Advancing bovine in vitro fertilization through 3D printing: the effect of the 3D printed materials.

Nowadays there is an increasing demand for assisted reproductive technologies due to the growth of infertility problems. Naturally, fertilization occurs in the oviduct, where the oviductal epithelial cells (OECs) secrete many molecules that affect the embryo's metabolism and protect it from oxidative stress. When the OECs are grown in 3D culture systems, they maintain a great part of their functional characteristics, making them an excellent model for in vitro fertilization (IVF) studies. In this work, we aimed to evaluate the suitability of different 3D-printing processes in conjunction with the corresponding set of commercially available biomaterials: extrusion-based processing using polylactic acid (PLA) and polycaprolactone (PCL) and stereolithography or digital-light processing using polyethylene-glycol-diacrylate (PEGDA) with different stiffness (PEGDA500, PEGDA200, PEGDA PhotoInk). All the 3D-printed scaffolds were used to support IVF process in a bovine embryo assay. Following fertilization, embryo development and quality were assessed in terms of cleavage, blastocyst rate at days 7 and 8, total cell number (TCN), inner cell mass/trophectoderm ratio (ICN/TE), and apoptotic cell ratio (ACR). We found a detrimental effect on cleavage and blastocyst rates when the IVF was performed on any medium conditioned by most of the materials available for digital-light processing (PEGDA200, PEGDA500). The observed negative effect could be possibly due to some leaked compound used to print and stabilize the scaffolds, which was not so evident however with PEGDA PhotoInk. On the other hand, all the extrusion-based processable materials did not cause any detrimental effect on cleavage or blastocyst rates. The principal component analysis reveals that embryos produced in presence of 3D-printed scaffolds produced via extrusion exhibit the highest similarity with the control embryos considering cleavage, blastocyst rates, TCN, ICN/TE and ACR per embryo. Conversely, all the photo-cross linkable materials or medium conditioned by PLA, lead to the highest dissimilarities. Since the use of PCL scaffolds, as well as its conditioned medium, bring to embryos that are more similar to the control group. Our results suggest that extrusion-based 3D printing of PCL could be the best option to be used for new IVF devices, possibly including the support of OECs, to enhance bovine embryo development.

Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro.

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.

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.

Update on mammalian sperm capacitation: how much does the horse differ from other species?

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.

The Importance of the Periconception Period: Immediate Effects in Cattle Breeding and in Assisted Reproduction Such as Artificial Insemination and Embryo Transfer.

In livestock breeding, the successful outcome is largely depending on the "periconception environment" which, in a narrow sense, refers to the genital tract, where gametogenesis and embryogenesis occur. During these early stages of development, gametes and embryos are known to be particularly sensitive to alterations in their microenvironment. However, as the microenvironment somehow reflects what is going on in the external world, we must widen our definition of "periconception environment" and refer to all events taking place around the time of conception, including metabolic state and health and nutrition of the dam. In modern dairy cows that have to manage an optimal reproductive performance with continued growth and high milk yield, the periconception period is particularly challenging. The metabolic priority for growth and lactation is known to generate adverse conditions hampering optimal ovarian function, oocyte maturation, and development of embryo/fetus. In addition, by using artificial reproductive technologies (ARTs), gametes and/or embryos of livestock are exposed to unnatural conditions outside the male and female genital tract. Artificial insemination, the most widely used technique, is currently yielding pregnancy rates similar to natural mating, and calves produced by AI are equally viable after natural mating. In contrast, other ART, such as multiple ovulation and embryo transfer, have been reported to induce changes in gene expression and DNA methylation patterns with potential consequences for development.Finally, the "periconceptional" environment has been shown to not only influence the successful establishment of pregnancy but also the long-term health and productivity of the offspring. Hence, the optimization of management around the time of conception might open doors to improve animal production and product quality.

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

BACKGROUND: Global 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. RESULTS: We 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. CONCLUSIONS: In 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.

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.

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.

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

BACKGROUND: Since 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. RESULTS: We 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. CONCLUSIONS: Serum 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.

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.

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

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.

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.

DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes.

Immunostaining is the preferred technique to assess differences in methylation and hydroxymethylation status of both pronuclei in single zygotes. DNA counterstaining is needed to delimitate the pronuclear area for quantification purposes. For a correct epitope retrieval of 5-methylcytosine and 5-hydroxymethylcytosine in bovine zygotes, 1h of denaturation with 4N HCl is needed. However, DNA stains are sensitive to denaturation. Therefore, four DNA stains were tested after 1h of denaturation with 4N HCl in this study. After this treatment, DAPI (4',6-diamidino-2-phenylindole) and Hoechst failed to bind DNA, but both propidium iodide and ethidium homodimer-2 successfully bound it and both pronuclei were stained.