Etsushi Kaneko

Insulin-like growth factor-I (IGF-I) system during follicle development in the bovine ovary: relationship among IGF-I, type 1 IGF receptor (IGFR-1) and pregnancy-associated plasma protein-A (PAPP-A).

Insulin-like growth factor-I (IGF-I) system that is exerted mainly through the type 1 IGF receptor (IGFR-1) and releasing of free IGF-I is regulated by the proteases of IGF-binding proteins (IGFBPs), an important factor in follicle development of bovine ovary. The aims of the present study were to examine the mRNA expressions of IGF-I, IGFR-1 and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells and theca tissues during bovine follicular development and the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of these genes in cultured bovine granulosa cells. Follicles were classified into four groups such as small follicle (SF), estrogen inactive dominant follicle (EID), estrogen active dominant follicle (EAD) and preovulatory follicle (POF). The concentration of free IGF-I in follicular fluid of POF was significantly higher than those in EID, whereas the total IGF-I in follicular fluid did not change at all developmental stages. The expression of IGF-I mRNA was not detected in the granulosa cells at all at any developmental stages but the expression was detected in the theca tissues. The amount of IGFR-1 mRNA in granulosa cell showed the constant level at all developmental stages except EID. The expressions of IGFR-1 and PAPP-A in cultured bovine granulosa cells were stimulated with FSH but not with E2. The PAPP-A mRNA expression was stimulated by FSH in presence of 1 ng/ml E2. These results indicate that IGF-I in follicular fluid is mainly derived from the circulation and that FSH is an inducer for the expression of IGFR-1 and PAPP-A genes in granulosa cells. Therefore, we suggest that PAPP-A stimulated with FSH play a crucial role for IGF-I system in bovine follicular development.

Involvement of the bone morphogenetic protein/receptor system during follicle development in the bovine ovary: Hormonal regulation of the expression of bone morphogenetic protein 7 (BMP-7) and its receptors (ActRII and ALK-2).

Bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. Among the BMP ligands, BMP-7 which use ActRII as their type II receptor, strongly bind to ALK-2 as their type I receptor. However, whether their receptors are expressed and the regulatory mechanisms controlling their expression during the process of bovine follicle development are still unknown. The aim of the present study was to clarify the involvement of the receptor system for BMP-7 in follicular selection by examining the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the regulation of ActRII and ALK-2 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression, follicles were obtained from heifers and GCs were classified into two groups: pre-selection follicles (PRF; follicles with an average diameter of 7 mm and low E2) and post-selection follicles (POF; follicles with an average diameter of 15 mm and high E2). The theca cell (TC) layer and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbeccos modified Eagles/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10,100 ng/ml), FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses (1, 5, 10 ng/ml). Total RNA was extracted from GCs and the mRNA expression of ActRII and ALK-2 was estimated by the quantitative PCR method using LightCycler. The expression of BMP-7 mRNA in TCs did not differ between the PRF and POF. ActRII and ALK-2 expression was detected in GCs from bovine antral follicles and was higher in the GCs of POF than in those of PRF, while the expression of the ActRII and ALK-2 genes in the TCs was not different between PRF and POF. Treatment of GCs with E2 (10 ng/ml) alone increased the expression of both ActRII and ALK-2 mRNAs, whereas FSH alone had no effect. However, ActRII and ALK-2 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). The results of the present study provide the first evidence that FSH and E2 regulate the expression of the ActRII and ALK-2 genes in bovine GCs. Thus, our data suggest that the BMP7/ActRII/ALK-2 system may be critically involved in the process of selection of bovine follicles.

Excess estrogen sulfoconjugation as the possible cause for a poor sign of parturition in pregnant cows carrying somatic cell clone fetuses

We conducted this study to elucidate a factor causing a poor sign of parturition and prolonged gestation, which is frequently observed in cows carrying somatic clone fetuses. Pre-partum rises in concentrations of plasma estrone and estradiol-17beta in the recipient cows pregnant with clones were subtle. By contrast, the plasma concentration of estrone sulfate in clone pregnancies increased gradually from pre-initiation of parturition induction whereas control cows that received in vivo-derived embryos showed a significant increase at parturition. Therefore, in clone pregnancies, the ratio of estrone/estrone sulfate was low during the pre-partum period compared with control. Messenger RNA expression of estrogen sulfotransferase (SULT1E1) in the placenta at parturition was significantly higher in clone pregnancies than control pregnancies and was localized in binucleate cells (BNC). SULT1E1 mRNA abundance was negatively and positively correlated with concentrations of maternal estrone and estrone sulfate at parturition respectively. Messenger RNA expressions of estrogen sulfatase (STS) and aromatase (CYP19) were similar between clone and control pregnancies and were localized in BNC and caruncular epithelial cells. STS and CYP19 mRNA abundances showed positive correlations with maternal estradiol-17beta concentration. The population of BNC in the placenta did not differ between clone and control pregnancies. Plasma cortisol concentration of vaginally delivered newborn clone calves was comparable with those of control, although cesarean section delivered clone calves showed a low concentration. These results suggest that excess estrogen sulfoconjugation is the reason for the perturbed low ratio of active to inactive estrogens and the resulting hormonal imbalance contributes to the lack of overt signs of readiness for parturition in cows pregnant with clones.

Improvement of Preimplantation Development of In Vitro- Fertilized Bovine Zygotes by Glucose Supplementation to a Chemically Defined Medium

ABSTRACT The influences of glucose supplementation on early development of bovine embryos in BSA-free synthetic oviduct fluid were examined. Among the groups supplemented with 1.5, 2.0, 4.0 or 5.6 mM glucose either at 0, 72 or 144 hr after fertilization, blastocysts yield significantly increased in the group supplemented with 4.0 mM glucose 144 hr after fertilization compared to the controls without glucose supplementation. The results suggest that appropriate amounts of glucose supplemented to the medium at the specific stage of embryo culture may be useful for the production of bovine blastocysts.

252 THE ROLE OF INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) IN DEVELOPMENT OF AN ESTROGEN-ACTIVE DOMINANT FOLLICLE DURING THE FIRST FOLLICULAR WAVE POSTPARTUM IN DAIRY COWS

Recent studies have shown that IGF-1 is a crucial factor for ovarian follicular development in mammals. In postpartum (pp) dairy cows, plasma IGF-1 and estradiol (E2) levels in ovulatory cows at the first follicular wave pp are higher than in anovulatory cows. However, the plasma IGF-1 profile in an ovulatory or anovulatory dominant follicle (DF), which have different E2 production, at the first follicular wave pp have not yet been elucidated. Thus, we investigated the changing profile of plasma IGF-1 levels during first follicular wave pp. In 22 multiparous Holstein cows, blood samples were obtained 2 times/week from 4 weeks prepartum to 3 weeks pp, and the first follicular wave was monitored by ultrasound 2 times/week from 7 days pp to ovulatory phase. Detailed IGF-1 profiles in blood were determined during DF growth and maturation 4 times/day from 10 days pp to 7 days after the first ovulation in 5 ovulatory cows and to 20 days pp in 4 anovulatory cows; the data were analyzed by repeated measures ANOVA, and Students t-test. There was no interaction between groups and time within the prepartum or the pp period. The ovulatory cows (n = 13/22) with an estrogen-active dominant (EAD: high plasma E2 level with peak) follicle showed higher IGF-1 levels than anovulatory cows (n = 9/22) with an estrogen-inactive dominant (EID: low plasma E2 level without peak) follicle during the prepartum (117 ± 8 vs. 91 ± 5 ng mL-1; P < 0.05) and the pp (91 ± 4 vs. 64 ± 4 ng mL-1; P < 0.001) period. Especially noteworthy, during the first follicular wave pp in ovulatory cows, the plasma IGF-1 levels were maintained at a high level until E2 levels increased, followed by an LH surge. We observed that the EAD follicle in ovulatory cows ovulated. To further examine the IGF-1 system in the intra-follicular environment, we used the EAD and EID follicles from ovaries of dairy cows obtained at a slaughterhouse. The EAD and EID follicles were classified on the basis of follicle diameter and E2 concentrations in follicular fluid (FF). The significant differences of factors between EAD and EID were analyzed by Students t-test. The expression of IGF-1 mRNA was not detected in follicular cells in either EAD and EID, suggesting that IGF-1 in FF is mainly derived from liver. The free IGF-1 levels in FF in EAD (4.8 ± 0.5 ng mL-1) were higher than those in EID (2.7 ± 0.1 ng mL-1; P < 0.05). In addition, the expression of type 1 IGF receptor (IGFR-1) mRNA in EAD was higher than hat in EID (P < 0.0001). From the results of the present study, it is apparent that the EAD follicle during the first follicular wave pp in ovulatory cows sufficiently expressed IGFR-1, and a liver-derived IGF-1 stimulates E2 production in the follicle to ovulate. In conclusion, our data suggest that a high concentration of IGF-1, secreted from the liver, during the peripartum period may be one of important factors for the appearance of an ovulatory follicle during the first follicular wave pp cows.

42 PREPARTUM HORMONAL CHANGES IN RECIPIENT COWS FOR SOMATIC CELL CLONED FETUSES

Placental estrogens are associated with fetal growth and development, and play important roles in the initiation of the parturition process. In the delivery of somatic cell-cloned (SC) calves, recipient cows show a weak and unclear symptom of parturition, although large offspring syndrome (LOS) frequently occurs. The mortality of calves by dystocia decreases the efficiency of SC cattle production. In this study, we examined peripheral estrone (E1), estradiol-17² (E2), estrone sulfate (E1S), and progesterone (P4) levels during preparturition in recipients of SC and in vivo calves. Recipients were administered 20 mg dexamethasone (DEX) at Days 276-288 of pregnancy, followed by 0.75-1 mg cloprostenol (PG) and 20 mg estriol 24 h later. Calves (Japanese black cattle) were delivered 2 or 3 days after the DEX administration. SC calves were delivered by cesarean section (C-sec) when LOS was suspected by rectal palpation. Blood samples of recipients [vaginal delivery of SC (Vag): n = 13; C-sec of SC: n = 8; vaginal delivery of in vivo calves (Control): n = 4] were taken at Days 257 and 271 of pregnancy, and just before DEX (-2P), PG (-1P), and parturition (0P). Blood samples of calves were taken just after delivery to analyze cortisol level. The statistical significance was analyzed by the Steel-Dwass test. Birth weight of calves was the heaviest (P 2156 ± 599 pg mL-1). E1 levels tended to be lower in Vag (<1968 ± 299 pg mL-1) and C-sec (<1268 ± 385 pg mL-1) calves at -2P, -1P, and 0P; E1 levels in C-sec calves were significantly lower (P < 0.05) than in Control calves at -2P (512 ± 85 pg/mL-1) and -1P (725 ± 91 pg mL-1). The E2 level in Control calves increased and reached a plateau at -2P. Vag and C-sec calves showed lower E2 levels, except that the Vag level at 0P was similar to that in Control calves. The E2 level of C-sec calves (61.8 ± 25.3 pg mL-1) at 0P was significantly lower (P < 0.05) than that of Control calves (247.5 ± 102.8 pg mL-1). E1S levels in Vag and C-sec calves increased progressively from -2P to 0P, whereas the E1S level in Control calves increased at 0P. The E1S level in Vag calves (41.1 ± 4.1 ng mL-1) was significantly higher (P < 0.05) than in Control calves (19.4 ± 5.1 ng mL-1) at -1P. P4 levels decreased from -2P in Vag and Control calves, and from -1P in C-sec calves. The cortisol level in C-sec calves (60.1 ± 19.1 ng mL-1) tended to be low compared with that in Vag (104.4 ± 23.1 ng mL-1) and Control (93.4 ± 15.0 ng mL-1) calves. This study revealed fetoplacental dysfunction of estrogen synthesis in the SC fetus during preparturition. Elevated E1S levels in recipients of the SC fetus, which correlated with high birth weight and agreed with previous reports for normal or plural pregnancy, might cause the reduction of E1 level. The comparable level of cortisol in Vag and Control calves indicated that SC calves had normal adrenal cortex function. Further analysis on placental estrogen synthesis and cortisol secretion in the SC fetus is necessary to clarify the cause of the prolonged gestation.