Improves Oocyte Developmental Competence Research Articles

266 THE SHORT TIME TREATMENT WITH SODIUM BUTYRATE ON GERMINAL VESICLE STAGE OOCYTES IMPROVES OOCYTE QUALITY AND DEVELOPMENTAL COMPETENCE IN PIGS

Oocyte maturation is a complex process during which the epigenetic modifications are dramatically changed, especially the histone acetylation and phosphorylation. Sodium butyrate (NaBu) is a histone deacetylase inhibitor that results in a more open structure of DNA. The aim of the present study was to analyse the role of NaBu in the meiosis of porcine oocytes and the subsequent embryonic developmental competence. Cumulus–oocyte complexes (COC) were collected from ovaries obtained at a local slaughterhouse. The COC were randomly divided into 3 groups and matured in vitro in medium (Hao et al. 2006) supplemented with 1 μM NaBu for 2 h [germinal vesicle (GV) stage, group 1] or for 22 h [GV to GV breakdown (GVBD) stage, group 2] or without treatment (control, group 3). After 44 h of in vitro maturation, the oocytes were denuded by 0.2% hyaluronidase, and oocytes with evenly dark ooplasm and visible first polar bodies were considered matured. The cortical granule distribution of matured oocytes was examined with immunostaining. The relative expression of CyclinB and Cdc2 of 3 group oocytes was determined with real-time PCR. Some matured oocytes from each group were collected and stimulated with electric pulse (2 direct current pulses of 1.2 kV cm–1 for 30 μs). The rate of parthenogenetic blastocyst was recorded, and cell number of each blastocyst was determined under an inverted fluorescence microscope after staining with 10 μg mL–1 of Hoechst 33342. The following results were found. 1) Compared with the control group (n = 70, 67.74 ± 1.64), oocyte maturation rates of group 1 and group 2 decreased significantly along with the extended treatments (n = 70, 59.57 ± 5.29 and 46.99 ± 1.22, respectively; P < 0.05). 2) The long time (22 h) treatment with NaBu inhibited the developmental competence (blastocyst rate) of oocytes (n = 30, 15.33 ± 3.47 v. 27.16 ± 2.10 P < 0.05), and the short time (2 h) treatment with NaBu on GV-stage oocytes inhibited the meiotic process slightly but improved the blastocyst rate (n = 30, 33.93 ± 2.51 v. 27.16 ± 2.10; P < 0.05). 3) The short time (2 h) treatment resulted in the migration of more cortical granules into the plasmasmic membrane and formed a monolayer with the membrane (compared with the control). 4) The exposure to NaBu from GV to GVBD stage induced the expression of the CyclinB and Cdc2 in the matured oocytes (4.68 ± 0.45 and 5.80 ± 0.58, respectively; P < 0.05) compared with the control. 5) Short time (2 h) exposure to NaBu on GV-stage oocytes inhibited the expression of the Cdc2 but increased the expression of the CylinB in the matured oocytes (0.43 ± 0.06 and 1.65 ± 0.26, respectively; P < 0.05) compared with the control. In conclusion, results of this study demonstrate that exposure to NaBu inhibits porcine oocyte meiosis in proportion to treatment length. However, a 2-h treatment with 1 μM NaBu improves oocyte developmental competence to the blastocyst stage. These results are useful for improving the developmental competent of oocytes for IVF and in vitro embryo production. This work was supported by a grant (No. 2009CB941001) from the National Basic Research Program of China.

Increased Beta-Oxidation, ATP Levels, and Improved Oocyte Developmental Competence in Response to L-Carnitine.

Increased Beta-Oxidation, ATP Levels, and Improved Oocyte Developmental Competence in Response to L-Carnitine.

FSH at time of hCG trigger improves oocyte developmental competence: a double blind randomized control trial

OBJECTIVE: Improvements in IVF success rates have largely been due to optimization of embryo culture and stimulation protocols; less attention has been directed toward methods to improve induction of final oocyte maturation. Normal ovulation is preceded by a surge in both LH and FSH. We hypothesize that modification of ovulation trigger toward a more physiologic process will improve the developmental competence of the oocyte as defined by fertilization and pregnancy outcomes.

The Cumulus Cell-Secreted Factor Neuregulin 1 Maintains the Cumulus Cell Function in Ovulated COCs To Enhance Fertilization and Developmental Competence in Porcine Oocytes.

It has been known that cumulus cells support oocyte meiotic and cytoplasmic maturation. Our previous study showed that the EGF like factors (amphiregulin, epiregulin and betacellulin) are expressed in granulosa cells and cumulus cells and act on cumulus cells to induce cumulus cell-oocyte complex (COC) expansion and oocyte maturation in mice and in pigs. Additionally, when ovulated mouse COCs were cultured with amphiregulin, this factor activated/phosphorylated the EGF receptor (ErbB1) and maintained cumulus expansion and the expression of specific genes when expression of endogenous amphiregulin is decreased markedly. Moreover, we have shown recently that the maintenance of the COC matrix and the secretion of specific chemokines from cumulus cells enhance successful fertilization via supporting sperm motility and induction of sperm capacitation. These data suggested that additional ligand members for the ErbB family are required to act to maintain cumulus cell function after ovulation. However, there is little information about what mechanisms regulate cumulus cells in oviductal COCs. Therefore, this study was designed to determine the kinetic expression patterns of ErbB family and the ligand in cumulus cells in vivo and in vitro, and then activate the ErbB signaling pathway to stimulate oocyte maturation in COCs in culture. For this, intact follicles and cumulus cells were collected at early, antral and ovulatory stages of follicle, or from COCs in the oviduct at specific time intervals following eCG and hCG treatment of gilts. FSH/eCG increased levels of EGF receptor and ErbB3 protein in cumulus cells, whereas ErbB2 remained constant and ErbB4 was down regulated. Immuno-histochemistry localized ErbB3 to cumulus cells after eCG and hCG stimulation. Because we found high expression of neuregulin 1 (Nrg1) mRNA in cumulus cells from our rat COC micro-array database, and because RT-PCR analyses showed that Nrg1 expression in cumulus cells of porcine COCs was increased after hCG stimulation and reached maximum levels after ovulation, we sought to determine if NGR1 might regulate cumulus cell functions via ErbB3. When porcine COCs were cultured in vitro with FSH alone, the expression of Nrg1 in cumulus cells did not increase. Therefore, to determine if NRG1 might enhance the actions of FSH, NRG1 was added to FSH-containing medium at 24 hr of culture and COC expansion and oocyte maturation were analyzed after an additional 20-hr culture (total 44 hr culture period). The addition of NRG1 strongly induced the phosphorylation of ErbB3 but not ErbB1, which resulted in the activation of both ERK1/2 and PI 3-kinase-PKB pathways in cumulus cells. The COCs exhibited improved cumulus cell survival (no apoptosis) and sustained expansion. The developmental competence of fertilized oocytes to the blastocyst stage was also enhanced by NGR1. From these results, we show for the first time that NRG1 is a novel COC secreted EGF-like factor that acts via autocrine mechanisms to activate ErbB3 expressed on cumulus cells within COCs after ovulation, and thereby helps maintain cumulus cell function to improve oocyte developmental competence, maybe due to suppress oocyte aging. Supported in part by JSPS-18688016 (MS). (poster)

The follicular phase in pigs: Follicle populations, circulating hormones, follicle factors and oocytes,

Abstract The predominant pattern of follicle development in pigs is characterized by continuous activation, slow growth to the antral stage, and rapid growth to 4 to 5 mm followed by atresia. The only time that this pattern is broken is when a small portion of the follicle population is selected for ovulation. The mechanisms that regulate the selection of ovulatory follicles are not well understood. However, the ovulatory cohort shifts from FSH to LH dependence at the expense of the nonovulatory follicles as indicated by the following: 1) decreased secretion of FSH, and 2) decreased expression of the FSH receptor and increased expression of the LH receptor. The selection of ovulatory follicles may be dependent on the interaction of members of the intraovarian IGF system to maintain a high level of IGF-I bioavailability. The maintenance of a proliferating population of antral follicles is critically dependent on circulating FSH. A naturally or experimentally induced increase in circulating FSH levels results in an increase in antral follicles; conversely, decreased secretion of FSH is followed by a decrease in the number and in health status of antral follicles. Gonadotropin treatment with eCG or PG600 triggers selection of ovulatory follicles, and although these treatments do not increase litter size, they are beneficial for treatment of anestrus and, in conjunction with hCG or GnRH analogs, provide better control of the time of ovulation. The use of porcine FSH has not increased ovulation rate or improved oocyte developmental competence. To improve reproductive efficiency in the future, research should be directed toward obtaining more knowledge about genetic and physiological regulation of ovulatory follicle selection and the effect of follicle development on oocyte developmental competence.

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly ( P<0.01) larger than NCL ovaries (width: 22.3±0.9 mm versus 15.9±0.4 mm, length: 33.2±1 mm versus 24.1±0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1±1.8 mm versus 46.8±2.2 mm), they had a similar number of follicles 3–8 mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5±0.1 mm versus 3.3±0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159±1.3 μm versus 146.1±1.5 μm, zona free: 124.7±1.8 μm versus 113.1±1.6 μm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6±3.2% versus 92.4±3.2%), cleavage (88.4±11% versus 90.7±2.6%) and blastocyst formation (21.0±3.7% versus 23.7±5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.

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

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&amp;lt;0.01; a,bP&amp;lt;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).

17Beta-estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys.

Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.

Oocyte maturation.

Primary oocytes recovered from small and growing follicles of > or = 3 mm in the ovaries of untreated women, can be matured in vitro, will fertilize and develop in vitro, and when transferred to the patient, develop to term. However, the implantation rate of cleaved embryos has been disappointingly low and when embryos are allowed to develop beyond the 4-cell in vitro, retardation of development and blockage is frequently observed, with relatively few embryos developing to blastocysts. We have devised new culture systems for human embryos to enable high rates of development of in-vivo matured oocytes to blastocysts within 5-6 days of culture, and high implantation rates of these blastocysts when they are transferred to the patients' uterus. These culture systems are now being used for in-vitro matured oocytes. In order to determine whether embryo developmental competence could be improved, a number of factors were examined. Treatment of patients with pure follicle stimulating hormone (FSH) early in the follicular phase, or treatment with oestrogen prior to oocyte recovery, had no apparent effect on any parameters of oocyte developmental competence. There was no indication that a medium made specifically for human oocyte maturation improved oocyte developmental competence. Nuclear and cytoplasmic changes in oocytes matured in vitro appear to be similar to that in vivo, although some lack of synchronization in completing maturation is evident. It is possible that follicles of < 10 mm diameter in the human contain developmentally-incompetent oocytes. However, the development to term and birth of normal babies from germinal vesicle stage oocytes recovered from small follicles and matured in vitro, suggests that further research will identify the factors necessary to improve embryo developmental competence. The application of immature oocyte collection (IOC) and in vitro maturation (IVM) as an alternative to ovulation stimulation with high doses of gonadotrophins for in-vitro fertilization (IVF), remains a priority for research in human medicine.