240OVINE PREPUBERTAL OOCYTE SHOWS ALTERATE GENE EXPRESSION
AND LOW DEVELOPMENTAL COMPETENCE

The aim of this work was to evaluate developmental competence and gene expression of prepubertal and adult ovine oocytes. GV prepubertal and adult oocytes were matured, fertilized and cultured in vitro until blastocyst stage;; the time (days) needed to reach this stage was recorded. Blastocysts developed on different days were cultured for hatching to evaluate their quality in relation to cleavage rate. Adult and prepubertal GV oocytes and blastocyst-stage embryos produced, respectively, at 6 and 7 days were compared for quantitative expression of poly(A) polymerase (polyA-P), glucose transporter I (Glut-I), desmocollin II (desmoII), plakofilin (plako) and heat shock protein 70.1 (HSP70) genes. Confirming previous results (Ledda et al., 1996 Zygote 4, 343–348), fertilized prepubertal ovine oocytes developed to blastocyst stage at lower rates than the adult ones (19.9 v. 51.3%, respectively, P<0.001) and this stage was delayed 24h in prepubertal compared to adult embryos (P<0.01), reflecting a lower quality (Fenwick et al., 2002 Hum. Reprod. 17, 407–412) of the former. In fact, 44.7, 25.0, 30.3 and 0% of adult blastocysts were obtained after 6, 7, 8 and 9 days, respectively, of postfertilization culture compared to 0, 48.4, 34.3 and 17.2% of prepubertal ones. Faster-developed blastocysts showed higher hatching rate in both prepubertal (54.8%, 7 days of culture) and adult (89.8%, 6 days). Hatching rate dropped to 18.2% when blastocysts were obtained at 8–9 days in prepubertal and to 54.5% and 32.5% at 7 and 8 days, respectively, in adult embryos. Analysis of gene expression showed that HSP70, plako and desmo genes were not expressed in GV oocytes, and Glut-I mRNA was lower in prepubertal GV oocytes than in the adult ones (P<0.01). All genes were expressed in blastocysts;; we found that Glut-I was at lower levels (P<0.01) in prepubertal-derived blastocysts whereas HSP70 was highly expressed (P<0.05) in prepubertal blastocysts than in those derived from adult oocytes. In conclusion this work shows that prepubertal ovine oocytes have a lower developmental competence compared to the adult ones, correlated to an altered gene expression during the growth phase of the oocyte and early embryonic development. Supported by MIUR (cofin).

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Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽

10.1017/s0967199413000580 ◽

2014 ◽

Vol 23 (3) ◽

pp. 327-335 ◽

Cited By ~ 5

Author(s):

Hruda Nanda Malik ◽

Dinesh Kumar Singhal ◽

Shrabani Saugandhika ◽

Amit Dubey ◽

Ayan Mukherjee ◽

...

Keyword(s):

Culture Media ◽

Combined Treatment ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cleavage Rate ◽

Cell Stage ◽

Quantitative Expression ◽

Pattern Of Variation ◽

Better Than

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

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53 EFFECT OF ADDITION OF L-CARNITINE AND CONJUGATED LINOLEIC ACID DURING BOVINE EMBRYO CULTURE ON CRYOSURVIVAL, LIPID CONTENT, AND GENE EXPRESSION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab53 ◽

2015 ◽

Vol 27 (1) ◽

pp. 119

Author(s):

A. Ruiz ◽

P. J. Hansen ◽

J. Block

Keyword(s):

Gene Expression ◽

Lipid Metabolism ◽

Linoleic Acid ◽

Conjugated Linoleic Acid ◽

Lipid Content ◽

Embryo Culture ◽

Blastocyst Stage ◽

Hatching Rate ◽

Bovine Embryo

The objective was to determine the effects of addition of l-carnitine (LC) and trans-10, cis-12 conjugated linoleic acid (CLA) during bovine embryo culture on cryosurvival, lipid content, and gene expression. For all experiments, embryos were produced in vitro using abattoir-derived oocytes. Following insemination, presumptive zygotes were randomly assigned in a 2 × 2 factorial to be cultured in SOF-BE1 supplemented with or without 3.03 mM LC and 100 μM CLA until Day 7. For Exp. 1, blastocyst- and expanded-blastocyst-stage embryos (n = 777) were slow-frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. There was no effect of LC on post-thaw re-expansion rates, but CLA reduced (P < 0.05) and tended (P < 0.08) to reduce re-expansion rate at 24 and 48 h, respectively (76.5 ± 2.5 v. 70.4 ± 2.5 and 79.5 ± 2.2 v. 76.0 ± 2.2, respectively). Whereas hatching rate at 72 h tended (P < 0.08) to be higher for embryos cultured with LC (67.8 ± 2.5 v. 74.4 ± 2.5), treatment with CLA reduced (P < 0.05) hatching rate at 48 h (62.3 ± 2.6 v. 54.9 ± 2.6). In Exp. 2, to determine lipid content, expanded blastocyst-stage embryos (n = 263) were harvested and stained using Nile Red. Embryos were examined for fluorescence using an epifluorescence microscope, and intensity of fluorescence per unit area was quantified using ImageJ software (NIH, Bethesda, MD, USA). There was a significant interaction (P < 0.01) between LC and CLA affecting embryo lipid content. Whereas addition of CLA during culture increased lipid, treatment with LC and the combination of LC and CLA reduced lipid (22.8 ± 1.1 v. 19.1 ± 1.1 v. 28.4 ± 1.1 v. 19.2 ± 1.2 for no additive, +LC, +CLA, and +LC and CLA, respectively). For Exp. 3, the effect of LC and CLA on the relative abundance of genes involved in lipid metabolism (ELOVL6, SCD1, SQLE, HMGCS1, CYP51A1, FDPS, FDFT1, LDLR, and SC4MOL) was determined. Pools of 5 expanded blastocyst-stage embryos from each treatment were collected across 5 replicates. The RNA was purified and synthesised into cDNA for RT-qPCR analysis. The SDHA, GAPDH, and YWAZ were used as housekeeping genes. Addition of LC during culture reduced (P < 0.05) the abundance of 4 of the 9 genes analysed (SQLE, HMGCS1, CYP51A1, and FDPS) and tended (P < 0.08) to reduce a fifth (FDFT1). In addition, there was a tendency (P < 0.08) for LC to increase the abundance of SCD1. Addition of CLA during culture had minimal effects on transcript abundance. In particular, CLA treatment reduced (P < 0.01) ELOVL6 and tended (P < 0.08) to increase SCD1. In contrast to previous studies, post-thaw cryosurvival was not significantly improved by treatment with LC or CLA. Results indicate that reduced embryo lipid content caused by LC treatment is due, in part, to an alteration in the abundance of genes involved in lipid metabolism. Further research is still necessary to determine whether in vivo survival following transfer of cryopreserved embryos can be enhanced by treatment with LC or CLA.Support was provided by USDA AFRI Grant 2010–85122–20623.

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123 OPTIMIZATION OF CULTURE CONDITIONS FOR IN-VITRO-PRODUCED BOVINE EMBRYOS TO ENHANCE BLASTOCYST YIELD AND SURVIVAL FOLLOWING VITRIFICATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab123 ◽

2008 ◽

Vol 20 (1) ◽

pp. 142

Author(s):

J. Block ◽

L. Bonilla ◽

P. J. Hansen

Keyword(s):

Bovine Serum ◽

Culture System ◽

Blastocyst Stage ◽

Culture Conditions ◽

Expansion Rate ◽

Hatching Rate ◽

Static System ◽

Cleavage Rate ◽

Static Culture

Objectives were to identify modifications in culture conditions that improve blastocyst yield and cryosurvival. The objective of Experiment 1 was to determine effects of sequential culture and fructose on blastocyst yield. Embryos were cultured in modified SOF with 4 mg mL–1 bovine serum albumin (BSA) and 1.0 mm alanyl-glutamine in 5% (v/v) oxygen with or without 0.5 mm fructose in either a static or sequential culture system. For the sequential system, embryos >4 cells were selected and placed in fresh drops of medium at day 3 after insemination. Culture system and fructose did not affect cleavage rate or the proportion of embryos >4 cells on day 3. The proportion of >4 cell embryos that developed to the blastocyst stage was higher (P < 0.04) for static culture than for sequential culture (41.6 � 1.2 v. 30.6 � 1.2%) and there was a trend (P = 0.1) for the proportion of oocytes that developed to blastocyst at day 7 to be greater for static culture (26.8 � 1.2 v. 20.9 � 1.2%). In both culture systems, fructose increased (P < 0.03) blastocyst yield from embryos >4 cells (32.5 � 1.2 v. 39.7 � 1.2%) and tended (P < 0.06) to improve blastoocyst yield from oocytes (21.8 � 1.1 v. 25.3 � 1.1%). The objective of Exp. 2 was to evaluate whether blastocyst yield and survival after cryopreservation would be enhanced by BSA and hyaluronan. Embryos produced in vitro were cultured in 5% oxygen using a static system of modified SOF with or without 4 mg mL–1 BSA and with 0, 0.1, 0.5, or 1 mg mL–1 hyaluronan. Blastocyst and expanded blastocyst stage embryos on day 7 were vitrified (Campos-Chillon LF et al. 2006 Theriogenology 65, 1200–1214). Vitrified embryos were thawed and then cultured for 72 h in modified SOF containing 10% (v/v) fetal bovine serum and 50 µm dithiothreitol. Re-expansion rate was recorded at 24 and 48 h, and the proportion of embryos that hatched by 72 h of culture was recorded. There was no effect of BSA or hyaluronan on cleavage rate. Blastocyst yield from oocytes was increased (P < 0.0005) by BSA (15.3 � 1.1 v. 20.9 � 1.1%). Addition of hyaluronan at 1 mg mL–1 improved (P < 0.04) blastocyst yield (16.2 � 1.7 v. 21.2 � 1.7%), but there was no effect at lower concentrations. There were no interactions between BSA and hyaluronan. Re-expansion rate at 24 and 48 h after thawing was reduced (P < 0.007) by BSA (24 h: 39.1 � 3.6 v. 17.0 � 3.6%; 48 h: 45.6 � 3.8 v. 18.7 � 3.7%), and BSA tended (P < 0.06) to reduce hatching rate at 72 h (22.3 � 3.0 v. 9.8 � 3.0%). Treatment of embryos with hyaluronan did not affect re-expansion rate at 24 h but tended (P < 0.08) to increase re-expansion at 48 h. Moreover, hyaluronan increased (P < 0.05) hatching rate at 72 h after thawing (0 mg mL–1 – 9.8 � 4.2; 0.1 mg mL–1 – 16.9 � 4.5; 0.5 mg mL–1 – 23.4 � 4.1; 1.0 mg mL–1 – 14.2 � 4.1%). In conclusion, blastocyst yield was improved by addition of fructose, BSA, and hyaluronan to culture medium and by use of a static culture system. Hyaluronan also enhanced cryosurvival, but BSA was detrimental to blastocyst survival after vitrification. Support: USDA NRI 2006-55203-17390, BARD US-3551-04.

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17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab17 ◽

2019 ◽

Vol 31 (1) ◽

pp. 134

Author(s):

D. Veraguas ◽

C. Aguilera ◽

D. Echeverry ◽

D. Saez-Ruiz ◽

F. O. Castro ◽

...

Keyword(s):

Gene Expression ◽

Somatic Cell ◽

Blastocyst Stage ◽

Domestic Cat ◽

Developmental Competence ◽

Relative Expression ◽

Standard Curve ◽

Morula Stage ◽

Leopardus Guigna

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P<0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P<0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P<0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P>0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P>0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P>0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

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133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab133 ◽

2007 ◽

Vol 19 (1) ◽

pp. 184 ◽

Cited By ~ 3

Author(s):

T. Somfai ◽

M. Ozawa ◽

J. Noguchi ◽

H. Kaneko ◽

K. Ohnuma ◽

...

Keyword(s):

Polar Body ◽

Antioxidant Defense System ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Cleavage Rate ◽

Sperm Penetration ◽

Second Polar Body ◽

Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

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25 THE DEVELOPMENTAL COMPETENCE AND PLURIPOTENT GENE EXPRESSION OF PORCINE SOMATIC CELL NUCLEUS TRANSFER EMBRYOS IMPROVED BY m-CARBOXYCINNAMIC ACID BISHYDROXAMIDE

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab25 ◽

2012 ◽

Vol 24 (1) ◽

pp. 124

Author(s):

S.-H. Park ◽

S.-Y. Kim ◽

M.-R. Lee ◽

H.-J. Eun ◽

S.-K. Baik ◽

...

Keyword(s):

Gene Expression ◽

Cell Nucleus ◽

Histone Deacetylase Inhibitor ◽

Blastocyst Stage ◽

Developmental Competence ◽

Deacetylase Inhibitor ◽

Somatic Cell Nucleus ◽

Nucleus Transfer ◽

Somatic Cell Nucleus Transfer

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic state after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Miyamoto et al. 2010 JBC), use of m-carboxycinnamic acid bishydroxamide (CBHA), a histone deacetylase inhibitor, during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA on the in vitro developmental competence and pluripotent gene expression of porcine SCNT embryos. The porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h) and then were assessed to cleavage rates and development to the blastocyst stage. In addition, pluripotent gene expression of SCNT embryos was analysed by RT-PCR. All data were analysed by chi-square. Following 4 replicates (207 and 124 for NT and CBHA-treated NT embryos, respectively), no significant difference was observed between NT and CBHA-treated NT embryos for cleavage rate (83.9 vs 82.2%). However, the developmental competence to the blastocyst stage differed significantly between NT and CBHA-treated NT embryos (6.8 vs 18.6%; P < 0.05). In addition, pluripotent transcription factors including Oct4, Nanog and Sox2 were higher expressed in the cloned embryos treated with CBHA (P < 0.05). The results of the present study suggest that treatment with CBHA as a histone deacetylase inhibitor significantly increased the developmental competence as well as the pluripotent gene expression of porcine SCNT embryos. This work was partly supported by a grant from the NRF (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.

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Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation of post-thaw in vitro development and gene expression

Reproduction Fertility and Development ◽

10.1071/rd10203 ◽

2011 ◽

Vol 23 (4) ◽

pp. 585 ◽

Cited By ~ 14

Author(s):

E. Siqueira Filho ◽

E. S. Caixeta ◽

C. Pribenszky ◽

M. Molnar ◽

A. Horvath ◽

...

Keyword(s):

Gene Expression ◽

Hydrostatic Pressure ◽

Transcript Abundance ◽

Developmental Competence ◽

Hatching Rate ◽

In Vitro Development ◽

Stress Treatment ◽

Pressure Stress ◽

Vitro Development

Sublethal stress treatment has been reported to enhance gametes’ performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60 min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2 h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72 h in vitro culture or were stored at –80°C until gene expression analysis. Re-expansion and hatching rates after vitrification–warming were significantly (P < 0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1 h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos.

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Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification

Zygote ◽

10.1017/s0967199418000606 ◽

2018 ◽

Vol 27 (1) ◽

pp. 36-45

Author(s):

Jaqueline Sudiman ◽

Alice Lee ◽

Kheng Ling Ong ◽

Wu Zi Yuan ◽

Sarah Jansen ◽

...

Keyword(s):

Cell Number ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cortical Granule ◽

Blastocyst Development ◽

Cleavage Rate ◽

Parthenogenetic Activation ◽

Mouse Oocytes ◽

Different Response

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

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