127 DEVELOPMENT AND QUALITY OF BOVINE MORULAE CULTURED IN SERUM-FREE MEDIUM WITH RETINOIC RECEPTOR SPECIFIC AGONISTS

In the cell, all-trans retinoic acid (ATRA), a vitamin A metabolite, binds to retinoic acid-receptor (RAR), whereas the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). Synthetic compounds such as LG100268 (LG; Ligand Laboratories) are highly specific to bind RXR, which allows to differentially study the RAR and RXR pathways. In previous work morulae treated with LG for 48 h showed to improve blastocyst development and to activate pro-apoptotic genes (in press), whereas ATRA for 24 h increased cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodr�guez et al. 2006 Hum. Reprod. 21, 2149–2157). However, LG and ATRA were never both compared for 24 in medium with BSA, which is thought to be more appropriate to produce embryos for cryopreservation than serum-containing medium. In this work we analyze development, quality, and viability of morulae cultured with RAR and RXR agonists. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in synthetic oviduct fluid (SOF) +3 gL–1 BSA. On day 6, morulae were treated for 24 h with ATRA 0.7 µm, LG 0.1 µm, or no additives. Blastocyst development was monitored up to day 8. Differential cell counts were made on hatched blastocysts on days 7 and 8. Apoptosis and necrosis (TUNEL + nuclear histology) were made on day 8 expanded and hatched blastocysts. Data were analyzed by GLM and Duncan's test, expressed as LSM � SE, and development rates were expressed as percentages of cultured morulae (replicates [R] = 14 for development; R = 9 for cell counts; R = 4 for apoptosis; n = 1647 morulae). ATRA yielded more blastocysts on day 8 than LG and controls (72.2 � 2.2 v. 60.0 � 2.3 and 65.6 � 2.4, respectively; P < 0.02), and more expanded blastocysts than LG (48.6 � 2.3 v. 36.6 � 2.4; P < 0.02), but no more than controls (43.5 � 2.5). Day-7 and day-8 hatched blastocysts cultured with ATRA showed more total cells than day-7 controls (163.5 � 8.0 and 161.5 � 5.4 v. 137.7 � 8.9, respectively; P < 0.05). However, in the presence of ATRA, day-8 blastocysts showed a strong cell reduction in the inner cell mass (ICM), whereas their day-7 counterparts conserved ICM/total cells proportions comparable to day-7 controls (11.0 � 1.2 v. 19.7 � 1.7 and 20.6 � 1.9, respectively; P < 0.03). The LG increased apoptotic index (AI) and necrotic index (NI) in the ICM (AI: 14.5 � 2.4 v. 6.4 � 1.5 and 6.4 � 1.4; NI: 5.0 � 1.2 v. 0.9 � 0.8 and 1.6 � 0.7; for LG, ATRA, and controls, respectively; P < 0.02). Embryos produced with ATRA showed improved development and cell distribution without increasing apoptosis and necrosis. Vitrification of excellent day-7 and day-8 blastocysts is in course to evaluate cryosurvival and further embryo transfer to determine full developmental competence. Grant Support: MEC, project AGL2005-04479. M. Muñoz is sponsored by FICYT.

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127 BINDING RETINOID RECEPTORS BY SPECIFIC AGONISTS AFFECTS THE BOVINE BLASTOCYST DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab127 ◽

2006 ◽

Vol 18 (2) ◽

pp. 172 ◽

Cited By ~ 1

Author(s):

E. Gómez ◽

A. Rodríguez ◽

C. Alonso-Montes ◽

N. Caamaño ◽

L. J. Royo ◽

...

Keyword(s):

Retinoic Acid ◽

Inner Cell Mass ◽

Retinoic Acid Receptor ◽

Cell Mass ◽

Cell Counting ◽

Retinoid Receptor ◽

Blastocyst Development ◽

Cell Counts ◽

Development And Differentiation

Production of embryos in vitro with improved inner cell mass (ICM) and high ICM per total cell rate is a major objective in reproductive biotechnology. Exogenous all-trans retinoic acid (ATRA), a vitamin A metabolite, and endogenous retinoid regulate development and differentiation during bovine morula to blastocyst transition in vitro. ATRA binds to retinoic acid-receptor (RAR), and the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). The unspecific binding of 9-cis-RA to receptors makes it difficult to study RXR transactivation. Therefore, in this work we studied blastocyst development and cell counts by using a specific synthetic RXR agonist [LG100268 LG; a gift of Ligand Laboratories] as opossed to the effect exerted by ATRA upon RAR binding. Cumulus-oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in B2 medium with Vero cells until 139 h post-insemination (Day 6), the time at which embryos [morulae (e90%) + early blastocysts] underwent treatments for 48 h in 400 �L of SOFaaci + 5% FCS. Data (5 replicates per experiment) were analyzed by CATMOD for effects, processed by GLM and Duncan's test, and expressed as LSM � SE (a,b,c P d 0.05). After a LG dose-response experiment (n = 480 morulae), blastocysts rates from LG 1 �M on Day 7 were higher than LG 10 �M, LG 0.1 �M, and LG 0 �M (Day 7: 42.8 � 4.1 vs. 34.4 � 3.7, 36.8 � 3.7, and 32.4 � 3.7, respectively). On Day 8, LG 1 �M also yielded more blastocysts than LG 0.1 �M (50 � 4.2 vs. 44.4 � 3.7, respectively). By differential cell counting (n = 113 blastocysts), hatched blastocysts with LG 10 �M showed proliferation in the ICM, while trophectoderm (TE) cells decreased conversely to LG concentration. These effects were not obvious in expanded blastocysts. In a subsequent experiment (n = 340 morulae), ATRA led to blastocysts rates on Day 8 that were higher than negative, untreated controls, but not different from LG 1 �M (42.4 � 2.4 vs. 33.1 � 2.0 and 36.0 � 2.4, respectively). ATRA and LG 1 increased TE in expanded blastocysts (n = 42) (102 � 13.2 and 96.23 � 13.2, respectively vs. 72.8 � 10.9 in the untreated group) but not in their hatched counterparts (n = 44). There were no differences in the ICM; but percentages of ICM per total cells were higher in hatched blastocysts cultured with ATRA than in expanded LG 1 �M blastocysts and expanded controls (39.5 � 5.5 vs. 24.2 � 5.7, and 20.9 � 4.7, respectively). Manipulation of retinoid receptor-specific pathways make it possible to control blastocyst development and differentiation, leading to embryos of improved quality and viability. Work is in progress to analyze gene expression in these blastocysts. This work was supported by grant MCYT, project AGL-2005-04479.

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58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab58 ◽

2011 ◽

Vol 23 (1) ◽

pp. 134

Author(s):

I. M. Saadeldin ◽

B. H. Kim ◽

B. Roibas da Torre ◽

O. J. Koo ◽

G. Jang ◽

...

Keyword(s):

Nuclear Transfer ◽

Inner Cell Mass ◽

Quantitative Polymerase Chain Reaction ◽

Cell Mass ◽

Donor Cell ◽

Developmental Competence ◽

Control Group ◽

Embryonic Cells ◽

Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

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Modified Spirulina maxima Pectin Nanoparticles Improve the Developmental Competence of In Vitro Matured Porcine Oocytes

Animals ◽

10.3390/ani11092483 ◽

2021 ◽

Vol 11 (9) ◽

pp. 2483

Author(s):

Pantu-Kumar Roy ◽

Ahmad-Yar Qamar ◽

Bereket-Molla Tanga ◽

Seonggyu Bang ◽

Gyeonghwan Seong ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Culture Conditions ◽

Developmental Competence ◽

Spirulina Maxima ◽

Blastocyst Development ◽

Parthenogenetic Activation ◽

Molecular Approaches

Molecular approaches have been used to determine metabolic substrates involved in the early embryonic processes to provide adequate culture conditions. To investigate the effect of modified Spirulina maxima pectin nanoparticles (MSmPNPs) on oocyte developmental competence, cumulus–oocyte complexes (COCs) retrieved from pig slaughterhouse ovaries were subjected to various concentrations of MSmPNPs (0, 2.5, 5.0, and 10 µg/mL) during in vitro maturation (IVM). In comparison to the control, MSmPNPs-5.0, and MSmPNPs-10 groups, oocytes treated with 2.5 µg/mL MSmPNPs had significantly increased glutathione (GSH) levels and lower levels of reactive oxygen species (ROS). Following parthenogenetic activation, the MSmPNPs-2.5 group had a considerably higher maturation and cleavage rates, blastocyst development, total cell number, and ratio of inner cell mass/trophectoderm (ICM:TE) cells, when compared with those in the control and all other treated groups. Furthermore, similar findings were reported for the developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos. Additionally, the relative quantification of POU5F1, DPPA2, and NDP52 mRNA transcript levels were significantly higher in the MSmPNPs-2.5 group than in the control and other treated groups. Taken together, the current findings suggest that MSmPNP treatment alleviates oxidative stress and enhances the developmental competence of porcine in vitro matured oocytes after parthenogenetic activation and SCNT.

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113 RETINOIC ACID DOES NOT PROTECT AGAINST THE SELECTIVE DAMAGE TO THE BOVINE INNER CELL MASS INFLICTED BY VITRIFICATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab113 ◽

2007 ◽

Vol 19 (1) ◽

pp. 174

Author(s):

C. Díez ◽

A. Rodríguez ◽

C. De Frutos ◽

J. N. Caamaño ◽

N. Facal ◽

...

Keyword(s):

Retinoic Acid ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Counting ◽

Bovine Embryo ◽

Embryo Survival ◽

Binding Effect ◽

Cell Counts ◽

Before And After

Successful cryopreservation of in vitro-produced embryos is a major objective in reproductive biotechnology. It was reported that in vitro culture with high BSA concentrations improved bovine embryo survival after vitrification (D�ez et al. 2005 Reprod. Dom. Anim. 40, 384). All-trans retinoic acid (ATRA) increases cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodr�guez et al. 2006 Hum. Reprod. 21, 2149–2157). This work analyzed the effect of ATRA on bovine embryo development, survival to vitrification, and cell allocation before and after cryopreservation. Bovine cumulus–oocyte complexes were matured and fertilized in vitro, and presumptive zygotes cultured in SOF + 20 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 917 morulae + early blastocysts were cultured for 24 h with: (1) 1.4 �M ATRA, (2) 0.7 �M ATRA, and (3) no ATRA (control). Embryos were subsequently cultured up to Day 9 in SOF + 20 g L-1 BSA. Development was recorded and differential cell counting was performed on Day 8 and 9 hatched blastocysts. Simultaneously, Day 7 and 8 expanded blastocysts were vitrified (OPS; Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). After warming, blastocysts were cultured for 72 h in B2 + 5% FCS with Vero cells, and cell counts were performed in fully expanded or hatched blastocysts. Data (7 replicates for cell counts before and 4 after vitrification) were processed by GLM and Duncan's test, and were expressed as LSM � SE (x,y: P = 0.01; a,b: P < 0.05; α,β: P < 0.002). Developmental rates did not differ among groups. Blastocysts cultured in 0.7 �M ATRA survived vitrification at rates similar to those of controls, and only hatching rates 24 h post-warming were significantly lower than those of controls (4.0 � 8.2a vs. 31.2 � 8.2b). ATRA at 1.4 �M was detrimental to survival of Day 7 embryos, whereas differences were not detected in Day 8 blastocysts. In all groups, the vitrification procedure significantly reduced the cells of the ICM (1.4 �M ATRA: 28.3 � 3.1α vs. 8.6 � 4.1β; 0.7 �M ATRA: 27.7 � 3.5α vs. 2.2 � 4.1β; Control: 31.3 � 3.1α vs. 7.0 � 5.1β). Total cell counts were: 1.4 �M ATRA: 160.0 � 9.8a vs. 130.0 � 12.2b; 0.7 �M ATRA: 165.3 � 8.8a vs. 123.2 � 11.7b; Control: 161.2 � 9.2a vs. 131.0 � 15.1b. The ratios of ICM/TE cells were: 1.4 �M ATRA: 16.9 � 2.7x vs. 6.1 � 3.2y; 0.7 �M ATRA: 17.2 � 2.3x vs. 2.0 � 3.0y; Control: 20.6 � 2.4x vs. 4.3 � 3.9y. All values are before and after vitrification, respectively. When considered together, the differences in the cell counts before and after vitrification were highly significant (*P < 0.0001): 1.4 �M ATRA: 29.2 � 1.9* vs. 5.9 � 2.6; 0.7 �M ATRA: 162.5 � 5.5* vs. 127.2 � 7.6; Control: 18.3 � 1.5* vs. 4.2 � 2.0. Our results show that ATRA did not improve the embryo survival to vitrification. Although 1.4 �M ATRA was used to avoid a 'binding effect' related to an elevated protein level (Klaassen et al. 1999 Biochim. Biophys. Acta 1427, 265–275), the BSA concentrations used in culture could mask any ATRA effect. The vitrification procedure used in this study produced a selective damage within the ICM cells, which can explain the reduced survival rates obtained after warming. This work was supported by Grant AGL2005-04479.

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Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes

Reproduction ◽

10.1530/rep-12-0318 ◽

2013 ◽

Vol 145 (4) ◽

pp. 345-355 ◽

Cited By ~ 29

Author(s):

F Moulavi ◽

S M Hosseini ◽

M Hajian ◽

M Forouzanfar ◽

P Abedi ◽

...

Keyword(s):

Cell Fate ◽

Embryo Development ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Developmental Competence ◽

Blastocyst Development ◽

Cleavage Rate ◽

Nuclear Remodeling

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep.In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances ofHSP90AA1(HSP90),NPM2andATPasegenes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents ofPOU5F1(OCT4) with the ZI-NT and ZF-NT methods and ofPAPOLA(PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

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Melatonin Alleviates the Toxicity of High Nicotinamide Concentrations in Oocytes: Potential Interaction with Nicotinamide Methylation Signaling

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/5573357 ◽

2021 ◽

Vol 2021 ◽

pp. 1-16

Author(s):

Marwa El-Sheikh ◽

Ahmed Atef Mesalam ◽

Seok-Hwan Song ◽

Jonghyeok Ko ◽

Il-Keun Kong

Keyword(s):

Inner Cell Mass ◽

Active Form ◽

Cell Mass ◽

Potential Interaction ◽

Developmental Competence ◽

Ros Scavenging ◽

Vitamin B3 ◽

Blastocyst Development ◽

Preimplantation Embryo Development

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10−7 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.

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Biological differences between in vitro produced bovine embryos and parthenotes

Reproduction ◽

10.1530/rep-08-0220 ◽

2009 ◽

Vol 137 (2) ◽

pp. 285-295 ◽

Cited By ~ 36

Author(s):

Enrique Gómez ◽

Alfonso Gutiérrez-Adán ◽

Carmen Díez ◽

Pablo Bermejo-Alvarez ◽

Marta Muñoz ◽

...

Keyword(s):

De Novo ◽

Inner Cell Mass ◽

Relative Proportion ◽

Cell Mass ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Blastocyst Development ◽

Cell Counts ◽

Biological Differences

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes andin vitroproduced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage.In vitromatured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-relatedPOU5F1and the methylationDNMT3Agenes were downregulated in parthenotes. Among pregnancy recognition genes,TP-1was upregulated in parthenotes, whilePGRMC1andPLAC8did not change. Expression ofp66shcandBAX/BCL2ratio were higher, andp53lower, in parthenotes. Among metabolism genes,SLC2A1was downregulated, whileAKR1B1,PTGS2,H6PD, andTXNwere upregulated in parthenotes, andSLC2A5did not differ. Among genes involved in compaction/blastulation,GJA1was downregulated in parthenotes, but no differences were detected withinATP1A1andCDH1. Within parthenotes, the expression levels ofSLC2A1,TP-1, andH6PD, and possiblyAKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, throughp66shcandp53respectively, and in their mechanisms to control pluripotency andde novomethylation.

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156 IN VITRO DEVELOPMENT OF BOVINE MORULAE PRODUCED AND/OR CULTURED WITH ACTIVIN

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab156 ◽

2010 ◽

Vol 22 (1) ◽

pp. 236 ◽

Cited By ~ 1

Author(s):

B. Trigal ◽

E. Gómez ◽

C. Diez ◽

J. N. Caamaño ◽

I. Molina ◽

...

Keyword(s):

Embryo Development ◽

Inner Cell Mass ◽

Cell Mass ◽

Total Cell ◽

Blastocyst Development ◽

Cell Counts ◽

Morula Stage ◽

Vitro Development ◽

And Control

We reported that the presence of activin during in vitro culture improves embryo development without changing the cell distribution in the blastocyst (Díez et al. 2009 AETE in press). In the present work, we aimed to analyze the morula stage as a putative milestone to activin exert differential effects. Day -5 morulae were produced with IVMFC oocytes from abattoir ovaries, using SOF with amino acids, myo-inositol, and 3 g L-1 of BSA as a culture medium. Embryo culture contained 10 ng mL-1 or 0 ng mL-1 of activin from Day -3 to Day -5. Early morulae (n = 543 out of 1099 cultured oocytes) were selected and subsequently cultured with or without 10 ng mL-1 of activin up to Day -8. Embryo development was daily monitored and cells differentially counted in Day -8 expanded blastocysts. (Thouas et al. 2001 Reprod. Biomed. 2001 3, 25-29). Data were analyzed by general linear model and presented as least squares means ± SEM. Activin from Days 3 to 5 did not change Day -5 morulae rates (P > 0.8). In morulae produced without activin (Days 5 to 8 and control), a treatment with activin from Days 5 to 8 improved total blastocyst rates v. controls, both in Day -7 and Day -8 (50.9 ± 3.6 v. 32.6 ± 3.6 and 60.8 ± 2.9 v. 42.3 ± 2.9, respectively; P < 0.01). Similarly, Day -7 expansion rates with activin (Days 5 to 8) were higher than controls (14.6 ± 1.8 v. 8.6 ± 1.8; P < 0.03). However, the above effects were not the same as those observed in morulae produced with activin (Days 3 to 5 and Days 3 to 8), where blastocyst development between activin treatment and controls only significantly differed in expansion rates on Day -7 (14.9 ± 1.8 v. 5.8 ± 1.8, respectively; P < 0.03). Morulae treated with activin (Days 5 to 8) yielded Day -7, total and expanded blastocyst rates, higher than morulae produced with activin (Days 3 to 5) (50.9 ± 3.6 v. 37.4 ± 3.6 and 14.6 ± 5.8 v. 5.8 ± 1.8, respectively; P < 0.03). Expansion rates on Day -8 were numerically higher within morulae produced and/or treated with activin (Days 3 to 8, Days 5 to 8, and Days 3 to 5) (values between 26.7 ± 2.6 and 27.4 ± 2.6) than in controls without activin at any time (19.2 ± 2.6) (P > 0.05). Trophectoderm (TE) cell numbers were reduced in embryos produced and/or treated with activin (Days 3 to 8, Days 3 to 5, and Days 5 to 8) (values between 109.4 ± 7.6 and 115.3 ± 7.9) as compared with untreated controls (141.2 ± 10.1) (P < 0.05). In morulae produced without activin, total cell counts were lower with activin being present from Day -5 to Day -8 (154.0 ± 8.8 v. 128.4 ± 7.2; P < 0.05). Inner cell mass (ICM) and ICM/total cell ratio were not affected by the presence of activin (P > 0.05). Activin did not change Day -5 morulae rates, although subsequent blastocyst development was in part affected by the presence of activin before the morula stage. Interestingly, improvements in blastocyst development, including expansion rates, triggered by activin led to reduced TE and unaltered ICM cell counts, suggesting that activin inhibits TE differentiation. Support: Cajastur (B. Trigal). MCINN: M. Muñoz (RYC08-03454); D. Martín (PTA2007-0268-I); INIA (I. Molina); Project HF2007-0126.

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Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid

Reproduction ◽

10.1530/rep.0.1250409 ◽

2003 ◽

pp. 409-416 ◽

Cited By ~ 38

Author(s):

CO Hidalgo ◽

C Diez ◽

P Duque ◽

N Facal ◽

E Gomez

Keyword(s):

Retinoic Acid ◽

In Vitro Maturation ◽

Developmental Competence ◽

Blastocyst Development ◽

Developmental Potential ◽

Vitro Fertilization ◽

Cell Counts ◽

Bovine Oocytes ◽

Number Of Cells

Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization and culture in simple medium, blastocyst development and hatching rates increased in oocytes matured with 9-cis-retinoic acid. Addition of ethanol (used as a solvent for 9-cis-retinoic acid) resulted in higher cell counts and proportions of cells in the inner mass of day 7 blastocysts. Day 8 blastocysts represented most differences observed in the number of cells. In these embryos, ethanol and 9-cis-retinoic acid increased both the number of cells and proportions in the inner mass. However, while ethanol treatment reduced the number of cells in the trophectoderm, 9-cis-retinoic acid did not. The total number of cells was unaffected by treatment within 1 day, although untreated oocytes lead to day 8 blastocysts with reduced total cell counts. Once transferred to recipients, both fresh and vitrified-warmed blastocysts derived from oocytes matured with 9-cis-retinoic acid yielded more pregnancies at day 60. Modifications of retinoid metabolism affect development and trophectoderm differentiation, and in vitro maturation with 9-cis-retinoic acid increased the developmental competence of the oocyte.

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