Relationship between donor animal age, follicular fluid steroid content and oocyte developmental competence in the pig

The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73%; P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL−1; P < 0.005) and androstenedione (70 v. 16 ng mL−1; P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17β-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed ‘oocyte capacitation’. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.

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Cerium oxide nanoparticles (CeO2 NPs) improve the developmental competence of in vitro-matured prepubertal ovine oocytes

Reproduction Fertility and Development ◽

10.1071/rd15521 ◽

2017 ◽

Vol 29 (5) ◽

pp. 1046 ◽

Cited By ~ 8

Author(s):

F. Ariu ◽

L. Bogliolo ◽

A. Pinna ◽

L. Malfatti ◽

P. Innocenzi ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Cumulus Cells ◽

Developmental Competence ◽

Nuclear Maturation ◽

Maturation Medium ◽

Group A ◽

Chromatin Configuration ◽

Development In Vitro

The present study investigated whether supplementation with different doses of cerium dioxide nanoparticles (CeO2 NPs) during in vitro maturation (IVM) of prepubertal ovine oocytes influenced their embryonic development in vitro. Cumulus–oocyte complexes derived from the ovaries of slaughtered prepubertal sheep underwent IVM with CeO2NPs (0, 44, 88 or 220 µg mL–1). Matured oocytes were fertilised in vitro and zygotes were cultured for 7 days. The results demonstrated that CeO2NPs were internalised in the cumulus cells and not in the oocyte. The treatment with CeO2NPs did not affect nuclear maturation or intracellular levels of reactive oxygen species of the oocytes. The percentage of oocytes with regular chromatin configuration and cytoskeleton structures when treated with 44 µg mL–1 CeO2NPs was similar to oocytes matured in the absence of CeO2NPs and significantly higher than those treated with 88 or 220 µg mL–1 CeO2NPs. The relative quantification of transcripts in the cumulus cells of oocytes matured with 44 µg mL–1 CeO2NPs showed a statistically lower mRNA abundance of BCL2-associated X protein (BAX), B-cell CLL/lymphoma 2 (BCL2) and superoxide dismutase 1 (SOD1) compared with the 0 µg mL–1 CeO2 NPs group. A concentration of 44 µg mL–1 CeO2NPs significantly increased the blastocyst yield and their total, inner cell mass and trophectoderm cell numbers, compared with the 0 and 220 µg mL–1 groups. A low concentration of CeO2NPs in the maturation medium enhanced in vitro embryo production of prepubertal ovine oocytes.

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Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo

Reproduction ◽

10.1530/rep-20-0311 ◽

2021 ◽

Vol 161 (4) ◽

pp. 353-363

Author(s):

Mun-Hyeong Lee ◽

Pil-Soo Jeong ◽

Bo-Woong Sim ◽

Hyo-Gu Kang ◽

Min Ju Kim ◽

...

Keyword(s):

Oxygen Tension ◽

Early Phase ◽

Inner Cell Mass ◽

Formation Rate ◽

Cell Mass ◽

Female Reproductive Tract ◽

Developmental Competence ◽

Early Embryos ◽

Developmental Parameters

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0–2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2–4 days) and late phases (4–6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

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178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab178 ◽

2006 ◽

Vol 18 (2) ◽

pp. 197 ◽

Cited By ~ 2

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.

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342 DISTINCT EFFECTS OF FOLLICULAR FLUID ON CUMULUS CELL APOPTOSIS AND PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab342 ◽

2007 ◽

Vol 19 (1) ◽

pp. 286

Author(s):

C. G. Grupen ◽

T. S. Hussein ◽

S. J. Schulz ◽

D. T. Armstrong

Keyword(s):

Oxidative Stress ◽

Follicular Fluid ◽

Cell Apoptosis ◽

Polar Body ◽

Cumulus Cell ◽

Developmental Competence ◽

Potent Inducer ◽

Oocyte Developmental Competence ◽

Cytoplasmic Maturation

Supplementing medium with follicular fluid (FF) during in vitro maturation (IVM) enhances the developmental competence of porcine oocytes, indicating that factors present in FF are beneficial to cytoplasmic maturation. Previous findings suggest that porcine FF contains high levels of superoxide dismutase activity and exerts a beneficial effect on cytoplasmic maturation by protecting oocytes from oxidative stress (Tatemoto et al. 2004 Biol. Reprod. 71, 1150–1157). Since oxidative stress is a potent inducer of apoptosis, the aim of the present study was to examine the temporal effects of FF during IVM on cumulus cell apoptosis and oocyte developmental competence. Ovaries of prepubertal pigs were collected from a local abattoir and antral follicles, 3 to 7 mm in diameter, were aspirated. Cumulus–oocyte complexes (COCs) with at least 3 uniform layers of compact cumulus cells (CCs) were recovered, washed, and transferred to maturation medium (MM) with or without 25% FF. At 22 h of IVM, COCs from each group were washed and transferred to fresh MM with or without 25% FF, forming 4 groups: -FF/-FF, -FF/+FF, +FF/-FF, and +FF/+FF. Cohorts of COCs were TUNEL stained at 22 and 44 h of IVM using the In Situ Cell Death Detection kit (Roche Diagnostics, Castle Hill, NSW, Australia) according to the manufacturer's instructions, and apoptotic CCs were visualized using confocal microscopy. Oocytes denuded at 44 h, that had a polar body, were treated with ionomycin and 6-dimethylaminopurine to induce parthenogenetic development, and were cultured for 7 days in NCSU-23 medium at 38.5°C in 5&percnt; O2, 5&percnt; CO2, and 90&percnt; N2. Data were subjected to ANOVA and Tukey's post-hoc test. At 22 h of IVM, the presence of FF reduced the proportion of apoptotic CCs in COCs (2.1&percnt; vs. 4.6&percnt;). COCs matured with FF from 22 to 44 h of IVM had much lower proportions of apoptotic CCs (+FF/+FF: 0.9&percnt;; −FF/+FF: 2.6&percnt;) compared with those matured without FF (+FF/−FF: 10.3&percnt;; −FF/−FF: 17.8&percnt;). The rate of maturation to the metaphase-II stage was greater when oocytes were matured with FF from 0 to 22 h of IVM (−FF/−FF: 68.6&percnt;; −FF/+FF: 72.8&percnt;; +FF/−FF: 89.2&percnt;; +FF/+FF: 86.2&percnt;). Maturation without FF for the entire IVM interval reduced the proportion of activated oocytes that formed blastocysts compared with the other groups (−FF/−FF: 25.1&percnt;; −FF/+FF: 44.6&percnt;; +FF/−FF: 46.6&percnt;; +FF/+FF: 47.3&percnt;). Despite a 4-fold difference in the proportion of apoptotic CCs between COCs of the +FF/−FF and −FF/+FF groups, exposure to FF for the first or second half of IVM was as beneficial to oocyte developmental competence as exposure to FF for the entire IVM interval. This suggests that the protective effect of FF in reducing oxidative stress on oocytes during IVM is distinct from the effect on oocyte developmental competence.

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148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab148 ◽

2017 ◽

Vol 29 (1) ◽

pp. 182

Author(s):

S. M. Bernal-Ulloa ◽

A. Lucas-Hahn ◽

P. Aldag ◽

D. Herrmann ◽

U. Baulain ◽

...

Keyword(s):

Inner Cell Mass ◽

Mammalian Species ◽

Cell Mass ◽

Phosphodiesterase Inhibitor ◽

Developmental Competence ◽

Differential Staining ◽

Final Phase ◽

Cell Numbers ◽

Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

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Morphology, developmental stages and quality parameters of in vitro-produced equine embryos

Reproduction Fertility and Development ◽

10.1071/rd19257 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1758 ◽

Cited By ~ 1

Author(s):

Elaine M. Carnevale ◽

Elizabeth S. Metcalf

Keyword(s):

Embryo Development ◽

Developmental Stages ◽

Inner Cell Mass ◽

Early Stage ◽

Cell Mass ◽

Quality Parameters ◽

Early Embryo ◽

Developmental Competence ◽

Limited Information

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

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Endocrine-disrupting chemicals in human follicular fluid impair in vitro oocyte developmental competence

Human Reproduction ◽

10.1093/humrep/der448 ◽

2012 ◽

Vol 27 (4) ◽

pp. 1025-1033 ◽

Cited By ~ 52

Author(s):

Evi M.L. Petro ◽

Jo L.M.R. Leroy ◽

Adrian Covaci ◽

Erik Fransen ◽

Diane De Neubourg ◽

...

Keyword(s):

Follicular Fluid ◽

Endocrine Disrupting Chemicals ◽

Developmental Competence ◽

Human Follicular Fluid ◽

Oocyte Developmental Competence ◽

Endocrine Disrupting

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53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab53 ◽

2007 ◽

Vol 19 (1) ◽

pp. 144

Author(s):

Y. U. Kim ◽

D. P. Bhandari ◽

M. S. Hossein ◽

S. M. Park ◽

E. Lee ◽

...

Keyword(s):

Somatic Cell ◽

Inner Cell Mass ◽

Cell Mass ◽

Defined Medium ◽

Culture Conditions ◽

Developmental Competence ◽

Differential Staining ◽

In Vitro Production ◽

Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

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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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192 PREMATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS AFFECTS BOTH OOCYTE AND BLASTOCYST ULTRASTRUCTURE

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab192 ◽

2016 ◽

Vol 28 (2) ◽

pp. 227

Author(s):

E. Razza ◽

H. Pedersen ◽

L. Stroebech ◽

M. Machado ◽

M. Nogueira ◽

...

Keyword(s):

Inner Cell Mass ◽

Smooth Endoplasmic Reticulum ◽

Cell Mass ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Developmental Competence ◽

Cortical Granules ◽

Cytoplasmic Vesicles ◽

Globally Distributed

Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosine monophosphate (cAMP) elevating agents have been used for artificial blocking of meiotic resumption (pre-IVM) to allow the oocyte to prepare for maturation, potentially increasing its developmental competence. However, the ultrastructural effects of this pharmacological approach on oocytes and embryos remain to be addressed. We assessed the effects of pre-IVM with cAMP modulators in oocytes (10 for each group) at the end of IVM and in blastocyst (10 for each group) after 7 days of culture. Cumulus-oocyte complexes (COC) were subjected to pre-IVM for 2 h with forskolin (Sigma, St. Louis, MO, USA; 100 μM) and 3-isobutyl-1-methylxanthine) (IBMX, Sigma, 500 μM) followed by 24 h of IVM with FSH-enriched media (IVF Vet Solutions, Adelaide, Australia). Simultaneously, another group of COC was subjected to conventional IVM (con-IVM) for 24 h (EmbryoTransBiotech, Copenhagen, Denmark) with BSA (4 mg mL–1, Sigma), gentamycin (50 mg mL–1), and FSH (0.1 IU mL–1). Matured oocytes were collected for qualitative ultrastructural analysis or followed to IVF. The morphology was carefully evaluated on serially sectioned oocytes and embryos, where each serial section (~60.2-μm section per oocyte/embryo) was analysed under light microscopy. Subsequently, the equatorial section from each oocyte and the section giving the optimal representation of the inner cell mass in each blastocyst was re-embedded and sectioned for electron microcopy as previously described (Hyttel and Madsen 1987 Acta Anat. 129, 12–14). Blastocyst rates did not differ between groups. Ultrastructural analyses revealed subtle ultrastructural differences between pre-IVM and con-IVM conditions. In both groups, oocytes had matured to metaphase II. The perivitelline space of pre-IVM oocytes was significantly narrower than con-IVM. The cytoplasmic vesicles were more abundant and globally distributed in pre-IVM oocytes, whereas at con-IVM a vesicle-free periphery of the ooplasm was frequent, except for cortical granules and clusters of mitochondria associated with smooth endoplasmic reticulum (SER). We observed typical hooded mitochondria and cortical granules either clustered in the periphery or solitarily distributed in the cortical ooplasmic region for both groups. In the blastocysts, differences were noted with respect to especially distribution of ribosomes. In pre-IVM blastocysts, ribosomes were mostly organised in free clusters (polysomes) and peripherally located in cells of the inner cell mass. Con-IVM blastocysts showed ribosomes preferentially associated with the rough ER and often associated with mitochondria. Lipid droplets and rounded mitochondria were observed in both groups as well as apically located tight junctions and desmosomes between adjacent trophectoderm (TE) cells. Pleomorphic and elongated mitochondria were abundant in the TE of pre-IVM blastocysts, whereas the mitochondrial population was more homogenous at con-IVM. These findings suggest that pre-IVM for 2 h affects oocyte and blastocyst ultrastructure. Research was supported by grants 12/50533-2 and 12/23409-9 from FAPESP.

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