286 DEVELOPMENTAL CAPACITY OF PREPUBERTAL BOVINE OOCYTES CULTURED WITH CYCLIC AMP MODULATORS

Prepubertal bovine donors are currently used for commercial breeding to accelerate the genetic gain and decrease the generation interval. Nevertheless, it has been reported that their oocyte developmental competence is lower than in adult females. Addition of cAMP regulators during in vitro maturation (IVM) has been suggested to enhance blastocysts rates (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). Here, we evaluated the effects of the cAMP modulators forskolin, 3-Isobutyl-1-methylxanthine (IBMX), and cilostamide during extended IVM on the developmental capacity of oocytes from prepubertal and adult bovine females. A total of 1851 oocytes from 24 lactating cows (>2 lactations) and 24 prepubertal donors (6–10 mo old) were collected by transvaginal oocyte recovery (OPU) twice per week and divided into 3 experiment groups: (1) TCM24 (OPU medium: PBS; 24 h of IVM; standard protocol/control); (2) cAMP30 [OPU medium: PBS-IBMX (500 μM); 2 h pre-IVM culture using forskolin (100 μM)-IBMX (500 μM) and 30 h of IVM adding cilostamide (20 μM)], and (3) DMSO30 [cAMP modulators are diluted in DMSO)/vehicle control; OPU medium: PBS-DMSO (46.3 mM); 2 h pre-IVM culture (280 mM DMSO) and 30 h IVM (5.6 mM DMSO)]. Following IVM, oocytes were either submitted to in vitro fertilization and embryo culture or fixed in 1% glutaraldehyde at 9, 20, 24, and 30 h after IVM and stained with Hoechst to evaluate their nuclear status. One-way ANOVA was implemented to evaluate recovered oocytes and meiotic stages. The Glimmix procedure from SAS/STAT was performed to compare blastocyst and cleavage rates. Total number of oocytes and IVM-suitable oocytes per donor per OPU session were similar in adult and prepubertal donors (total number/IVM suitable; prepubertal donors: 6.7/4.2, 6.4/4.0, 6.5/3.8; cows: 6.2/4.7, 6.2/4.4, 6.2/4.5 for TCM24, cAMP30 and DMSO30, respectively). At 9 h, cAMP regulators were able to maintain meiotic arrest in prepubertal and adult donors (GV: 80.0 and 40.9%, respectively) compared to standard IVM (GV: 61.1 and 31.2%) and DMSO30 (GV: 40.0 and 26.6%) protocols (P < 0.05). Using the cAMP30 protocol, the percentage of oocytes that reached MII stage at 20 h was lower in adult (4.5%) and prepubertal donors (5.26%) compared to the DMSO30 (50.0 and 42.8%, respectively) and TCM24 (56.2 and 44.4% respectively) protocols. Metaphase II rates after either 24 or 30 h were similar among treatments (prepubertal donors: 88.2, 70.5, and 84.2%; cows: 71.4, 85.7, and 81.2% for TCM24, cAMP30, and DMSO30, respectively; P > 0.05). Cleavage rates (prepubertal donors: 63.4, 54.9, and 52.1%, cows: 56.1, 57.8, and 51.6% for TCM24, cAMP30, and DMSO30, respectively) and blastocysts/presumptive zygotes rates (prepubertal donors: 26.2, 19.6, and 16.2%; cows: 27.5, 28.1, and 21.5% for TCM24, cAMP30, and DMSO30, respectively) did not show significant differences (P > 0.05). Although cAMP modulators delayed the progression through meiosis in adult and prepubertal oocytes, similar blastocysts rates were obtained. Our results suggest so far that oocyte retrieval and competence in prepubertal donors can be similar to that of the adult donors with and without addition of cAMP modulators.

Comparative effects of estradiol, methyl-piperidino-pyrazole, raloxifene, and ICI 182 780 on gene expression in the murine uterus

Selective estrogen receptor modulators (SERMs) are potentially useful in treating various endometrial disorders, including endometrial cancer, as they block some of the detrimental effects of estrogen. It remains unclear whether each SERM regulates a unique subset of genes and, if so, whether the combination of a SERM and 17β-estradiol has an additive or synergistic effect on gene expression. We performed microarray analysis with Affymetrix Mouse Genome 430 2.0 short oligomer arrays to determine gene expression changes in uteri of ovariectomized mice treated with estradiol (low and high dose), methyl-piperidino-pyrazole (MPP), ICI 182 780, raloxifene, and combinations of high dose of estradiol with one of the SERM and dimethyl sulfoxide (DMSO) vehicle control. The nine treatments clustered into two groups, with MPP, raloxifene, and high dose of estradiol in one, and low dose of estradiol, ICI + estradiol, ICI, MPP + estradiol, and raloxifene + estradiol in the second group. Surprisingly, combining a high dose of estradiol with a SERM markedly increased (P<0.02) the number of regulated genes compared with each individual treatment. Analysis of expression for selected genes in uteri of estradiol and SERM-treated mice by quantitative (Q)RT-PCR generally supported the microarray results. For some cancer-associated genes, including Klk1, Ihh, Cdc45l, and Cdca8, administration of MPP or raloxifene with estradiol resulted in greater expression than estradiol alone (P<0.05). By contrast, ICI 182 780 suppressed more genes governing DNA replication compared with MPP and raloxifene treatments. Therefore, ICI 182 780 might be superior to MPP and raloxifene to treat estrogen-induced endometrial cancer in women.