Masafumi Tetsuka

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.

Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum

Glucocorticoid (GC) acts as a modulator of physiological functions in several organs. In the present study, we examined whether GC suppresses luteolysis in bovine corpus luteum (CL). Cortisol (an active GC) reduced the mRNA expression of caspase 8 (CASP8) and caspase 3 (CASP3) and reduced the enzymatic activity of CASP3 and cell death induced by tumor necrosis factor (TNF) and interferon gamma (IFNG) in cultured bovine luteal cells. mRNAs and proteins of GC receptor (NR3C1), 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), and HSD11B2 were expressed in CL throughout the estrous cycle. Moreover, the protein expression and the enzymatic activity of HSD11B1 were high at the early and the midluteal stages compared to the regressed luteal stage. These results suggest that cortisol suppresses TNF-IFNG-induced apoptosis in vitro by reducing apoptosis signals via CASP8 and CASP3 in bovine CL and that the local increase in cortisol production resulting from increased HSD11B1 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells.

Improvement on in vitro maturation, fertilization and development of minke whale ( Balaenoptera acutorostrata ) oocytes

The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mares serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.

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.

Comparison of two ultrasonic methods for multiple pregnancy diagnosis in sheep and indicators of multiple pregnant ewes in the blood

Single and multiple pregnancies in sheep were diagnosed to compare the accuracy at day 80–95 of gestation by an ultrasonic Doppler method (Doppler) and a real-time linear ultrasound scanning method (Scanning). In order to investigate indicator(s) of multiple pregnant ewes, plasma total protein, total cholesterol, glucose and progesterone were measured in 37 pregnant and 26 non-pregnant ewes. For predicting single or multiple (⩾ 2 fetuses) pregnancy, the Doppler method resulted in a higher rate of accuracy in both single (82.6%) and multiple (92.9%) diagnoses than those by the Scanning method (68.2% and 66.7%, respectively). However, there were no significant differences between the accuracy of the methods, and between single and multiple diagnoses. Plasma total protein and total cholesterol concentrations did not significantly differ between non-pregnant and pregnant ewes, and between single- and multiple-pregnant ewes. Plasma glucose concentrations in pregnant ewes were significantly lower than in non-pregnant ewes (2.3 ± 0.05 vs 2.6 ± 0.07 mmol/1; P<0.01). Also, glucose concentrations were significantly lower in multiple-pregnant ewes compared with single-pregnant ewes (2.2 ± 0.08 vs 2.4 ± 0.07 mmol/1; P<0.01). On the contrary, plasma progesterone concentrations in pregnant ewes were significantly higher than in non-pregnant ewes (4.9 ± 0.38 vs 1.7 ± 0.22 ng/ml; P<0.01). Concentrations of plasma progesterone in multiple pregnant ewes were significantly higher than in single-pregnant ewes (5.8 ± 0.53 vs. 4.2 ± 0.49; P<0.05). There was a high correlation coefficient (r=0.783) between concentrations of plasma glucose and progesterone in pregnant ewes bearing triplets, but this relationship was non-significant. The only inverse relationship was found in non-pregnant ewes. The present study indicates that the Doppler method may be useful for multiple pregnancy diagnosis at day 80–95 of gestation, and that both plasma glucose and progesterone concentrations are indicators of pregnant ewes bearing multiple fetuses.

Plasma and pituitary concentrations of gonadotropins (FSH and LH) in minke whales (Balaenoptera acutorostrata) during the feeding season.

This study investigated plasma and pituitary concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and steroid hormones (progesterone: P4, testosterone:T, estradiol-17beta: E2) by enzyme-immunoassay (EIA) in minke whales (Balaenoptera acutorostrata) captured during the feeding season (December to March) in the Antarctic Ocean. Plasma FSH and LH levels in female minke whales were higher (P <0.05) than in male whales. Although the pituitary weight was not significantly different between male and female whales, pituitary FSH and LH levels were higher in females than in males (P<0.01) and mature whales than immature whales (P<0.05). Plasma levels of FSH, T and E2 were not significantly different between immature and mature male whales, but plasma LH and pituitary FSH and LH levels were higher (P<0.05) in mature than in immature whales. In both immature and mature whales regardless of gender, pituitary FSH and LH levels were correlated significantly (r=0.69: P<0.01). In mature male whales, plasma T and E2 levels (r=0.60: P<0.01), and testis weight and plasma T levels (r=0.46: P <0.05) were correlated. In immature female whales, plasma FSH and LH levels were highly correlated (r=0.68: P<0.001), but were not for mature female whales. The results show that gender and maturity influence gonadal and pituitary function of minke whales during the feeding season.

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.

Areolae of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

Abstract In this study, we examined the existence and structure of areolae and the steroidogenesis of areolar trophoblast cells in the Antarctic minke whale placenta morphologically and immunohistochemically. Placentas were collected from the 15th, 16th and 18th Japanese Whale Research Program under Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. The opening and cavity of fetal areolae formed by taller columnar trophoblast cells (areolar trophoblast cells) with long microvilli and a bright cytoplasm, as compared with the trophoblast cells of the chorionic villi interdigitating with the endometrial crypts, were recognized in observations of serial sections. The opening of the areolar cavity was hidden by chorionic villi with areolar trophoblast cells. Furthermore, a closed pouch-like structure lined by tall columnar cells similar to areolar trophoblast cells within the stroma of chorionic villi was noticed and continued to the areolar cavity, with the opening seen on serial sections. In a surface investigation of the chorion and endometrium by SEM, maternal (endometrial) areolae irregularly surrounded by endometrial folds were obvious. Moreover, we distinguished areolar trophoblast cells with long microvilli attached with many blebs from trophoblast cells. In our immunohistochemical observations, a steroidogenic enzyme, cytochrome P450 side chain cleavage enzyme (P450scc), was detected with strong immunoreactivity in trophoblast cells. However, areolar trophoblast cells showed weak or no immunoreactivity for P450scc.