Sonia Heras

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.

Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts

BackgroundSince the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production.ResultsWe used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism.ConclusionsSerum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.

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.

Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism

ABSTRACT Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1–5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage.

Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI

BackgroundGlobal epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining.ResultsWe were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes.ConclusionsIn conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development.

Asymmetric histone 3 methylation pattern between paternal and maternal pronuclei in equine zygotes.

Hoechst staining has traditionally been used to evaluate fertilization and parental origin of pronuclei. However, prevalence of parthenogenetic activation cannot be distinguished accurately by this protocol, and variation of relative pronuclear size and position makes it impossible to determine parental origin. We demonstrate that in equine zygotes, the epigenetic modification histone 3 lysine 9 trimethylation (H3K9me3) shows an asymmetric pattern between maternal and paternal pronuclei. H3K9me3 immunostaining appears to be a robust technique to identify the parent of origin of equine pronuclei; it can be used in combination with 5-methylcytosine and 5-hydroxymethylcytosine immunostaining and applied to evaluate fertilization.

Determination of the parental pronuclear origin in bovine zygotes: H3K9me3 versus H3K27me2-3

To study the dynamics of 5-methylcytosine and 5-hydroxymethylcytosine in zygotes, the parental origin of the pronuclei needs to be determined. To this end the use of the asymmetric distribution of histone modifications in pronuclei is becoming more popular. Here, we demonstrated that histone 3 lysine 27 di-tri-methylation shows a stable pattern being present in the maternal but not in the paternal pronucleus of bovine zygotes, even in late stages of pronuclear development. In contrast, the pattern of histone 3 lysine 9 tri-methylation is very variable, and therefore cannot be used to reliably determine the parental origin of bovine pronuclei.

UROKINASE-TYPE PLASMINOGEN ACTIVATOR DOES NOT AFFECT IN VITRO BOVINE EMBRYO DEVELOPMENT AND QUALITY

The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems.

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