Effect of elevated circulating progesterone concentration on bovine blastocyst development and global transcriptome following endoscopic transfer of in vitro produced embryos to the bovine oviduct.

Elevated concentrations of circulating progesterone in the immediate postconception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the uterine tissues. Using state-of-the-art endoscopic techniques, this study examined the effect of elevated progesterone on the development of in vitro produced bovine zygotes transferred to the oviducts of heifers with high or normal circulating progesterone concentrations and on the transcriptome of blastocysts developing under such conditions. Simmental heifers (n = 34) were synchronized using a controlled internal drug release (CIDR) device for 8 days, with a prostaglandin F(2 alpha) analogue administered 3 days before removal of the CIDR device. Only animals exhibiting a clear standing estrus (Day 0) were used. To produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the estrous cycle; the PRID was left in place until embryo recovery. All animals were sampled for blood daily from Day 0 to Day 7. Cleaved embryos were transferred by endoscopy to the ipsilateral oviduct of each recipient on Day 2 and then recovered by nonsurgically flushing the oviduct and the uterus on Day 7. The number of embryos developing to the blastocyst stage was recorded at recovery and following overnight culture in vitro. Potential effects of elevated progesterone on transcript abundance were examined using the Affymetrix GeneChip Bovine Genome Array. Insertion of a PRID on Day 3 resulted in a significant elevation of progesterone concentration (P < 0.05) from Day 3.5 until Day 6. Elevated progesterone did not affect the proportion of embryos developing to the blastocyst stage. Genomewide gene expression analysis identified 194 differentially expressed genes between embryos collected from heifers with normal or elevated progesterone, and quantitative real-time PCR validation with a subset of selected genes and an independent sample confirmed the microarray results. Interaction network analysis indicated a significant interaction between progesterone-regulated genes in the blastocyst and in the maternal endometrium. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo but do result in subtle changes to the transcriptome of the embryo that may be associated with advanced elongation posthatching.

Carter F ，Rings F ，Mamo S ，Holker M ，Kuzmany A ，Besenfelder U ，Havlicek V ，Mehta JP ，Tesfaye D ，Schellander K ，Lonergan P ... -  《-》

Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium?

The steroid hormone progesterone (P(4)) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P(4) in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-tau production and higher pregnancy rates in cattle. Using in vitro and in vivo models and approximately 8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P(4) affects bovine embryo development in vitro and in vivo. mRNA for P(4) receptors was present at all stages of embryo development raising the possibility of a direct effect of P(4) on the embryo. Exposure to P(4) in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P(4) in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P(4) resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P(4)-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P(4) in order to exhibit advanced elongation.

Clemente M ，de La Fuente J ，Fair T ，Al Naib A ，Gutierrez-Adan A ，Roche JF ，Rizos D ，Lonergan P ... -  《-》

Transcriptome changes at the initiation of elongation in the bovine conceptus.

The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of this study was to examine the temporal changes in transcriptional profile as the embryo develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo- and in vitro-derived blastocysts that may be associated with embryonic survival/mortality using the bovine Affymetrix microarray. All embryos were produced either in vitro by in vitro fertilization or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen, and the remainder were transferred (n = 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n = 25 per pool) and Day 13 conceptuses (n = 5 per pool) were used for microarray analysis. In Day 7 blastocysts, 50 genes were found to be differentially expressed (P < 0.05), of which 19 were up-regulated and 31 down-regulated in the in vivo compared to in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (P < 0.05), of which 133 were up-regulated and 155 down-regulated in the in vivo compared to in vitro embryos. The comparison between Day 7 and Day 13 embryos revealed significant temporal changes in transcript profile with 1806 and 909 transcripts differentially expressed in the in vitro- and in vivo-derived embryos, respectively. Across the three array comparisons between Day 7 and Day 13 embryos, 444 genes were consistently exclusively present in the in vivo embryos, whereas 1341 were exclusively present in the in vitro embryos. Regardless of the origin of the embryo, 465 differentially expressed genes between Day 7 and 13 were common to both in vivo- and in vitro-derived embryos; these genes are likely critical for the transition between the blastocyst (Day 7) and ovoid conceptus (Day 13) stages of embryo development. In order to validate the microarray findings, differences in the expression of six genes (CYP51A1, FADS1, TDGF1, HABP2, APOA2, and SLC12A2) were confirmed by quantitative real-time PCR on in vivo- and in vitro-derived embryos on Day 7 and Day 13 using independent samples from those used for the microarray. Subsequent mapping of these differentially expressed genes into relevant functional groups and pathways identified important pathways involved in conceptus elongation in cattle. In conclusion, this analysis has identified genes and pathways crucial for the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).

Clemente M ，Lopez-Vidriero I ，O'Gaora P ，Mehta JP ，Forde N ，Gutierrez-Adan A ，Lonergan P ，Rizos D ... -  《-》

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro-produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)-releasing intravaginal device (PRID) with the administration of a prostaglandin F2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro-produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%-55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of "rescuing" poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.

O'Hara L ，Forde N ，Kelly AK ，Lonergan P ... -  《-》

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction.

In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

Gad A ，Besenfelder U ，Rings F ，Ghanem N ，Salilew-Wondim D ，Hossain MM ，Tesfaye D ，Lonergan P ，Becker A ，Cinar U ，Schellander K ，Havlicek V ，Hölker M ... -  《-》