1,720,995 research outputs found
Oviduct-on-a-chip: Creating an in vitro oviduct to study bovine gamete interaction and early embryo development
No full textThe oviduct is host to the period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of epigenetic marks as differentiation proceeds. This reprogramming period is vulnerable to changes in environmental conditions, such as compromised maternal health or diet. Likewise, ART related factors, such as culture medium composition, light, temperature and oxygen tension, also affect the epigenetic of early developing embryos. In this respect, we demonstrated that conventional IVM and IVF markedly alter the dynamics of DNA (de)methylation and the expression of genes related to DNA (de)methylation in bovine zygotes. We were not able to determine whether the differences are due primarily to IVM or also to the IVF procedure. It is generally believed that, at the zygote stage, control of embryo development depends primarily on maternal mRNA transcripts since embryonic transcription (genome activation) doesn’t begin until between the 4-8 cell stages in bovine in vivo embryos and between the 8-16 cell stages in bovine in vitro embryos. However, a minor embryonic genome activation event has also been described to take place at in vivo the 2 cell stage. We designed and successfully 3D-printed an oviduct-on-a-chip model using a stereo-lithographic technique. Bovine oviduct epithelial cells (BOECs) cultured in the device regained and maintained their ciliated and cuboidal to columnar epithelium for a period of at least 6 weeks, with a mixed population of ciliated and secretory cells comparable to that in the in vivo oviduct epithelium. In this system, the oviduct cells in culture conditioned the apical medium, which in turn supported physiological sperm-oocyte interaction and fertilization, and completely abolished polyspermic fertilization and parthenogenetic activation of oocytes. Since our first oviduct-on-a-chip prototype was found to release components (phthalates and polyethylene-glycol) toxic to developing embryos, we identified a non-toxic material (PDMS) to create an improved oviduct-on-a-chip. BOECs grown in the new oviduct-on-a-chip were responsive to steroid hormone stimulation, mimicking the luteal- and pre-ovulatory phases. Moreover, the improved oviduct-on-a-chip supported not only fertilization, but also embryo development up to the blastocyst stage. The zygotes resulting from on chip (CH) culture were more similar to their in vivo counterparts (VV) than to conventional in vitro (VT) zygotes, in terms of their global DNA methylation level and transcriptome. Interestingly, VV and CH zygotes exhibited lower global DNA methylation levels than VT zygotes, which is presumably a factor of the up-regulation of genes related to DNA demethylation in 80% of the VV and 50% of the CH zygotes (G2). This reduced level of DNA methylation seems to be essential to the minor embryonic genome activation event, since an up-regulation of genes related to transcription and translation initiation was observed in G2 zygotes. Suggesting that standard IVP conditions delay zygote minor transcriptome activation, but that the delay can be partially ameliorated using our oviduct-on-a-chip platform. Finally, our results highlight the importance of using a more in vivo-like environment to study pathways related to normal fertilization and embryo development in vitro
Roles of the reproductive tract in modifications of the sperm membrane surface
No full textSuccessful fertilization requires viable and functional spermatozoa to recognize and fuse with the oocyte. In most mammalian species, mature spermatozoa are not capable of fertilizing the oocytes immediately after ejaculation. However, unlike somatic cells, spermatozoa, after leaving the testis, are transcriptionally and translationally silent; therefore, upon completion of spermiogenesis, spermatozoa carry only a minimal amount of essential proteins on their membranes as well as within their restricted volume of cytoplasm. To develop into a fully functional and competent sperm that is capable of successful fertilization, modifications of the sperm membrane surface during its transit in the reproductive tracts is critical. These post-spermatogenesis modifications advance the maturation of epididymal spermatozoa. In addition, components secreted into the lumen of the reproductive tracts that are later added onto the sperm membrane surface also regulate (inhibit or activate) the functions of the spermatozoa. This acquisition of additional proteins from the reproductive tracts may compensate for the inactivity of morphologically mature spermatozoa. In this review, we discuss the contributions of the male and female genital tracts to modifications of the sperm membrane surface at different stages of fertilization
Transcriptome profiling of liver of non-genetic low birth weight and long term health consequences
Get PDFBACKGROUND: It is believed that the main factors of low prenatal growth in mammals are genetic and environmental. We used isogenic mice maintained in standard conditions to analyze how natural non-genetic microsomia (low birth weight) is produced in inbred mice and its long term effect on health. To better understand the molecular basis of non-genetic microsomia, we undertook transcriptome profiling of both male and female livers from small and normal size mice at birth. RESULTS: Naturally occurring neonatal microsomia was defined as a gender-specific weanling weight under the 10th percentile of the colony. Birth weight variation was similar in inbred and outbred lines. Mice were phenotyped by weight, size, blood pressure, organ size, their response to a glucose challenge, and survival rates. Regardless of diet, adult mice born with microsomia showed a significantly lower body weight and size, and differences in the weight of several organs of microsomic adult mice compared to normal birth weight adults were found. After a high-fat diet, microsomic mice were less prone to obesity, showing a better glucose tolerance and lower blood pressure. Through a transcriptome analysis, we detected a different pattern of mRNA transcription in the liver at birth comparing male vs female and microsomic vs normal mice, noting some modifications in epigenetic regulatory genes in females and modifications in some growth factor genes in males. Finally, using embryo transfer of embryos of different quality and age, we identified a putative preimplantation origin of this non-genetic microsomia. CONCLUSIONS: (1) neonatal microsomia is not always a risk factor for adult metabolic syndrome, (2) neonatal non-genetic microsomia displays changes in the expression of important epigenetic genes and changes in liver mRNA transcription profile at birth, exaggerating sexual dimorphism, and (3) random preimplantation phenotypic variability could partially explain body birth weight variation in isogenic lines
An update on post-ejaculatory remodeling of the sperm surface before mammalian fertilization
Get PDFThe fusion of a sperm with an oocyte to form new life is a highly regulated event. The activation-also termed capacitation-of the sperm cell is one of the key preparative steps required for this process. Ejaculated sperm has to make a journey through the female uterus and oviduct before it can approach the oocyte. The oocyte at that moment also has become prepared to facilitate monospermic fertilization and block immediately thereafter the chance for polyspermic fertilization. Interestingly, ejaculated sperm is not properly capacitated and consequently is not yet able to fertilize the oocyte. During the capacitation process, the formation of competent lipid-protein domains on the sperm head enables sperm-cumulus and zona pellucida interactions. This sperm binding allows the onset for a cascade reaction ultimately resulting in oocyte-sperm fusion. Many different lipids and proteins from the sperm surface are involved in this process. Sperm surface processing already starts when sperm are liberated from the seminiferous tubules and is followed by epididymal maturation where the sperm cell surface is modified and loaded with proteins to ensure it is prepared for its fertilization task. Although cauda epididymal sperm can fertilize the oocyte IVF, they are coated with so-called decapacitation factors during ejaculation. The seminal plasma-induced stabilization of the sperm surface permits the sperm transit through the cervix and uterus but prevents sperm capacitation and thus inhibits fertilization. For IVF purposes, sperm are washed out of seminal plasma and activated to get rid of decapacitation factors. Only after capacitation, the sperm can fertilize the oocyte. In recent years, IVF has become a widely used tool to achieve successful fertilization in both the veterinary field and human medicine. Although IVF procedures are very successful, scientific knowledge is still far from complete when identifying all the molecular players and processes during the first stages the fusion of two gametes into a new life. A concise overview in the current understanding of the process of capacitation and the sperm surface changes is provided. The gaps in knowledge of these prefertilization processes are critically discussed
Prion protein in ESC regulation
No full textA large number of studies have analysed the putative functions of the prion protein (PrP(C)) in mammals. Although its sequence conservation over a wide range of different animals may indicate that this protein could have a key role in prion diseases, an absolutely accepted involvement has not been found so far. We have recently reported that PrP(C) regulates Nanog mRNA expression, the first non-redundant function of PrP(C) in embryonic stem cells (ESC), which translates into control of pluripotency and early differentiation. Contrary to what it is believed, the other two members of the prion protein family, Doppel and Shadoo, cannot replace the absence of PrP(C), causing the appearance of a new embryoid body (EB) population in our in vitro culture. The similarities between EB and an early post-implantation embryo suggest that this might also occur in vivo, enhancing the importance of this finding. On the other hand, our data may support the hypothesis of a relationship between the loss of PrP(C) function and neuronal degeneration in prion diseases. A reduction in brain stem cells pluripotency after PrP(C) is misfolded into the pathological conformation (PrP(Sc)) could lead to a delay or a disappearance of the normal brain damage recovery
The involvement of epididymal secretory proteins in the selection processes of sperm cells
No full textNovel methods to detect capacitation-related changes in spermatozoa
Get PDFPrior to interaction with the oocyte, spermatozoa must undergo capacitation, which involves a series of physio-chemical transformations that occur in the female tract. As capacitation is a pre-requisite for successful fertilisation, it is a topic of great interest for sperm biologists, but the complexity of the numerous biochemical and biophysical processes involved make it difficult to measure. Capacitation is an extremely complex event that encompasses numerous integrated processes that can occur concurrently during this window of time. The identification of techniques to accurately assess and quantify capacitation is therefore crucial to gain a meaningful insight into this fascinating sperm maturation event. Whilst there are extensive reviews in the literature that focus on the functional changes to spermatozoa during capacitation, few have examined the methods required to measure these changes. The aim of this review is to highlight frequently used methods to quantify different stages of capacitation and identify promising novel techniques. Factors that are able to modulate various capacitation processes will also be discussed. The overall outcome is to provide researchers with a toolbox of methods that can be used to gain a deeper understanding of the intricacies of capacitation in spermatozoa
Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation
Get PDFTo investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na(+)/K(+) gradient, which is dependent on an Na(+)-K(+) antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility
Cumulus cells protect the oocyte against saturated free fatty acids
No full textIn the cow a major characteristic of metabolic stress is an elevated level of plasma free fatty acid, due to increased lipid mobilization from adipose tissue. Elevated levels of free fatty acids in blood (complexed to albumin) are associated with increased lipotoxicity in non-adipose tissue. An overview is provided on the negative impact of free fatty acids and the metabolic stress imposed on the oocyte and early embryo and thus on bovine fertility. There is increasing evidence that in vitro as well as in vivo the elevated levels of free fatty acids in blood during metabolic stress can severely hamper oocyte and embryo development. However, fatty acids do also form an essential nutrient source for the oocyte and embryo, which indicates that these good and bad effects of fatty acids should be in subtle balance to optimize the developmental competence of the oocyte and embryo
- …
