Whole transcriptome sequencing reveals that non-coding RNAs are related to embryo morphogenesis and development in rabbits.

The roles of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in embryonic development remain unclear. We performed a comprehensive analysis of lncRNA and circRNA profiles in rabbit embryos at different stages by whole transcriptome sequencing. We identified 719 lncRNAs and 744 circRNAs that were differentially expressed between stages S1, S2 and S3. A total of 241 differentially expressed lncRNAs and 166 differentially expressed circRNAs were significantly involved in embryonic morphogenesis and development. An RNA network was established and of the embryonic development-associated RNAs, the lncRNAs TCONS_00009253 and TCONS_00010436 were persistently downregulated, while circRNA_07129, circRNA_15209, and circRNA_12526 were persistently upregulated, and their co-expressed mRNAs TBX1, WNT3 and FGFR2 were persistently downregulated during embryonic development. These candidate RNAs were mainly involved in the Wnt, PI3K-Akt, and calcium signaling pathways. This study reports candidate lncRNAs and circRNAs that may be indispensable for the morphogenesis and development of rabbit embryos.

Early specification and development of rabbit neural crest cells.

The phenomenal migratory and differentiation capacity of neural crest cells has been well established across model organisms. While the earliest stages of neural crest development have been investigated in non-mammalian model systems such as Xenopus and Aves, the early specification of this cell population has not been evaluated in mammalian embryos, of which the murine model is the most prevalent. Towards a more comprehensive understanding of mammalian neural crest formation and human comparative studies, we have used the rabbit as a mammalian system for the study of early neural crest specification and development. We examine the expression profile of well-characterized neural crest markers in rabbit embryos across developmental time from early gastrula to later neurula stages, and provide a comparison to markers of migratory neural crest in the chick. Importantly, we apply explant specification assays to address the pivotal question of mammalian neural crest ontogeny, and provide the first evidence that a specified population of neural crest cells exists in the rabbit gastrula prior to the overt expression of neural crest markers. Finally, we demonstrate that FGF signaling is necessary for early rabbit neural crest formation, as SU5402 treatment strongly represses neural crest marker expression in explant assays. This study pioneers the rabbit as a model for neural crest development, and provides the first demonstration of mammalian neural crest specification and the requirement of FGF signaling in this process.

A single injection of corifollitropin alfa supplemented with human chorionic gonadotropin increases follicular recruitment and transferable embryos in the rabbit.

Superovulation protocols are designed to achieve maximum embryo yields. Nevertheless, ovarian response control and the quality of obtained embryos are still a challenge. On the other hand, to save the superovulated embryos until their subsequent use, it is usual to cryopreserve them, so it is also crucial to assess their cryotolerance. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (FSH-CTP) alone or supplemented with human chorionic gonadotropin (hCG) and to determine the impact of this stimulation on in vitro and in vivo development of fresh or devitrified embryos. Our outcomes showed that ovulation rate and recovered embryos were significantly increased when hCG was used. In vitro development of fresh and devitrified embryos and survival at birth were not significantly affected by superstimulation treatment. Results of this study suggest that a single injection of long-acting FSH-CTP supplemented with hCG can be effectively used in rabbits to elicit an increase in ovulation rate and number of recovered embryos. Furthermore, we demonstrated that hCG supplementation had no negative effects in embryo cryosurvival and development, showing similar survival rate at birth than FSH-CTP alone group.

Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system.

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms.

A Probability Analysis of Historical Pregnancy and Fetal Data from Dutch Belted and New Zealand White Rabbit Strains from Embryo-Fetal Development Studies.

Embryo-fetal development (EFD) studies, typically in pregnant rats and rabbits, are conducted prior to enrolling females of reproductive age in clinical trials. Common rabbit strains used are the New Zealand White (NZW) and Dutch Belted (DB). As fetal abnormalities can occur in all groups, including controls, Historical Control Data (HCD) is compiled using data from control groups of EFD studies, and is used along with each study's concurrent control group to help determine whether fetal abnormalities are caused by the test article or are part of background incidences. A probability analysis was conducted on 2014 HCD collected at Charles River Inc., Horsham PA on Covance NZW, Covance DB, and Charles River (CR) NZW rabbits. The analysis was designed to determine the probability of 2 or 3 out of a group of 22 does aborting their litter or of having a fetal abnormality by chance. Results demonstrate that pregnancy parameters and fetal observations differ not only between strains, but between sources of rabbits of the same strain. As a result the probability of these observations occurring by chance in two or three litters was drastically different. Although no one single strain is perfect, this analysis highlights the need to appreciate the inherent differences in pregnancy and fetal abnormalities between strains, and points out that an apparent isolated increased incidence of an observation in one strain will not necessarily be test-article related in another strain. A robust HCD is critical for interpretation of EFD rabbit studies, regardless of the rabbit strain used.

Effects of Female Dietary Restriction in a Rabbit Growth Line During Rearing on Reproductive Performance and Embryo Quality.

Maternal diet prior to mating has an effect on reproductive performance. We analysed the effect of maternal dietary restriction during rearing on reproductive performance, the embryo development and foetal growth. Females were categorized in two groups: (i) does with ad libitum access to feed or (ii) restricted. Two experiments were performed: (i) after 1 month, receptive females from both experimental groups were artificially inseminated and the reproductive performance was recorded during three reproductive cycles; at the first insemination, the body weight and perirenal fat thickness were recorded, and (ii) females from both experimental groups were inseminated, and 24 h later, embryos were recovered and transferred to recipient females from a maternal line. Later, embryonic implantation was assessed at day 14 by laparoscopy and foetal growth was monitored by ultrasound examination. In experiment 1, no differences in kindling rate was found, but prolificacy was showed to be higher in ad libitum does, which also were heavier than restricted ones. In experiment 2, no differences among does either in body weight, in perirenal fat thickness or in reproductive performance (ovulation rate and embryo recovery rate) were related to differences in feed intake. However, despite similar embryonic implantation losses, embryos from restricted females demonstrated higher foetal and gestational losses. Embryos from restricted does presented lower foetal growth than embryos from ad libitum does. Therefore, our results demonstrated that nutrition before first conception in a rabbit line selected for growth rate may impact on the embryo and results in a disturbance in gestational losses and foetal growth over all reproductive life.

Role of embryonic and maternal genotype on prenatal survival and foetal growth in rabbit.

The aim of this work was to evaluate the influence of maternal and embryonic genotype on prenatal survival and foetal growth during pregnancy. Embryos were recovered at 48 h of gestation from two different donor lines (R = 46 and A = 40) and transferred to nulliparous recipient does (26 R and 24 A). Each recipient doe received six embryos into one oviduct from line R, and six embryos form line A into the other. Laparoscopy was performed at Day 14 to determine implantation rate. Recipient females were slaughter at Days 14, 24 and 30 (12, 24, and 14, respectively) to determine the number of live foetuses and the weight of live foetuses, foetal placenta and maternal placenta. A transcriptome analysis was performed to search for differences between foetal placentas at Days 14 and 24 of development. Prenatal survival at Days 14, and 24 was affected by embryonic genotype and determined by maternal genotype at Day 30. Foetal weight at Day 14 was influenced by both genotypes, being the weight higher for group A/A (0.29 ± 0.01 g vs 0.19 ± 0.01 g, for group R/R). However, both genotypes were determinant for foetal placenta weight at Day 24, while those genotypes affected maternal placenta weight at Day 30. Nevertheless, no differences in foetal placenta at transcriptome level and progesterone and IGF-I plasma levels in recipient does were found. In conclusion, results indicate that the influence of embryo and maternal genotype on the prenatal survival and growth seems to be changing over gestation.

First steps of in vitro gastrulation in rabbit vitrified embryos.

BACKGROUND: The in vitro rabbit embryo production and their cryopreservation methodologies such as vitrification generate less viable embryos, and occasionally, with significant differences from those that are not subjected to any treatment. Besides, in vitrified rabbit embryos little information is available about exactly when and where begin to emerge the first differences that finally result in foetal losses comparing with non-vitrified embryos. OBJECTIVE: The aim of this study was to evaluate the vitrification effects on the early in vitro gastrulation events. MATERIALS AND METHODS: After oviductal transfers of vitrified and non-vitrified embryos (control) in rabbit recipients, blastocysts from 144h (6-day-old) were recovered and cultured into TCM199 supplemented with rabbit homologous serum media for 48 hours. Gastrula stage and measures of perimeter and area of blastocyst and gastrula were noted. Moreover, eight independent pools consisting of six embryos each one were generated for each experimental group (control and vitrified) and total RNA was isolated to study the OCT4 gene expression. RESULTS: Of 151 control and 164 vitrified morulae transferred, 69.5 % and 70.1 % developed in vivo to 6-day-old blastocyst respectively. After 24 hour of in vitro culture, 41.8 % of vitrified blastocyst had begun the neurulation (stage 5-) versus 22.8 % of control group. Nevertheless, the vitrified group showed the highest percentage of collapsed blastocyst at 48 hours (26.8 %). Non morphometric differences differences were observed in perimeter and area of blastocyst and gastrula between control and vitrified group at 0 and 24 hours. By contrast, perimeter and gastrula areas were slightly higher for the vitrified group than those for the control group at 48 hours of in vitro culture. CONCLUSION: The study reveal the existence of the first morphological differences in vitrified blastocysts of 7 and 8-day-old, marked by a further development of gastrulation in the vitrified group.

A pictorial essay on fetal rabbit anatomy using micro-ultrasound and magnetic resonance imaging.

INTRODUCTION: With this pictorial essay, we aimed to provide gestational age specific reference ranges of relevant fetal structures using micro-ultrasound, as well as its correlation with postmortem MRI and whole body sections. MATERIAL AND METHODS: Time-mated pregnant rabbits (n = 24) were assessed once at various gestational ages in the second half of pregnancy (15, 17, 21 to 23, 25 to 28, and 30 to 31 days; term = 31 days). We obtained biometric data, together with Doppler flow patterns in the ductus venosus, umbilical artery and umbilical vein. After euthanasia, micro-ultrasound images were compared with images obtained by 9.4 Tesla MRI and whole body paraffin sections at 15, 23, 26, and 28 days. RESULTS: We constructed biometric normative curves, which showed a significant correlation with gestational age. The pulsatility index (PI) in the umbilical artery decreased with gestation (PI = 5.746-0.2969(GA) + 0.004931(GA)(2) ; R(2) = 0.30), whereas pulsatility index for veins (PIV) in the ductus venosus remained constant (median PIV = 0.82 (0.60-1.12)). In this report, we provide an anatomical atlas of fetal thoracic development using both micro-ultrasound and MRI. CONCLUSION: We describe anatomical fetal leporine development as can be visualized by micro-ultrasound and MR imaging. The reported reference ranges may be useful for researchers using the fetal rabbit model.

Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development.

To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1ß and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.

Quality of rabbit vitrified/thawed transgenic embryos.

The aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous-hFVIII or exogenous-enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

Development of interspecies cloned embryos reconstructed with rabbit (Oryctolagus cuniculus) oocytes and cynomolgus monkey (Macaca fascicularis) fibroblast cell nuclei.

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

Effect of different culture systems on mRNA expression in developing rabbit embryos.

The rate of zygotes in vitro developed to hatched blastocyst stage was evaluated between two different commercial media (TCM-199 and Ham's F10) and two different culture systems (renewal and non-renewal single medium) to determine the effects of culture conditions on rabbit embryo preimplantation development. The relative transcript abundances of OCT4, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor 3 (erbB3) of resultant blastocysts were also analysed and compared with in vivo developed blastocysts. Results showed an important divergence in mRNA expression between embryos developed under in vivo and in vitro conditions despite there being no significant difference in hatching blastocyst rates between different culture systems and different media. For OCT4, transcript abundance of in vitro culture embryos differs from their in vivo chronological counterparts, but, when the medium is renewed, mRNA expression seemed similar to in vivo developed 4-day-old embryos. In addition, VEGF and erbB3 expression showed marked variation between different in vitro conditions. Therefore, the study of specific transcript abundance in rabbit blastocyst provides a more detailed description of which alterations in gene expression occur due in vitro conditions, and further studies should be carried out to reduce current limitations of long-term culture of rabbit pre-implantation embryos.

Effect of embryonic and maternal genotype on embryo and foetal survival in rabbit.

The aim of this work was to study the influence of embryonic and maternal genotype of two lines of rabbits selected by growth rate (line R) and litter size at weaning (line A) on prenatal survival. Embryos were recovered at 48 h of gestation from R and A donors (39 and 35 does, respectively) and reciprocally transferred to the oviducts of recipient does to the R (n = 15) and A (n = 14) lines. Each recipient doe received six embryos from line R into one oviduct and six embryos from line A into the other. Recipient does were examined by laparoscopy to determine implantation rate on day 14 and slaughtered on day 25 of gestation to determine the number of live foetuses and the weight of foetuses and placentas. No differences were found between lines in fertilization rate and stage of embryo development at 48 h post-insemination. Implantation rate was affected by both the embryonic and maternal genotype. While embryos from donor line A had the highest implantation rate (0.78 ± 0.032 vs 0.65 ± 0.036 for line R), recipient line R had a better implantation rate (0.78 ± 0.033 vs 0.64 ± 0.036 for line A). Foetal survival was affected by the embryonic genotype. Embryos from donor line A had a higher foetal survival rate than embryos from donor line R (0.65 ± 0.036 vs 0.53 ± 0.038, respectively) but lower foetal and placenta weights. In conclusion, while embryonic genotype influenced both implantation and foetal survival rate, R embryos had the lowest rates, maternal genotype affected the implantation rate and R recipients may show a greater uterine receptivity during implantation period. Moreover, it must be observed that foetal and placenta weights were significantly affected by embryonic genotype and heavier for R line.

Detection of glycosylated proteins in rabbit oviductal isthmus and uterine endometrium during early embryo development.

In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of oviductal isthmus and uterine endometrium in rabbits during early embryo development. Rabbit embryos are surrounded not only by the zona pellucida but also by tubal secretion-derived mucinous coat material, the mucin coat. Twenty sexually mature females were euthanized at 0 (pre-ovulatory group) and 24, 72 and 96 h after insemination (pseudopregnancy group). The following lectin-binding agents were used: Arachis hypogaea, Peanut (PNA) to label galactosyl (ß-1,3)N- acetyl-galactosamine, Dolichos biflorus Agglutinin (DBA) to label galactosyl (ß-1,4)N- acetyl-galactosamine, Lens curinaris (LCA) to label α--mannose, α-d-glucose and Pisum sativum agglutinin (PSA) to label α-d-mannose, α-d-glucose. Blood was collected by cardiac puncture, and direct enzyme immunoassay technique was used to measure progesterone concentration. A significant increase in total plasma progesterone concentrations was detected at 96 h post-ovulation when compared with 0, 24 and 72 h post-ovulation (2.9 ± 0.5 vs 0.5 ± 0.15, 1.6 ± 0.5 and 1.5 ± 0.4 ng/ml, at 96 h vs 0, 24 and 72 h post-ovulation, respectively). No differences between pre-ovulatory and pseudopregnant females were observed for glycoprotein localization in isthmus. In contrast, in the endometrium, differences in the glycoprotein detection between pre-ovulatory and pseudopregnant stages were detected. PNA to label galactosyl (ß-1,3)N- acetyl-galactosamine was not detected at the pre-ovulatory stage, but its presence was detected at 24 h after ovulation. Both PSA and LCA to label α-d-mannose, α-d-glucose were only detected at 72 h after ovulation. DBA detection was similar for all stages of the reproductive cycle. Therefore, N-acetyl-galactosamine secreted from isthmus could be involved in the formation of the embryonic mucin coat. d-galactose (PNA), d-glucose and d-mannose (PSA and LCA) might be crucial for the implantation period.

Up-regulation of insulin-like growth factor I and uteroglobin in in vivo-developed parthenogenetic embryos.

Parthenote embryos are being considered as an alternative source of embryonic stem cells. However, as there is still a dearth of knowledge of this kind of embryos, a better understanding of their biology is needed for their application. In this work, we studied the differences and similarities between parthenotes and normal embryos at the blastocyst stage in vivo developed. We analysed the expression of factor OCT-4, vascular endothelial growth factor (VEGF), insulin-like growth factor I (IGF-I) and uteroglobin (UG) by real-time PCR. To do so, oocytes were recovered and after activation procedure were transferred by ventral middle laparoscopy to receptive does to undergo completely in vivo development. Does were slaughtered 6 days post-ovulation induction, and parthenote and normal embryos were recovered for mRNA expression analysis. Our results reported that parthenotes and normal embryos showed similar mRNA expression for OCT-4 and VEGF. However, IGF-I and UG showed to be over-expressed in parthenote embryos. Thus, our study highlights that despite the in vivo development of parthenotes, they still seem to have an altered expression and, therefore, to be different to normal embryos. The altered expression pattern of parthenote embryos suggests that these embryos should be studied carefully before future application.

Maternal exposure to high temperatures disrupts OCT4 mRNA expression of rabbit pre-implantation embryos and endometrial tissue.

We examined the effect of prolonged high heat stress on reproductive performance and its relationship with gene expression in pre-implantation embryos and endometrial tissue. In experiment 1, primiparous rabbit does were divided into two environments: control does (maintained between 14 and 22°C) and heat-treated does housed in a climatic chamber (maintained between 25 and 35°C). Females were reproducing, and the litter size and live born kits were assessed at 2nd and 3rd partum. In heat-treated does, lower litter size (9.7 ± 0.48 and 11.4 ± 0.50) and fewer live born kits (7.2 ± 0.55 and 10.2 ± 0.57) were observed, although similar ovulation rates and numbers of pre-implantation embryos were noted. In experiment 2, after 3rd partum multiparous non-lactating does from each experimental group were used to obtain pre-implantation embryos and endometrial tissue. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-É£, HSP70 and HSP90 were analysed by real-time qPCR. Higher values of OCT-4 expression were observed in embryos and endometrial tissue in females reproduced under heat conditions. Moreover, elevated temperatures have been shown to up-regulate VEGF in embryos and down-regulate Ifn-É£ in endometrial tissue. The findings suggest a deleterious temperature effect on litter size and live born kits as a consequence of variation in gene expression pattern of the pre-implantational embryo and the endometrium associated with proliferation and differentiation and probably with implantation and uterine and foetal development during gestation.

Effects of slow freezing procedure on late blastocyst gene expression and survival rate in rabbit.

Studies of embryo cryopreservation efficiency have focused mainly on technical and embryo factors. To determine how a slow freezing process affects embryo and fetal development, we studied in vivo development ability after the freezing procedure by assessing blastocyst development at Day 6, implantation, and birth rates. A transcriptional microarray study was also performed to compare gene expression of 6-day-old rabbit embryos previously frozen and transferred into recipient rabbit females to their in vivo counterparts. Our goal was to study which alteration caused by the freezing procedure still remained in late blastocyst stage just at the time when the implantation process began. A microarray specifically designed to study rabbit gene expression profiling was used in this study. Lower implantation and birth rates were obtained in frozen embryos than in the control group (29.9% and 25.7% vs 88.5% and 70.8% for frozen and control embryos, respectively). Likewise, differences were also observed in gene expression profiles. Compared to 6-day-old in vivo-derived embryos, viable frozen embryos presented 70 differentially expressed genes, 24 upregulated and 46 downregulated. In conclusion, our findings showed that the slow freezing process affected late blastocyst development, implantation, and birth rates and that the gene expression alterations identified at late blastocyst stage could be useful in understanding the differences in developmental potential observed and the deficiencies that might hinder implantation and fetal development.

Rabbit as a reproductive model for human health.

The renaissance of the laboratory rabbit as a reproductive model for human health is closely related to the growing evidence of periconceptional metabolic programming and its determining effects on offspring and adult health. Advantages of rabbit reproduction are the exact timing of fertilization and pregnancy stages, high cell numbers and yield in blastocysts, relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structured similarly to the human placenta. To understand, for example, the mechanisms of periconceptional programming and its effects on metabolic health in adulthood, these advantages help to elucidate even subtle changes in metabolism and development during the pre- and peri-implantation period and during gastrulation in individual embryos. Gastrulation represents a central turning point in ontogenesis in which a limited number of cells program the development of the three germ layers and, hence, the embryo proper. Newly developed transgenic and molecular tools offer promising chances for further scientific progress to be attained with this reproductive model species.

Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos.

Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.