Balanced Notch-Wnt signaling interplay is required for mouse embryo and fetal development

Reproduction ◽  
2021 ◽  
Vol 161 (4) ◽  
pp. 385-398
Author(s):  
Mariana R Batista ◽  
Patrícia Diniz ◽  
Daniel Murta ◽  
Ana Torres ◽  
Luís Lopes-da-Costa ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Fetal Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Embryonic Cell ◽  
Differentiation Markers ◽  
Mouse Early Embryo ◽  
Carry Over ◽  
Embryo Kinetics

This study investigated the role of Notch and Wnt cell signaling interplay in the mouse early embryo, and its effects on fetal development. Developmental kinetics was evaluated in embryos in vitro cultured from the 8-16-cell to the hatched blastocyst stage in the presence of signaling inhibitors of Notch (DAPT) and/or Wnt (DKK1). An embryo subset was evaluated for differential cell count and gene transcription of Notch (receptors Notch1-4, ligands Dll1, Dll4, Jagged1-2, effectors Hes1-2) and Wnt (Wnt3a, Lrp6, Gsk3β, C-myc, Tcf4, β-catenin) components, E-cadherin and pluripotency and differentiation markers (Sox2, Oct4, Klf4, Cdx2), whereas a second subset was evaluated for implantation ability and development to term following transfer into recipients. Notch and Wnt blockades had significant opposing effects on developmental kinetics – Notch blockade retarded while Wnt blockade fastened development. This evidences that Notch and Wnt regulate the pace of embryo kinetics by respectively speeding and braking development. Blockades significantly changed the transcription profile of Sox2, Oct4, Klf4 and Cdx2, and Notch and double blockades significantly changed embryonic cell numbers and cell ratio. The double blockade induced more severe phenotypes than those expected from the cumulative effects of single blockades. Implantation ability was unaffected, but Notch and double blockades significantly decreased fetal development to term. Compared to control embryos, Notch blockade and Wnt blockade embryos originated, respectively, significantly lighter and heavier fetuses. In conclusion, Notch and Wnt signaling interplay in the regulation of the pace of early embryo kinetics, and their actions at this stage have significant carry-over effects on later fetal development to term.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

197 DOES DURATION OF BOVINE OOCYTE MATURATION IN VITRO AFFECT THE SPEED OF EMBRYO DEVELOPMENT IN VITRO AND SEX RATIO AT THE TWO-CELL OR BLASTOCYST STAGE?

2008 ◽  
Vol 20 (1) ◽  
pp. 177
Author(s):  
P. Bermejo-Álvarez ◽  
A. Gutiérrez-Adán ◽  
P. Lonergan ◽  
D. Rizos
Keyword(s):  
Sex Ratio ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blastocyst Development ◽  
Maturation In Vitro ◽  
Maturational Stage ◽  
Development In Vitro ◽  
Oviduct Fluid

The faster-developing blastocysts in IVC systems are generally considered more viable and better able to survive following cryopreservation or embryo transfer than those that develop more slowly. However, evidence from several species indicates that embryos that reach the blastocyst stage earliest are more likely to be males than females. The aim of this study was to determine whether the duration of maturation could affect early embryo development and, furthermore, the sex ratio of early- or late-cleaved embryos and blastocysts. Cumulus–oocyte complexes were matured in vitro for 16 h (n = 2198) or 24 h (n = 2204). Following IVF, presumptive zygotes from each group were examined every 4 h between 24 and 48 h postinsemination (hpi) for cleavage, and all embryos were cultured to Day 8 in synthetic oviduct fluid to assess blastocyst development. Two-cell embryos at each time point and blastocysts on Days 6, 7, and 8 from both groups were snap-frozen individually for sexing. Sexing was performed with a single PCR using a specific primer BRY. There was a significantly lower number of cleaved embryos from the 16-h compared with the 24-h maturation group at 28 (10.0 � 1.51 v. 28.8 � 3.57%), 32 (35.3 � 1.48 v. 57.6 � 3.33%), 36 (54.8 � 1.76 v. 67.4 � 2.81%), 40 (63.3 � 1.82 v. 72.0 � 2.54%), and 48 (70.6 � 1.78 v. 77.1 � 2.18%) hpi, respectively (mean � SEM; P d 0.05). However, the blastocyst yields on Day 6 (17.1 � 3.11 v. 16.4 � 2.11%), 7 (30.6 � 4.10 v. 34.6 � 3.51%), or 8 (34.1 � 3.90 v. 39.4 � 4.26%) were similar for both groups (mean � SEM; 16 v. 24 h, respectively). Significantly more 2-cell early cleaved embryos (up to 32 hpi) were male compared with the expected 1:1 ratio from both groups (16 h: 1.24:0.76 v. 24 h: 1.17:0.83, P ≤ 0.05); however, the overall sex ratio among 2-cell embryos was significantly different from the expected 1:1 in favor of males only for the 16-h group (1.18:0.82, P ≤ 0.05). The sex ratio of blastocysts on Day 6, 7, or 8 from both groups was not different from the expected 1:1. However, the total number of male blastocysts obtained after 8 days of culture from the 24-h group was significantly different from the expected 1:1 (1.19:0.81, P ≤ 0.05) and approached significance in the 16-h group. These results show that the maturational stage of the oocyte at the time of fertilization has an effect on the kinetics of early cleavage divisions but not on blastocyst yield. Furthermore, irrespective of the duration of maturation, the sex ratio of early-cleaving 2-cell embryos was weighted in favor of males, and this observation was maintained at the blastocyst stage.

65 Improvement of bovine early embryo development in vitro by coculture with endometrial epithelial cells

2019 ◽  
Vol 31 (1) ◽  
pp. 158
Author(s):  
M. Sponchiado ◽  
W. F. A. Marei ◽  
P. E. J. Bols ◽  
M. Binelli ◽  
J. L. M. R. Leroy
Keyword(s):  
Epithelial Cells ◽  
Primary Culture ◽  
Epithelial Mesenchymal Transition ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Phenol Red ◽  
Functional Characteristics ◽  
Indirect Contact ◽  
Mesenchymal Transition

We optimized a bovine endometrial epithelial cell (BEEC) line as a valuable research model for the study of very early embryo-maternal interactions in vitro. In this study, we aimed to (1) characterise the BEEC monolayers along the primary culture and first passages with respect to the expression of epithelial and mesenchymal cell markers and abundance of functional key transcripts; (2) to test whether direct or indirect contact with endometrial cells alter the quality of the embryos in vitro; and (3) to test the specificity of the effect. In Exp. 1, after isolation from slaughterhouse uteri at the early luteal phase, BEEC were cultured in DMEM/F12 phenol red-free medium supplemented with 10% fetal bovine serum (FBS) from primary culture until subculture 3. Fixed samples were immunostained for cytokeratin and vimentin. Transcript abundances for cellular lineage markers (KRT18 and VIM), oestrogen receptor (ESR1), interferon α/beta receptor 1 (IFNAR1), and prostaglandin G/H synthase 1 (PTGS1) and 2 (PTGS2) were evaluated by real-time quantitative PCR. Statistical analyses were carried out by ANOVA and Tukey test. Immunofluorescence data revealed that the BEEC line co-expresses cytokeratin together with a mesenchymal marker (Vimentin). This indicates that these epithelial cells underwent an epithelial-mesenchymal transition in vitro. Gene expression data showed a 6-fold increased (P<0.001) abundance of VIM mRNA from the primary culture to the subculture 1, which remained constant until subculture 3; however, KRT18, ESR1, IFNAR1, PTGS1, and PTGS2 were similar between the passages, suggesting that the cells conserved their functional characteristics. In Exp. 2, groups of 15 morulas (Day 5.5) were cultured in SOF medium supplemented with 5% FBS in the absence (control) or in the presence (co-culture) of BEEC at passage 2, for 48h. Embryos were placed on direct or indirect contact with a BEEC monolayer using a 96-well insert containing 8μm pores. Developmental rates were compared by chi-square test and P-values were adjusted by Tukey’s test. The percentage of embryos that had developed from morula into blastocyst stage on Day 7.5 was significantly higher in the direct and indirect contact co-culture (65%; P<0.05) groups compared with the control (53%) group. Moreover, 63% of the blastocysts were expanded, hatching, or hatched in the co-culture groups, whereas a rate of 46% was found in the control counterparts (P<0.05). In Exp. 3, the same experimental conditions from Exp. 2 were used, but groups of 15 Day 5.5 morulas were cultured in control, or conditioned medium from BEEC (CondBEEC) or bovine fibroblasts (CondFib). Blastocyst development rate on Day 7.5 was higher in the CondBEEC group (71%; P<0.001) compared with the control (54%) and CondFib (50%) groups. In conclusion, based on the markers studied, BEEC monolayers undergo epithelial-mesenchymal transition in vitro but preserve functional characteristics after few passages. The co-culture system improves bovine embryonic development from morula into blastocyst stage. This support is BEEC specific and does not rely on a direct cell-to-embryo contact.

Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos

2002 ◽  
Vol 14 (5) ◽  
pp. 291 ◽  
Author(s):  
N. W. Kurniani Karja ◽  
Takeshige Otoi ◽  
Masako Murakami ◽  
Minori Yuge ◽  
Mokhamad Fahrudin ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Protein Supplementation ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
The Mean

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P<0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula or blastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P<0.05) and hatching blastocyst stages (P<0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P<0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

scholarly journals HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo

2018 ◽  
Author(s):  
Tristan Frum ◽  
Tayler Murphy ◽  
Amy Ralston
Keyword(s):  
Cell Fate ◽  
Early Embryo ◽  
Embryonic Cell ◽  
Hippo Signaling ◽  
Mammalian Development ◽  
Mouse Early Embryo ◽  
Cell Positioning ◽  
Apical Localization ◽  
Complex Components

AbstractDuring mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway segregates pluripotent and extraembryonic fates by controlling cell positioning as well as expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.

scholarly journals HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Tristan Frum ◽  
Tayler M Murphy ◽  
Amy Ralston
Keyword(s):  
Cell Fate ◽  
Early Embryo ◽  
Embryonic Cell ◽  
Hippo Signaling ◽  
Mammalian Development ◽  
Sox2 Expression ◽  
Mouse Early Embryo ◽  
Cell Positioning ◽  
Complex Components

During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway in controlling cell positioning and expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy

2019 ◽  
Vol 31 (12) ◽  
pp. 1830 ◽  
Author(s):  
Kaatje D. Ducheyne ◽  
Marilena Rizzo ◽  
Juan Cuervo-Arango ◽  
Anthony Claes ◽  
Peter F. Daels ◽  
...  
Keyword(s):  
Chromosomal Abnormalities ◽  
Morphological Characteristics ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Early Embryo ◽  
Embryo Morphology ◽  
In Vitro Production ◽  
Pregnancy Failure ◽  
Micronucleus Formation

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

185 PGI2 ANALOG INDUCES HIGH-QUALITY PORCINE BLASTOCYSTS IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 209 ◽  
Author(s):  
J.-S. Kim ◽  
G. Wee ◽  
B.-S. Song ◽  
J.-S. Park ◽  
X.-L. Jin ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mitogen Activated Protein Kinase ◽  
Blastocyst Formation ◽  
Prostaglandin I2 ◽  
Western Blots ◽  
Porcine Oocyte

Prostaglandin I2 (PGI2) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. In hatched embryos, prostaglandin I2 (PGI2) is related to implantation improvement, but its role during oocyte maturation and early embryo development remains controversial. Therefore, in this study, the effect of addition of a PGI2 analog during early porcine oocyte maturation on nuclear maturation, blastocyst formation, and pre-implantation embryonic quality was investigated. Porcine oocytes were matured in NCSU-23 medium supplemented with 10% (v/v) porcine follicular fluid, 10 ng mL-1 epidermal growth factor, 25 �M �-mercaptoethanol, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, 10 IU mL-1 hCG, and 1 �M PGI2 analog for 22 h, and then further cultured in maturation medium without PGI2 analog and hormones for 22 h. After fertilization in Tris-buffered (mTBM) medium for 6 h, presumptive porcine zygotes were cultured in the NCSU-23 medium supplemented with 4% BSA for 6 days. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). First, we confirmed that PGI2 analog-treated (90.0 � 2.6%) oocytes showed a higher proportion of the metaphase II stage than non-treated (65.7 � 1.4%) ones (P < 0.05). Thus, to confirm the activities of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK), Western blots were performed in matured oocytes by using specific antibodies such as anti-cdc2 and anti-ERK1/2. The activities of MPF and MAPK were increased in porcine oocytes treated with PGI2 analog. In the PGI2 analog-treated group, polyspermic rate (17.9 � 13.3%) was reduced as compared with that of the non-treated group (35.8 � 9.4%). Furthermore, the rate (25.3%, 40/158) of blastocyst formation in the PGI2 analog-treated group was higher than in the non-treated group (19.7%, 27/137; P < 0.05). Also, cell numbers of blastocysts were increased (29 � 2.5% vs. 39.6 � 1.4%) in the treated vs. the non-treated group. The numbers of fragmented DNA nuclei detected in the blastocyst stage by the TUNEL assay were decreased in the PGI2-treated group compared with the non-treated group (2.1% vs. 5.2%). In conclusion, direct roles of PGI2 during porcine oocyte maturation may involve reducing apoptosis and enhancing blastocyst quality.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

134 EFFECT OF ELEVATED CIRCULATING PROGESTERONE CONCENTRATION ON DEVELOPMENT OF IN VITRO PRODUCED BOVINE ZYGOTES IN VIVO

2008 ◽  
Vol 20 (1) ◽  
pp. 147 ◽  
Author(s):  
F. Rings ◽  
F. Carter ◽  
M. Hölker ◽  
A. Kuzmany ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Prostaglandin F2α ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Progesterone Concentration ◽  
Interferon Tau ◽  
Endoscopic Techniques ◽  
Embryo Recovery ◽  
Chi Square Analysis

Elevated concentrations of circulating progesterone in the immediate post-conception 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 tissues of the uterus. However, whether or not progesterone has a direct effect on the embryo also is unknown and, at least in vivo, in single ovulating animals, is difficult to assess. Using state-of-the-art endoscopic techniques, the objective of this study was to examine the effect of elevated progesterone on the development of IVP zygotes transferred to the oviducts of cattle with high or normal circulating progesterone concentrations. Simmental heifers (n = 14) were synchronized using a combination of 2 injections of a prostaglandin F2α analogue administered 11 days apart and gonadotropin-releasing hormone. Only animals exhibiting a clear standing oestrus (= day 0) were used. In order to produce animals with divergent progesterone concentrations, half of the animals received a PRID on day 3 of the oestrous cycle, which was left in place until embryo recovery. All animals were blood sampled daily from days 0 to 7. Cleaved embryos were transferred using endoscopy to the ipsilateral oviduct of each recipient on day 2 and recovered by non-surgically flushing the oviduct and the uterus on day 7. The number of embryos developing to the morula/blastocyst stage was recorded at recovery and following overnight culture in CR1aa medium. Data were analyzed by chi-square analysis. Insertion of a PRID on day 3 resulted in a significant elevation in progesterone concentrations from day 4 (2.36 ± 0.16 ng mL–1 v. 0.54 ± 0.10 ng mL–1, P < 0.001) until day 6 (1.98 ± 0.22 ng mL–1 v. 0.95 ± 0.17 ng mL–1; P < 0.01). The recovery rate was lower in animals that received a PRID (P < 0.05). However, there was no effect of progesterone on the proportion of embryos developing to the morula/blastocyst stage. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo and that the reported positive effect of high progesterone levels in terms of fertility are manifested after day 8. Table 1. Effect of elevated progesterone concentration on development of in vitro produced bovine zygotes in vivo

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