C. Díez

Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines

Bovine embryonic stem cells are of potentially big value in transgenic research and studies of lineage commitment and development. Nevertheless, key aspects of the establishment of bovine embryonic stem cells such as the identification of specific pluripotency markers need to be clarified to achieve successful results. Bovine blastocysts were produced in vitro and cultured for 8 days up to the expanded or hatched stage. The trophectoderm, the inner cell mass and its embryonic stem cell-derived lines, all showed a common positive immunocytochemical staining for stage-specific embryonic antigen-4, tumour-rejection antigen gp96 and NANOG proteins. The antigenic profile obtained partially agrees with previous data from bovine and other species. Until a validated pluripotent bovine stem cell marker can be identified, it might be advisable to combine the use of epiblast and trophoblast-specific markers to rule out the presence of early committed trophectoderm cells in bovine embryonic stem cell cultures.

Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.

Proteome of the Early Embryo–Maternal Dialogue in the Cattle Uterus

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.

Biological differences between in vitro produced bovine embryos and parthenotes

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66(shc) and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66(shc) and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm.

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.

Embryonic sex induces differential expression of proteins in bovine uterine fluid.

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.

Embryonic Stem Cells in Cattle

Because of the potential use of embryonic stem cells (ESC), especially for genetic modifications, there is great interest in establishing domestic animals-related ESCs. Unfortunately, despite considerable efforts, validated ESC lines in species other than mice and primates are yet to be isolated. In this paper, we will summarize the current knowledge on bovine ESCs in an attempt to understand why derivation of domestic animal ESC is still unsuccessful and we will discuss some promising future approaches.

Use of two replacements of serum during bovine embryo culture in vitro

This study evaluated the effect of two commercial serum replacements (Ultroser G and CPSR-3 on in vitro bovine embryo culture. In Experiment 1, zygotes were cultured in SOF+Ultroser G (2, 4 and 6%), SOF+CPSR-3 (2, 4 and 6%), and SOF+5% FCS (control). Blastocyst rates obtained after culturing with Ultroser G were lower than those with FCS. However, blastocyst rates for CPSR-3 were similar to those for serum. In addition, embryos produced in SOF+CPSR-3 had the same proportion inner cell mass number and total cell number as embryos cultured with FCS. In Experiment 2, a combination of serum replacements during different periods showed that treatment before the five-to eight-cell stages had no effect on further embryo development. However, treatments up to the morula stage affected blastocyst formation. The concentration of supplement and the timing of its inclusion in culture markedly affected embryo development. The serum replacement CPSR-3 can supplement embryo culture with blastocyst rates and quality similar to those for serum.

Effects of acetoacetate and d-β-hydroxybutyrate on bovine in vitro embryo development in serum-free medium

It is known that the ketone bodies acetoacetate and D-beta-hydroxybutyrate can be metabolized by the early bovine embryo for in vitro development. In the present work, we report experiments leading to the culture of bovine embryos in the absence of serum. In vitro-produced bovine zygotes were cultured in modified synthetic oviduct fluid medium supplemented with acetoacetate derivatives, acetoacetate and D-beta-hydroxybutyrate. Acetoacetate and its derivatives prevented blastocysts from forming in the absence of serum during the whole culture period. However, from Days 6 to 8 of culture in the absence of serum, acetoacetate did not affect development as compared to controls containing lactate and pyruvate or no substrate. Interestingly, D-beta-hydroxybutyrate stimulated blastocyst and expansion development, and allowed lipid mobilization. In feeder cells coculture, embryos produced with D-beta-hydroxybutyrate showed improved hatching. Embryos cultured in D-beta-hydroxybutyrate were viable upon transfer to recipients, although no pregnancies were confirmed later by ultrasonic scanning. The protective effect of serum upon embryos cultured in medium containing acetoacetate is apparently not required in the presence of D-beta-hydroxybutyrate.

Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate=52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80±0.053; plasma: 0.89±0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607±0.038 (CM, expanded blastocysts) and 0.672±0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.