Expression levels of FSHR, IGF1R, CYP11al and HSD3β in cumulus cells can predict in vitro developmental competence of bovine oocytes

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 425-431 ◽  
Author(s):  
Atchalalt Khurchabilig ◽  
Akane Sato ◽  
Shiori Ashibe ◽  
Asuka Hara ◽  
Rika Fukumori ◽  
...  
Keyword(s):  
Follicle Stimulating Hormone ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Expression Levels ◽  
Non Invasive ◽  
Production Technologies ◽  
Bovine Oocytes ◽  
Selection Of

SummaryThe efficiency of in vitro embryo production technologies would be improved by the development of suitable non-invasive biomarkers that allow the selection of good quality cumulus–oocyte complexes (COCs). The present study used whole, single oocyte culture to investigate whether the expression levels of follicle-stimulating hormone receptor (FSHR), insulin-like factor 1 receptor (IGF1R) and three steroidogenesis-related enzymes (CYP11al, CYP19al and HSD3β) in cumulus cells reflected the developmental competence of COCs. Cumulus cells were collected from single COCs before maturation culture and relative mRNA levels were assessed using real-time PCR. The analysis indicated that mRNAs for FSHR, IGF1R, CYP11al and HSD3β were present at higher levels in cumulus cells from COCs that failed to form blastocysts compared with cumulus cells from COCs that formed blastocysts. Moreover, FSHR and IGF1R mRNA levels were positively correlated with those of genes for steroidogenesis-related enzymes. In conclusion, poor developmental competence of COCs was related to higher expression of FSHR, IGF1R, CYP11al and HSD3β in cumulus cells, which may indicate the advanced differentiation of cumulus cells into granulosa cells.

scholarly journals 325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 282
Author(s):  
L.J. Royo ◽  
A. Rodriguez ◽  
A. Gutierrez-Adan ◽  
C. Diez ◽  
E. Moran ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Defined Medium ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Atp Production ◽  
Beneficial Effects ◽  
Oocyte Developmental Competence ◽  
Grant Support ◽  
Bovine Oocytes

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP<0.01; a,bP<0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

346 BENEFICIAL EFFECTS OF ACETYL-l-CARNITINE TREATMENT DURING IVM ON POST-FERTILIZATION DEVELOPMENT OF BOVINE OOCYTES IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 280 ◽  
Author(s):  
T. Yamada ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
Energy Production ◽  
Metabolic Regulation ◽  
Cellular Proliferation ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Acid Oxidation ◽  
Oocyte Cytoplasm ◽  
Peripheral Type ◽  
Bovine Oocytes

The lower competence of in vitro-matured oocytes for post-fertilization development is attributed to the lack of physiological factors in in vitro maturation (IVM) that regulate maturation events which occur exclusively in the cytoplasm of oocytes. It has been found recently that mitochondrial function plays an important role in regulation of oocyte developmental competence via metabolic regulation of energy production. Acetyl-l-carnitine (ALC) is known to enhance fatty acid oxidation and energy production in the mitochondria, and to exert enhancing effects on cellular proliferation and survival. In this experiment, we examined the effects of ALC on IVM and post-fertilization development of bovine oocytes. Cumulus-oocyte complexs (COCs) were aspirated from 2-5 mm follicles of ovaries from a slaughterhouse. COCs were cultured in IVM medium (mSOFaa+estradiol+hCG+BSA) with or without ALC (10 mM) for 24 h at 39�C under 5% CO2 in air, and then fertilized according to the conventional method. After 6 h of insemination, presumptive zygotes were freed from cumulus cells by repeated pipetting and cultured in mSOFaa with 1% FCS at 39�C under 5% CO2, 5% O2, and 90% N2. At 48 h post-fertilization, the rates of cleaved embryos were assessed. The cleaved embryos were transferred to mSOFaa with 5% FCS and cultured for additional 6 days at 39�C under 5% CO2, 5% O2, and 90% N2. The percentages of embryos developing to the blastcyst stage were assessed on Days 6, 7, and 8 (fertilization = Day 0), and the data were analyzed for statistically significant differences with the t-test. For examinination of mitochondrial organization in oocytes at different maturation stages, oocytes were stained for active mitochondria with MitoRed (1 �M in IVM medium for 2 h at 37�). When COCs were matured in medium without (control) or with ALC, although the rates of post-fertilization cleavage of oocytes were 60% to 70% despite the presence or absence of ALC, ALC significantly (P < 0.05) increased the rates of cleaved embryos forming blastcysts on Days 6, 7, and 8 (30%, 36%, 40%) compared with those in the control (13%, 21%, 34%). We next examined effects of ALC treatment during IVM on active mitochondria distribution in oocytes. In 75% of immature oocytes, active mitochondria localized in the periphery of the oocytes (peripheral type). After 24 h of IVM without ALC, while 17% of oocytes remained in a peripheral type, 44% showed some migration of active mitochondria toward the center of the oocytes (semiperipheral type) and 39% presented a diffused distribution of active mitochondria in the whole oocyte cytoplasm (diffused type). On the other hand, in ALC treated oocytes, 60% of the oocytes presented a diffused type, 25% exhibited a semiperipheral type, and 15% had still maintained a peripheral distribution. These results provide the first evidence that ALC treatment during IVM of bovine oocytes enhances their post-fertilization development to the blastcyst stage and enhances the frequency of oocytes that exhibit an extensive relocation (diffused type) of active mitochondria to the inner oocyte cytoplasm.

scholarly journals Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 303-310 ◽  
Author(s):  
Atef Ali ◽  
Marc-André Sirard
Keyword(s):  
Protein Kinase ◽  
Follicle Stimulating Hormone ◽  
Term Treatment ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Short Term ◽  
Short Term Treatment ◽  
Developmental Capacity ◽  
Bovine Oocytes

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus–oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 μM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1–0.5 μM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

230 ROLE OF PITUITARY HORMONES AND CUMULUS CELLS IN MODULATING THE DEVELOPMENTAL CAPACITY OF AGING BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 204
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
N. Zinovieva
Keyword(s):  
Embryonic Development ◽  
Tyrosine Kinases ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Prolonged Culture ◽  
Bovine Oocytes

The competence for embryonic development acquired during the oocyte maturation attenuates during the subsequent oocyte aging both in vivo and in vitro. Thus, the successful control of the female fertility requires information regarding factors responsible for the oocyte protection from early aging. The aim of the present research was to study the pattern and pathways of actions of two closely related pituitary hormones, prolactin (PRL), and growth hormone (GH), on the developmental potential of bovine oocytes during their aging in vitro. Therefore, we analysed (1) effects of PRL and GH during the prolonged culture of bovine oocytes on their subsequent development up to the blastocyst stage and (2) the role of cumulus cells (CC) and tyrosine kinases, the well-known mediators of PRL and GH signalling, in these effects. Bovine cumulus-enclosed oocytes (CEO) were cultured for 22 h in the following maturation medium: TCM 199 containing 10% fetal calf serum (FCS), 10 μg mL–1 of porcine FSH, and 10 μg mL–1 of ovine LH. After IVM, CEO or denuded oocytes (DO) were transferred to the aging medium consisting of TCM 199 supplemented with 10% FCS and cultured for 10 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL or 10 ng mL–1 recombinant bovine GH and/or 10 μg mL–1 genistein (a non-selective inhibitor of tyrosine kinases). Genistein was not applied in the case of aging DO, since their developmental potential was not affected by both hormones. Following the prolonged culture, oocytes underwent IVF and IVC. Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 8. The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data from 5 to 6 replicates using 135–184 oocytes per treatment were analysed by ANOVA. Aging of oocytes in the control medium had no effect on the cleavage rate, but caused the blastocyst yield to decline (P < 0.001) from 31.1 ± 2.3% (CEO fertilized immediately after maturation) to 10.5 ± 2.4% (aged CEO) and 7.9 ± 1.9% (aged DO). Cleavage rates of aging CEO and DO were unaffected by both PRL and GH. In the case of CEO, the addition of PRL (but not GH) to the aging medium raised the blastocyst yield from 8.2 ± 0.9% to 15.2 ± 2.1% (P < 0.05), whereas the removal of CC abolished this effect, reducing the yield up to 9.1 ± 2.7% (P < 0.05). At the same time, genistein did not influence the blastocyst yield in the PRL-treated group. The findings demonstrate that PRL can inhibit the attenuation of the developmental competence of bovine oocytes aging in vitro, with this effect being achieved via cumulus cells. Tyrosine kinases are unlikely to mediate the beneficial action of PRL on the CEO capacity for embryonic development. Meanwhile, closely related GH does not affect the developmental competence of aging bovine oocytes.This research was supported by RFBR (project No. 13-04-01888).

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

scholarly journals 94MEIOSIS INHIBITION OF BOVINE OOCYTES: EFFECTS ON SURVIVAL AFTER VITRIFICATION BY OPEN PULLED-STRAW METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 168
Author(s):  
C. Diez ◽  
M. Carbajo ◽  
L. Fernandez ◽  
C.O. Hidalgo ◽  
S. de la Varga ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
17Β Estradiol ◽  
Bovine Oocytes ◽  
Range Test

Mammalian oocytes remain one of the most difficult cell types to successfully cryopreserve. The in vitro-maturation protocols (IVM) have a large impact on the oocyte maturation. Consequently, inhibition of meiosis has been used to improve developmental competence of oocytes without reducing blastocyst rates. Moreover, the meiotic stage influences the ability of oocytes to survive cryopreservation. This work analyzes the effect of the inhibition of meiosis (prematuration) on the freezability of the bovine oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Inmature oocytes (I) with compact cumulus and evenly granulated cytoplasm were selected. Prematuration (PM) was performed by incubating COCs for 22h in TCM199 NaHCO3 and roscovitine 25μM. IVM was accomplished in TCM199 NaHCO3, 10% FCS, FSH-LH and 17β-estradiol. Oocytes were subjected to 5 treatments prior the vitrification (see table). COCs were partially denuded from cumulus cells and vitrified/warmed using the OPS system (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). Warmed oocytes were fertilized (Day 0) and presumptive zygotes having a normal morphological appearance were cultured in SOFaa+5% of FCS (Day 3); elements with degenerated appearance were discarded and recorded. Fresh oocytes submitted to IVM (c-M) or prematured and matured (c-PM+M) were fertilized and cultured as controls. Data were analyzed by ANOVA and Duncan’s multiple range test and expressed as LSM±SE. Developmental data are referred to the zygotes cultured. Only oocytes vitrified after IVM reached the blastocyst stage, but at lower rates than fresh controls. However, no differences were found between treatments at any developmental stage. Oocytes vitrified both as prematured+matured and immature oocytes showed increased proportions (P&lt;0.01) of degenerated oocytes (37.3±5.9 and 49.9±5.9, respectively), as compared with oocytes matured before vitrification (17.6±5.9). These results show that effects induced by incubation with roscovitine (Lonergan et al. 2003 Mol. Reprod. Dev. 64, 369–378) in combination with cryodamage compromise the oocyte developmental ability. Supported by CICYT, AGL2001-379.

138 THE EFFECT OF FOLLICLE SUPERSTIMULATION ON mRNA LEVELS IN BOVINE OOCYTES COLLECTED BY OVUM PICKUP

2012 ◽  
Vol 24 (1) ◽  
pp. 181
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hormone Treatment ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Dominant Follicle ◽  
Oocyte Collection ◽  
Light Cycler ◽  
Group 3 ◽  
Bovine Oocytes

A previous study revealed that follicle superstimulation significantly improved the developmental competence of immature bovine oocytes collected by ovum pickup (OPU; Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The aim of the present study was to investigate the effect of follicle superstimulation on the expression of developmentally important genes in bovine oocytes collected by OPU. Follicular oocytes were collected by OPU without (OPU group) or after follicle superstimulation by FSH (FSH/OPU group) by using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner according to Imai et al. (2008). In the FSH/OPU group, after dominant follicle removal from Holstein dry cows by OPU, a CIDR was inserted on Day 5 (dominant follicle removal = Day 0). Cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by IM injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). Oocyte collection by OPU was performed 48 h after PGF administration (Day 11) by the aspiration of follicles larger than 5 mm in diameter. In the OPU group, 3-to-6-mm follicles were aspirated without any previous hormone treatment. In vitro oocyte maturation (IVM) was performed according to Imai et al. (2006 J. Reprod. Dev. 52(Suppl), 19–29). Gene expression was assessed before (0 h IVM) and after IVM (22 h IVM) by RT quantitative PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1 and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using Qiagen RNeasy Micro kit (Qiagen, Valencia, CA, USA). Gene expression was analysed by a Roche Light Cycler 480 device. The relative expression of each gene was normalized to ACTB. Three replications were performed. Data were analysed by ANOVA. At 0 h IVM, PAP and DYNC 1/1 were found to be down-regulated (P < 0.05) in the FSH/OPU group compared with the OPU group, whereas the rest of the studied genes showed similar expression in the FSH/OPU and OPU groups. At 22 h IVM, PAP and DYNC 1/1 remained down-regulated in FSH/OPU oocytes. However, at this time the expression of GDF9 appeared significantly higher (P < 0.05) in FSH/OPU oocytes than in OPU oocytes. The expression of GDF9 was found to decrease during IVM in both groups; however, this decrease was less drastic in FSH/OPU oocytes. The results suggest that follicle superstimulation caused reduced expression of mRNA levels of PAP and DYNC 1/1 irrespective of maturation status and it also moderated the reduction of mRNA levels of GDF9 during IVM.

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