there is a sender, a message and a receiver that is able to decode the message. Embryos secrete a variety of autocrine and paracrine factors and, of these, extracellular vesicles have recently been implicated as putative messengers in embryo-embryo communication, as well as in communication of the embryo with the maternal tract. Extracellular vesicles (EVs) are membrane-bound vesicles that are found in biofluids and in culture media conditioned by the presence of embryos or cells. EVs carry and transfer regulatory molecules, such as microRNAs, mRNAs, lipids and proteins. We conducted a systematic search of the literature to review and present the currently available evidence regarding the possible roles of EVs in in vitro embryo communication and embryo development. It is important to note that there is limited information available on the molecular mechanisms and many of the biologically plausible functions of EVs in embryo communication have not yet been substantiated by conclusive experimental evidence. However, indirect evidence, such as the use of media conditioned by embryos or by somatic cells with improved embryo development as a result, may indicate that EVs can be an important asset for the development of tailor-made media, allowing better embryo development in vitro, even for single embryo culture.

The Importance of Adequate Fixation for Immunofluorescent Staining of Bovine Embryos

Immunofluorescent staining is often used to investigate the expression of specific proteins in pre-implantation embryos. The success of this method is determined by the specificity of the antibodies, but also by the protocol used for fixation and permeabilization of the samples. In this study, different fixatives are compared in combination with immunofluorescent staining of caudal-type homeobox 2 (CDX2), fibronectin 1 (FN1) and integrins (ITGs) on bovine blastocysts. For both CDX2 and the ITGs, the outcome of the staining was largely dependent on the fixation methods. Paraformaldehyde fixation was best for the intracellular CDX2 protein, whereas acetone fixation gave the best results for the transmembrane ITGs. No difference was observed for the FN1 staining between samples fixed with paraformaldehyde or acetone. These examples demonstrate that the choice of fixation and permeabilization agents is very important for the outcome of the experiment, and this choice is dictated by the (extra)cellular location of the protein under investigation. Inappropriate fixation and/or permeabilization methods can lead to erroneous conclusions regarding the site and amount of protein expression.

Alpha-linolenic acid protects the developmental capacity of bovine cumulus–oocyte complexes matured under lipotoxic conditions in vitro

Abstract Elevated concentrations of free fatty acids (FFAs), predominantly palmitic, stearic, and oleic acids (PSO), exert detrimental effects on oocyte developmental competence. This study examined the effects of omega-3 alpha-linolenic acid (ALA) during in vitro oocyte maturation (IVM) in the presence of PSO on subsequent embryo development and quality, and the cellular mechanisms that might be involved. Bovine cumulus–oocyte complexes (COCs) were supplemented during IVM with ALA (50 µM), PSO (425 µM), or PSO+ALA. Compared with FFA-free controls (P < 0.05), PSO increased embryo fragmentation and decreased good quality embryos on day 2 postfertilization. Day 7 blastocyst rate was also reduced. Day 8 blastocysts had lower cell counts and higher apoptosis but normal metabolic profile. In the PSO group, cumulus cell (CC) expansion was inhibited with an increased CC apoptosis while COC metabolism was not affected. Mitochondrial inner membrane potential (MMP; JC-1 staining) was reduced in the CCs and oocytes. Heat shock protein 70 (HSP70) but not glucose-regulated protein 78 kDa (GRP78, known as BiP; an endoplasmic reticulum stress marker) was upregulated in the CCs. Higher reactive oxygen species levels (DCHFDA staining) were detected in the oocytes. In contrast, adding ALA in the presence of PSO normalized embryo fragmentation, cleavage, blastocyst rates, and blastocyst quality compared to controls (P > 0.05). Combined treatment with ALA also reduced CC apoptosis, partially recovered CC expansion, abrogated the reduction in MMP in the CCs but not in the oocytes, and reduced BiP and HSP70 expression in CCs, compared with PSO only (P < 0.05). In conclusion, ALA supplementation protected oocyte developmental capacity under lipotoxic conditions mainly by protecting cumulus cell viability. Summary Sentence Alpha-linolenic acid protects cumulus cell viability and oocyte quality during in vitro maturation under lipotoxic conditions, which results in an improvement of early embryo quality and blastocyst development.

Alternative models for the study of embryo-maternal cross-talk and signaling molecules from fertilisation to implantation.

In nature, mammalian life is conceived inside the female genital tract, more specifically in the oviduct. Because the processes of fertilisation and early preimplantation development take place in one of the most inaccessible parts of the mammalian body, it has been studied predominantly in vitro. In static culture platforms, the environmental conditions to which the gametes and embryos are exposed are in sharp contrast to what is observed in vivo. In this Research Front on embryo–maternal interactions, we present four reviews that investigate model systems for the study of embryo–maternal cross-talk and the processes that are known to be important in the period between fertilisation and implantation. The Research Front is one of the outcomes of a European Union-funded COST (European Cooperation in Science and Technology) Action on ‘Maternal Interaction with Gametes and Embryos’ (FA0702; http://www.cost-gemini.eu, accessed 20 September 2011), which aims to promote understanding of this topic by bringing together researchers to share data from different species. In the female reproductive tract, embryos are surrounded by a constantly changing minimum amount of media and are constantly moved by ciliated epithelia. The preimplantation embryo, in vivo, develops in the absence of direct cell contact with the reproductive tract before implanting. It is free-floating, lacks a blood supply and is dependent on luminal secretions of the oviduct and uterus for its nutrition. The preimplantation embryo expresses several receptors for signalling ligands (O’Neill 2008). These signalling ligands are often paracrine factors, defined as factors that are secreted by one cell type and that execute their function on another cell type. They can originate from cells of the reproductive tract (e.g. cytokines) and have an effect on the embryo, or can be secreted by the embryo and have an effect on the oviduct or uterus (Orsi and Tribe 2008). It is clear that these paracrine factors are crucial in the embryo–maternal dialogue. The importance of normal embryo–maternal interaction is evidenced by the finding that exposure of ruminant embryos to a suboptimal environment can lead to the so-called large offspring syndrome: affected offspring show changes in phenotype, such as having twice to five times increased birthweights. In many cases breathing difficulties, reluctance to suckle and sudden perinatal death can occur and the severity of the syndrome is influenced by culture conditions and animal species (Farin et al. 2010). In humans, assisted reproduction has been associated with increased risk of imprinting diseases such as Beckwith– Wiedemann syndrome (Owen and Segars 2009). Abnormal development originates from epigenetic changes in imprinted genes and epigenetically sensitive alleles (for review, see Jammes et al. 2011), and hypothetically this can be caused by exposure to unwanted signalling molecules during a potential window of vulnerability in development. These cases of abnormal embryonic, fetal and neonatal development illustrate the pressing need to understand what happens at the time of fertilisation and during the first days and weeks of life. What is the best approach to study these signallingmolecules and in which species should they be studied? If we first focus on the species, it seems that the mouse has traditionally been the most popular model specific for the human (or even for mammals in general). This is based on the fact that mice are highly productive (reaching sexual maturity early (6–8 weeks) and producing many offspring per litter). They exhibit a similar placentation to humans (i.e. hemochorial) (Rosenfeld 2010). However, for studying signalling molecules in connection with embryo–maternal interactions, the mouse may not be the best choice after all. Most mouse strains are inbred, genetically almost identical and therefore not comparable to humans, which are markedly diverse, with genetic and epigenetic variability. Moreover, the mouse genome is dissimilar from that of humans, in that the number of unique orthologous groups is greater for rodents than for several othermammalian species includingman (Hansen 2010). In this respect, farm animals – such as cattle, pigs and even horses – are a much more interesting group of model species for research in (human) reproduction, especially when one wants to focus on signalling ligands. Another important recent development is that the advancement in molecular tools has led to the complete sequencing of the genomes of cattle (Larkin 2011), pigs (Fan et al. 2011) and horses (Chowdhary and Raudsepp 2008). The horse represents a valuable model for human infertility for several reasons: (1) breeding sport horses is often postponed to later ages, and this is associated with reduced fertility, both in mares and stallions, (2) such breeding horses can successfully be treated with intracytoplasmic sperm injection and embryo CSIRO PUBLISHING