176 EFFECT OF LEUKEMIA INHIBITORY FACTOR FROM HUMAN AND MOUSE ORIGIN ON THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 204
Author(s):  
C. De Frutos ◽  
A. Rodríguez ◽  
C. Díez ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Bovine Embryos ◽  
Cell Counts

Leukemia Inhibitory Factor (LIF) is a cytokine with potential to influence embryonic quality and proliferation within the inner cell mass (ICM). However, conflicting effects of LIF have been reported with in vitro-produced (IVP) bovine embryos, in spite of LIF receptor (LIFr) and gp130 transcripts being expressed at all stages during pre-implantation development (Niemann and Wrenzycki 2000 Theriogenology 53, 21–34). As there is no commercially available bovine LIF (bLIF), researchers have used human LIF (hLIF) because of its greater sequence homology compared to murine LIF (mLIF). However, mLIF has been not compared with hLIF in culture with bovine embryos; thus this was the aim of this study. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro and presumptive zygotes cultured in modified synthetic oviduct fluid with 6 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 423 morulae (>90%) and early blastocysts were cultured for 48 h with: (1) 100 ng mL-1 recombinant mLIF (Sigma-Aldrich Quimica SA, Madrid, Spain); (2) 100 ng mL-1 recombinant hLIF (Sigma); and (3) no LIF. Data (6 replicates) were processed by GLM and Duncan's test, and expressed as LSM � SE (ab: P < 0.05; xy: P < 0.01). Development was recorded up to the hatched blastocyst stage and cells were differentially counted in the ICM and trophectoderm (TE) following the method described by Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29). There were no differences within developmental rate on Day 7, but reduced blastocyst rates were observed on Day 8 between hLIF (42.0 � 3.9a and 27.2 � 3.3a) and controls (57.7 � 3.9b and 38.9 � 3.3b) at the medium and expanded stages, respectively, whereas mLIF had no effect (47.4 � 3.9 and 32.3 � 3.3). Contrary to development, Day 8 blastocysts showed decreased cell counts in both the ICM and the ICM/total cell proportions in the presence of mLIF (19.1 � 3.1x and 13.8 � 2.4x vs. 32.6 � 3.0y and 24.8 � 2.3y for controls, respectively), whereas hLIF had no effect (29.7 � 3.1y and 20.9 � 2.4y). No changes were seen in TE and total cell counts. The disparate effects exhibited by hLIF and mLIF during blastocyst formation may reflect the fact that these compounds are inappropriate to replace bLIF, and/or endogenous LIF probably suffices during bovine development. In fact, mouse embryonic development and blastocyst cell numbers decrease in murine embryos injected with LIF antisense nucleotides (Cheng et al. 2004 Biol. Reprod. 70, 1270–1276). Furthermore, embryonic stem (ES)-like cell derivation in bovine is possible with (Saito et al. 2003 Biochem. Biophys. Res. Com. 309, 104–113) and without (Mitalipova et al. 2001 Cloning 3, 59–67) exogenous LIF. Therefore, strategies to investigate LIF signalling in bovine embryos and stem cells should be reconsidered. This work was supported by Grant AGL2005-04479.

178 IN VITRO CULTURE OF BOVINE EMBRYOS WITH NEUROTROPHINS

2007 ◽  
Vol 19 (1) ◽  
pp. 205
Author(s):  
E. Gómez ◽  
A. Rodríguez ◽  
C. De Frutos ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
The Other ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Cell Counts

Neurotrophins (NTs) mediate human embryonic stem (hES) cell survival and may also improve methods for hES cell derivation (Pyle et al. 2006 Nature Biotech. 24, 344–350) and quality of the inner cell mass (ICM). We searched published microarray data sets for tyrosine kinase receptors (TRK) (geo data base: GSM27469, GSM27470, GSM27471). The analysis suggested that bovine embryos in vitro at unspecified stages express TRKA, for nerve growth factor (NGF); TRKC, for neurotrophin-3 (NT3); and TRKB, for both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF). NTs functionally cooperate among them and also with basic fibroblast growth factor (bFGF) (Pyle et al. 2006; Logan et al. 2006 Brain 129, 490–502). Experiments in progress include detection of TRK expression by RT-PCR at defined development stages, and analysis of embryonic development with NTs and without bFGF. In this work we cultured embryos matured and fertilized in vitro from slaughterhouse oocytes for 8 days in SOF medium with 6 g L-1 BSA and 2 ng mL-1 bFGF (negative control). Development was monitored and cells were differentially counted in the ICM and trophectoderm (TE) of expanded and hatched blastocysts. NTs were used during the whole culture at 20 ng mL-1 as single (4 experimental groups: NGF, NT3, NT4, and BDNF) or as pooled (1 group) NT compounds. Data (5 replicates; 1403 oocytes) were processed by GLM and Duncan's test, and expressed as LSM � SE (a,b: P < 0.05). At Day 3, no differences were found at the 5- to 8-cell stage, but NT3 and NT4 increased the proportions of embryos at the 8- to 16-cell stage (19.1 � 2.2 and 20.5 � 2.2, respectively, vs. 12.9 � 2.2 to 13.7 � 2.2 within the other groups). On Day 6, NT4 yielded more morulae than controls, BDNF, and NGF (35.3 � 2.7 vs. 26.1 � 2.7, 27.4 � 2.7, and 27.8 � 2.7, respectively), and did not differ from the other groups. NT4 produced more total Day 7 blastocysts than NT3 and BDNF (12.5 � 2.2 vs. 8.1 � 2.2 and 9.9 � 2.2, respectively), whereas there were no differences within medium and expanded blastocysts and Day 8 blastocysts. Proportions of morulae that formed blastocysts were appreciably lower than in concomitant experiments without bFGF. Pooled NTs showed decreased values as compared to some single NTs within the ICM [13.0 � 4.0 vs. 29.1 � 4.6 (NT3) and 24.9 � 4.3 (NGF)], the TE [89.0 � 8.4 vs. 120 � 11.9 (BDNF)], total cells [102.0 � 8.5 vs. 134.0 � 9.9 (NT3), and 140.0 � 12.1 (BDNF)], and tended to differ (P = 0.08) within ICM/total cells [13.1 � 3.1 vs. 21.6 � 3.6 (controls) and 22.2 � 3.6 (NT3)]. Controls differed from BDNF (TE: 88.1 � 9.8 vs. 120.2 � 11.9; total cells: 110.8 � 10.0 vs. 140.0 � 12.1, respectively), and from NT4 for ICM/total cells (21.6 � 3.6 vs. 11.5 � 2.9, respectively). NT4 is likely to exert a role during early embryonic development. However, these blastocysts showed decreased cell counts in the ICM, probably reflected in the pooled NTs group. Targeting proliferation stimuli specifically to the ICM is difficult to get when the ICM is enclosed in the embryo, in contrast with the isolated ICM or the derived stem cells. This work was supported by Grant AGL2005-04479.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

scholarly journals Leukemia Inhibitory Factor Stimulates Primitive Endoderm Expansion in the Bovine Inner Cell Mass

2021 ◽  
Vol 2 ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Primitive Endoderm ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Bovine Blastocyst

Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE), and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8, and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

scholarly journals A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 142
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Apoptosis Rate ◽  
Cell Counts ◽  
Hatching Rates

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

scholarly journals Leukemia inhibitory factor stimulates primitive endoderm expansion in the bovine inner cell mass

2021 ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Primitive Endoderm ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Bovine Blastocyst

Abstract Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE) and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8 and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

124 Cell Count of Bovine In Vitro-Produced Blastocysts After In Vitro Maturation in Glass versus Plastic Vials

2018 ◽  
Vol 30 (1) ◽  
pp. 202
Author(s):  
J. O. Secher ◽  
N. Hashem ◽  
J. H. Pryor ◽  
C. R. Long ◽  
J. Docherty ◽  
...  
Keyword(s):  
Cell Count ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Total Cell ◽  
Differential Staining ◽  
Atmospheric Air ◽  
Cell Counts ◽  
Blastocyst Rate

Optimal bovine in vitro oocyte maturation (IVM) is a prerequisite for subsequent optimal blastocyst rates. Ovum pick-up (OPU), by which cumulus–oocyte complexes (COC) are collected in vivo, is performed outside a laboratory and often requires IVM to take place during transportation from the farm to the IVF laboratory. Hashem et al. (2017 Reprod. Fertil. Dev. 29, 179) demonstrated that blastocyst rates are affected by type of vial (glass v. plastic), number of COC per vial, and volume of medium per vial. This was achieved by maturing more than 2500 COC from slaughterhouse material under contrasting conditions, followed by standardised IVF and in vitro culture (IVC) and observation of blastocyst rates, morphology (1: poor; 2: good; 3: excellent), and kinetics (1: blastocyst; 2: expanded blastocyst ; 3: hatching/hatched blastocyst). Here we examined differential staining of a subset of expanded blastocysts (XB) from the previous study to assess the influence of vial material, medium volume, and number of COC per vial on total cell count, number and ratio of inner cell mass (ICM), and trophectoderm (TE) cells. In experiment 1 (4 groups), oocytes were matured in different vials without lids in an incubator at 5.5% CO2 in humidified atmospheric air at 38.5°C to assess plastic toxicity. In experiment 2 (6 groups) and experiment 3 (6 groups), the 2 best performing vials-polypropylene cryovials (Sigma-Aldrich, St. Louis, MO, USA) and glass vials (VWR International, Radnor, PA, USA)-containing 50% (Exp. 2) or 95% (Exp. 3) medium volume per vial and 5, 20, or 45 COC per vial were tested. In experiments 2 and 3, the vials were closed and incubated in atmospheric air at 38.5°C. All groups were evaluated for blastocyst rates, kinetics, and morphology. Because kinetics (range 2.01–2.25) and morphology (range 2.15–2.50) were similar in all groups, only XB were collected from each group. These were fixed and stained with CDX2 antibody and Hoechst (Wydooghe et al. 2011 Anal. Biochem. 416, 228-230) and their ICM and TE cells were counted. The cells were counted manually in blinded groups using an inverted fluorescence microscope and 16× magnification. Counts of total, ICM, and TE cells were compared between treatments by a two-way ANOVA analysis. A total of 240 XB from the 16 different vial groups were counted in the 3 experiments, with average total cell counts of 139 (110–211) and ICM cell counts of 44 (28–75). Even though the blastocyst rates differed between some of the groups, the cell counts within the XB did not differ statistically significantly between groups. In fact, the highest cell count was found in the glass vial group with the lowest blastocyst rate (45 COC per vial in 50% medium volume; blastocyst rate 28%, total cells 211, ICM cells 75). We have previously demonstrated that the type of vial, number of COC per vial, and the volume of medium per vial influence the subsequent blastocyst rates. It is concluded, however, that the embryos able to proceed to the blastocyst stages seem to be of the same quality in all groups, assessed by kinetics, morphology, and cell counts within XB.

scholarly journals Biological differences between in vitro produced bovine embryos and parthenotes

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 285-295 ◽  
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.

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

2010 ◽  
Vol 22 (1) ◽  
pp. 236 ◽  
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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