scholarly journals Global transcriptomic response of bovine endometrium to blastocyst-stage embryos

Reproduction ◽  
2019 ◽  
Vol 158 (3) ◽  
pp. 223-235 ◽  
Author(s):  
C Passaro ◽  
D Tutt ◽  
S Bagés-Arnal ◽  
C Maicas ◽  
R Laguna-Barraza ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Subsequent Pregnancy ◽  
Rna Seq ◽  
Interferon Tau ◽  
Transcriptomic Response ◽  
Interferon Stimulated Genes ◽  
Induced Changes

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at −80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

scholarly journals 243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
P. Lonergan ◽  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
P.M. Moreira ◽  
B. Pintado ◽  
...  
Keyword(s):  
Growth Factor ◽  
Relative Abundance ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Cell Stage

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P<0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P<0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

scholarly journals Impact of the LH surge on granulosa cell transcript levels as markers of oocyte developmental competence in cattle

Reproduction ◽  
2012 ◽  
Vol 143 (6) ◽  
pp. 735-747 ◽  
Author(s):  
Isabelle Gilbert ◽  
Claude Robert ◽  
Christian Vigneault ◽  
Patrick Blondin ◽  
Marc-André Sirard
Keyword(s):  
Transcript Abundance ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Follicle Cells ◽  
Transcriptomic Response ◽  
Lh Surge ◽  
Oocyte Developmental Competence ◽  
The Difference

In the case of in vitro embryonic production, it is known that not all oocytes detain the developmental capacity to form an embryo. This capacity appears to be acquired through completion of folliculogenesis, during which the oocyte and follicular cells influence their respective destinies. The differentiation status of granulosa cells (GCs) could therefore offer an indicator of oocyte quality. The aim of this study was to compare mRNA transcript abundance in GCs associated with oocytes that subsequently reach or not the blastocyst stage. GCs were collected from cattle following an ovarian stimulation protocol that did or did not include the administration of LH. GCs were classified according to the developmental stage achieved by the associated oocytes. Transcript abundance was measured by microarray. Follicles (n=189) obtained from cows before and after the LH surge were essentially similar and the rates of oocytes reaching the blastocyst stage were not significantly different (52 vs 41%), but blastocyst quality was significantly better in the post-LH-surge group. In GCs from the pre-LH-surge group and associated with developmentally competent oocytes, 18 overexpressed and 22 underexpressed transcripts were found, including novel uncharacterized transcripts, whereas no differentially expressed transcripts were associated with developmentally different oocytes in the post-LH-surge group. The novel transcriptomic response associated with LH appeared to mask the difference. Based on oocyte developmental competence, the period prior to the LH surge appears best suited for studying competence-associated mRNA transcripts in bovine follicle cells.

scholarly journals Blastocyst-induced changes in the bovine endometrial transcriptome

Reproduction ◽  
2018 ◽  
Vol 156 (3) ◽  
pp. 219-229 ◽  
Author(s):  
C Passaro ◽  
D Tutt ◽  
D J Mathew ◽  
J M Sanchez ◽  
J A Browne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conditioned Medium ◽  
Direct Contact ◽  
Blastocyst Stage ◽  
Induced Responses ◽  
Interferon Stimulated Genes ◽  
Maternal Interaction ◽  
Induced Changes ◽  
Maximum Expression

The objectives of this study were (i) to determine whether blastocyst-induced responses in endometrial explants were detectable after 6- or 24-h co-culture in vitro; (ii) to test if direct contact is required between embryos and the endometrial surface in order to stimulate endometrial gene expression; (iii) to establish the number of blastocysts required to elicit a detectable endometrial response; (iv) to investigate if upregulation of five interferon-stimulated genes (ISGs) in the endometrium was specific to the blastocyst stage and (v) to test if alterations in endometrial gene expression can be induced by blastocyst-conditioned medium. Exposure of endometrial explants to Day 8 blastocysts in vitro for 6 or 24 h induced the expression of ISGs (MX1, MX2, OAS1, ISG15, RSAD2); expression of IFNAR1, IFNAR2, NFKB1, IL1B, STAT1, LGALS3BP, LGALS9, HPGD, PTGES, ITGB1, AKR1C4, AMD1 and AQP4 was not affected. Culture of explants in the presence of more than five blastocysts was sufficient to induce the effect, with maximum expression of ISGs occurring in the presence of 20 blastocysts. This effect was exclusive to blastocyst stage embryos; oocytes, 2-cell embryos or Day 5 morulae did not alter the relative abundance of any of the transcripts examined. Direct contact between blastocysts and the endometrial surface was not required in order to alter the abundance of these transcripts and blastocyst-conditioned medium alone was sufficient to stimulate a response. Results support the notion that local embryo–maternal interaction may occur as early as Day 8 of pregnancy in cattle.

scholarly journals Predictive value of bovine follicular components as markers of oocyte developmental potential

2014 ◽  
Vol 26 (2) ◽  
pp. 337 ◽  
Author(s):  
Satoko Matoba ◽  
Katrin Bender ◽  
Alan G. Fahey ◽  
Solomon Mamo ◽  
Lorraine Brennan ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Principal Component ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Thecal Cells ◽  
Total Fatty Acids

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (± s.e.m.) follicular concentrations of testosterone (62.8 ± 4.8 ng mL–1), progesterone (616.8 ± 31.9 ng mL–1) and oestradiol (14.4 ± 2.4 ng mL–1) were not different (P > 0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P = 0.023) and total fatty acids (P = 0.031) and significantly higher in linolenic acid (P = 0.036) than follicular fluid from incompetent oocytes. Significantly higher (P < 0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

214 EFFECT OF OVARIAN SUPERSTIMULATION ON EXPRESSION OF GENES ASSOCIATED WITH THE OOCYTE DEVELOPMENTAL COMPETENCE OF NELORE COWS

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
R. A. Satrapa ◽  
E. M. Razza ◽  
A. G. Pupulim ◽  
A. C. S. Castilho ◽  
B. Loureiro ◽  
...  
Keyword(s):  
Animal Health ◽  
Rna Extraction ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Oocyte Competence ◽  
Embryo Yield ◽  
Expression Of Genes

The P36 protocol has contributed to the genetic improvement of Brazilian herd through its successful use in embryo transfer programs. We aimed to investigate the effect of P36 protocol on embryo yield and mRNA expression of genes correlated with the competence of cumulus–oocyte complex (COC): receptors of FSH (FSHR), EGF (EGFR), and pentraxin 3 (PTX3) in cumulus cells; receptors of LH (LHR) and angiotensin 2 (AT2) in granulosa cells; and GDF9, BMP15, and histone H2A (H2A) in oocytes. Multiparous Nelore cows were allocated in control and P36 groups. Control group (non-superovulated, n = 15) received a progesterone intravaginal device (P4, 1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of oestradiol benzoate (EB, IM, BER-BE®, Syntex, Buenos Aires, Argentina) at a random day of the oestrous cycle (Day 0). A PGF2α analogue (150 mg d-cloprostenol, IM, Prolise®, RARS SRL) was administered (Day 8) and Primer® was removed. The P36 group (n = 10) received a Primer® and 2.0 mg of EB (Day 0). The FSH treatment (160 mg Folltropin®, Bioniche Animal Health, Ontario, Canada) was initiated at decreasing doses: 40, 30, 20, and 10% of the total dose twice daily for 4 days (Day 5). The PGF2α analogue was administered (Day 8) and after 36 h primer was removed. Animal slaughter to ovary collection was performed 12 h after Primer® removal (Day 9). Some of the oocytes were matured (TCM199), fertilized with Nelore semen (n = 6), and cultured (SOF-synthetic oviduct fluid) to the blastocyst stage. Embryos were removed from culture (Day 6), allocated in 5 pools with 5 embryos in each group, and subjected to RNA extraction. Remaining oocytes were denuded from cumulus and zona pellucida (vortex and Protease®, Sigma-Aldrich, St. Louis, MO, USA). Pools of 20 oocytes and of their respective cumulus cells (n = 6 pools; control group and n = 4 pools, P36 group) were subjected to RNA extraction (RNeasy kit, Qiagen, Valencia, CA, USA). Gene expression was performed by real-time RT-PCR using oligo-dT in reverse transcription and bovine-specific primers. Expression of cyclophilin A was used as endogenous control. Change to developmental rates to the blastocyst stage and transcript abundance were compared by t-test and significance was considered when P < 0.05. Blastocyst rates were also similar (P > 0.05) in groups P36 (40/99; 40%) and control (16/43; 37%). Expression of H2A, EGFR, FSHR, and PTX3 in cumulus cells did not differ (P > 0.05) among treatment groups. The expression of GDF9 and BMP15 in cumulus cells was higher (P < 0.05) in the P36 group, but in oocytes these transcripts were more expressed in the control group (P < 0.05). Although important genes (GDF9 and BMP15) were less expressed in oocytes from superstimulated cows, the maintenance of H2A in oocytes, as well as PTX3, EGFR, and FSHR, and the increases in GDF9 and BMP15 expression in cumulus cells do not seem to affect oocyte competence due to the similar embryo yield of both groups. Supported by FAPESP.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

scholarly journals iCLIP analysis of RNA substrates of the archaeal exosome

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Jochen Bathke ◽  
A. Susann Gauernack ◽  
Oliver Rupp ◽  
Lennart Weber ◽  
Christian Preusser ◽  
...  
Keyword(s):  
Rna Binding ◽  
Transcript Abundance ◽  
Global Scale ◽  
Circular Rnas ◽  
Rna Seq ◽  
Uv Crosslinking ◽  
Rna Targets ◽  
Nucleotide Resolution

Abstract Background The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. Results To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected. Conclusions In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.

267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 241
Author(s):  
D. Tesfaye ◽  
K. Nganvongpanit ◽  
F. Rings ◽  
M. Gilles ◽  
D. Jennen ◽  
...  
Keyword(s):  
Free Water ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
E Cadherin

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

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

279 CYTOPLASMIC POLYADENYLATION-REGULATED GENE EXPRESSION DURING BOVINE OOCYTE MATURATION

2015 ◽  
Vol 27 (1) ◽  
pp. 228
Author(s):  
J. M. Reyes ◽  
P. J. Ross
Keyword(s):  
Oocyte Maturation ◽  
Tail Length ◽  
Transcript Abundance ◽  
Bovine Oocyte ◽  
Rna Seq ◽  
Cytoplasmic Polyadenylation ◽  
Transcript Levels ◽  
Regulated Gene Expression ◽  
Exogenous Control

Using RNA-seq of GV and MII oocytes we have described changes in polyadenylated transcript abundance that occur during in vitro bovine oocyte maturation (Reyes J. M., J. L. Chitwood, and P. J. Ross. 2013. Deciphering regulation of transcript abundance in maturing bovine oocytes. Poster session presented at International Plant & Animal Genome XXI. San Diego, CA). These changes can be attributed to transcript degradation, transcription, or transcript polyadenylation levels. The objectives of the present study were to determine the extent of cytoplasmic polyadenylation (CP) by measuring total and polyadenylated transcript abundance and poly(A) tail length in GV and MII oocytes for 8 (CCNB1, CPEB4, DNMT3B, FBXO43, EZH2, GDF9, PRDX2, and PAIP2) genes selected based on RNA-seq results. Oocytes were obtained by aspiration of abattoir-derived ovaries (GV) and in vitro maturation for 24 h (MII). Four pools of 40 oocytes were collected per stage. Enhanced green fluorescent protein (EGFP) cRNA was spiked into each sample before RNA extraction using the PicoPure RNA Isolation Kit. Extracted RNA was equally divided for cDNA synthesis using either random hexamers or anchored oligo(dT) primers to detect total and polyadenylated transcripts, respectively. Quantitative PCR (qPCR) of target genes, EGFP (exogenous control), and PPIA (endogenous control) was performed in duplicate for each replicate and gene. Relative transcript abundance was calculated using the 2[–ΔΔC(T)] method and statistically analysed using the Student's t-test. Transcript poly(A) tail length was determined for all but 2 genes (DNMT3B and EZH2) at the GV and MII stages using rapid amplification of cDNA ends poly(A) test (RACE-PAT). Two replicates of GV (n = 100) and MII (n = 100) pairs were collected to perform RACE-PAT followed by fragment analysis on a Bioanalyzer DNA 1000 chip. Polyadenylated RNA abundance levels matched those of the RNA-seq study for 7/8 genes using both PPIA and EGFP to normalise qPCR data, demonstrating the validity of RNA-seq results. Furthermore, total transcript levels for 6/8 and 7/8 genes remained unchanged when normalized to an endogenous and exogenous control, respectively. Combined, the results suggest a significant role for CP considering changes in polyadenylated transcript levels occur without changes in total RNA abundance. Also, changes in transcript abundance corresponded to differences in poly(A) tail length at the specific stages as determined by RACE-PAT. In conclusion, CP is the predominant mechanism responsible for changes in transcript abundance during oocyte maturation at least for the majority of the examined genes, though more in-depth studies are required to determine the global extent of CP. This study improved the atlas of CP-regulated genes potentially providing researchers with critical knowledge to improve in silico tools that predict genes regulated by CP based on presence, position, and distribution of motifs within the 3′ untranslated region.

Sign in / Sign up

Export Citation Format

Share Document