Toshio Inaba

Effects of cortisol on the amounts of estradiol-17β and progesterone secreted and the number of luteinizing hormone receptors in cultured bovine granulosa cells

Abstract The direct effects of cortisol upon the amounts of estradiol-17β and progesterone secreted, and the number of luteinizing hormone (LH) receptors in cultured bovine granulosa cells were examined. Granulosa cells from small bovine follicles (diameter 3–7 mm) were successfully cultivated in a 1:1 mixture of Dulbeccos modified Eagles medium and Hams F-12 nutrient medium containing 10% fetal calf serum for the first day and subsequently incubated with follicle stimulating hormone and testosterone in serum-free medium for up to 8 days. The cells increased their output of both estradiol and progesterone during 9 days of culture. The addition of cortisol in the range 0.1–10 μM did not significantly change the total DNA content of the granulosa cells. Cortisol treatment caused a significant decrease in the amount of estradiol-17β secreted by granulosa cells (P These results demonstrate the direct inhibitory effect of cortisol on follicular secretion of estradiol-17β and LH receptor content. This observation is consistent with the hypothesis that cortisol released by stress may directly inhibit the functional maturation of the bovine follicles.

The effects of heparin-binding epidermal growth factor-like growth factor on preimplantation-embryo development and implantation in the rat.

This study examined the effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on preimplantation-embryo development and initiation of implantation in the rat. In vitro studies showed that HB-EGF improved the development of 8-cell embryos to the blastocyst stage in a concentration-dependent manner, and the growth factor had no effect on the cell number of the blastocyst developed. Intraluminal injection of an anti-HB-EGF antiserum into the uterine horns at 0600 h on day 5 of pregnancy decreased the number of implantation sites (blue dye reaction) at 0200 h on day 6. Intraluminal injection of 20 microl of HB-EGF solution (10 or 100 ng/ml) into each uterine horn induced implantation in about half of the ovariectomized progesterone-treated delayed implanting rats, and the number of implantation sites per rat increased dose-dependently. These results suggest that HB-EGF is involved in the preimplantation-embryo development and initiation of implantation in the rat.

INDUCTION OF FERTILE ESTRUS IN BITCHES USING A SUSTAINED-RELEASE FORMULATION OF A GnRH AGONIST (LEUPROLIDE ACETATE)

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.

Evaluation of transplantation of autologous bone marrow stromal cells into the cerebrospinal fluid for treatment of chronic spinal cord injury in dogs.

OBJECTIVE To evaluate effects of transplantation of bone marrow stromal cells (BMSCs) into the CSF for the treatment of chronic spinal cord injury in dogs that had not responded by 1 month after decompressive surgery. ANIMALS 23 dogs. PROCEDURES Dogs with paraplegia and loss of nociception in the pelvic limbs for at least 1 month after decompressive surgery were assigned to transplantation or control groups. Dogs in the transplantation group received BMSCs injected into the CSF 1 to 3 months after decompressive surgery. Dogs in the control group did not receive additional treatments. Improvements in gait, proprioceptive positioning, and nociception were evaluated by use of the Texas Spinal Cord Injury Scale for ≥ 6 months after BMSC transplantation. RESULTS 6 of 10 dogs in the transplantation group regained the ability to walk, whereas only 2 of 13 dogs in the control group regained the ability to walk. Scores for the Texas Spinal Cord Injury Scale in the transplantation group were significantly higher than scores in the control group at the endpoint of the study (6 months after BMSC transplantation or after decompressive surgery for the transplantation and control groups, respectively). Only 1 dog (transplantation group) recovered nociception. All dogs from both groups had fecal and urinary incontinence. No complications were observed in relation to BMSC transplantation. CONCLUSIONS AND CLINICAL RELEVANCE Injection of BMSCs into the CSF caused no complications and could have beneficial effects on pelvic limb locomotion in dogs with chronic spinal cord injuries.

Improved conception in timed-artificial insemination using a progesterone-releasing intravaginal device and Ovsynch protocol in postpartum suckled Japanese Black beef cows

The primary objective was to determine the effect of supplemental progesterone, administered via an intravaginal device (CIDR), on conception rates to timed-artificial insemination (timed-AI) in postpartum suckled Japanese Black beef cows treated with the Ovsynch protocol. A secondary objective was to compare the effects of treatments on plasma concentrations of progesterone and estradiol. Cows in the control group (Ovsynch, n=38) received a standard Ovsynch protocol (100 microg GnRH analogue on Day 0, 500 microg PGF2alpha analogue on Day 7, and 100 microg GnRH analogue on Day 9), with AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the treatment group (Ovsynch+CIDR; n=40) received a standard Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Plasma progesterone concentrations were determined on Days 0, 1, 7, 9, 10, and 17 and plasma estradiol-17beta concentrations were determined on Days 7, 9, 10, and 17. The odds ratio for likelihood of conception was 3.29 times greater (P=0.02) in the Ovsynch+CIDR group compared to Ovsynch group. The conception rate was greater (P=0.03) in the Ovsynch+CIDR group than in the Ovsynch group (72.5% versus 47.7%). Insertion of a CIDR device significantly increased plasma progesterone concentrations only on Days 1 and 7 (P<0.001 and P=0.05, respectively), but had no significant effect on plasma estradiol-17beta concentrations. Including a CIDR with the Ovsynch protocol significantly improved conception rates in postpartum suckled Japanese Black beef cows.

Enhancement of estrogen receptor gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

Estrogen receptor mRNA (ER mRNA) levels were measured in the mediobasal hypothalamus (MBH) of beagle bitches at different stages of the estrous cycle, and compared with levels in ovariectomized (OVX) estrogen-treated bitches. In cyclic bitches, the level of hypothalamic ER mRNA increased during the progression of anestrus and declined thereafter. Hypothalamic ER mRNA and plasma luteinizing hormone (LH) levels during anestrus and proestrus were positively correlated (r = 0.94, P < 0.001). In OVX bitches, levels of hypothalamic ER mRNA were low, and increased significantly after treatment with a low dose of estradiol benzoate. These results suggest that, during the course of anestrus in the bitch, hypothalamic ER mRNA expression increases, and may be up-regulated by estradiol.

Evidence for the involvement of transforming growth factor-α in implantation in the rat

Abstract This study was conducted to examine the possibility for participation of transforming growth factor-α (TGF-α), one of the epidermal growth factor (EGF) family of growth factors, in implantation in the rat. Immunostaining of TGF-α in the periim-plantation uterus showed distinct staining in the luminal and glandular epithelium and moderate staining in stromal and myometrial cells. After implantation decidual cells and embryos were also positive stained for immunostaining. Immunocytochemistry of EGF receptor showed distinct staining in the luminal and glandular epithelium during the preimplantation period, and after implantation decidua at implantation sites and embryos were stained. Intraluminal injection of anti-TGF-α antibodies into uterine horns at 0600 h on day 5 of pregnancy decreased the number of rats showing implantation (blue dye reaction) at 0200 h on day 6 in a dose-dependent manner. Intraluminal injection of 100 pg of TGF-α on day 5 of pseudopregnancy elicited a greater decidual response when compared with the vehicle-injected contralateral uterine horn. Intraluminal injection of 20 pg of TGF-α into each uterine horn induced implantation in 50% of the ovariectomized progesterone-treated delayed implanting rats. These results suggest that TGF-α is involved in the implantation process in the rat.

Roles of pulsatile release of LH in the development and maintenance of corpus luteum function in the goat

The roles of the pulsatile release of LH in the functional development and maintenance of the corpus luteum (CL) during the estrus cycle in the goat were examined using a potent GnRH antagonist. In Experiment 1, to assess the inhibitory effects of the GnRH antagonist on the release of LH during the estrus cycle, 9 goats were divided into 3 groups. Goats in Group I received only saline on Days 0 (day of ovulation), 5, 10 and 15. Goats in Group II received the GnRH antagonist (50 microg/kg, s.c.) on the days mentioned for Group I to inhibit endogenous LH during the periods of luteal development and maintenance. Goats in Group III received saline on Days 0 and 5 and then the GnRH antagonist on Days 10 and 15 to inhibit LH during the period of luteal maintenance. Serial blood sampling took place on Days 1, 3, 5, 8, 13 and 18 to characterize the LH pulses. The LH pulses were observed throughout the estrus cycle in Group I but were completely abolished in Group II. In Group III, the pulsatile release of LH was observed from Day 1 to 8, but the LH pulses were completely abolished on Days 13 and 18. In Experiment 2, 16 goats were divided into the same 3 groups as in Experiment 1 to examine the effects of the GnRH antagonist on the luteal function. The concentration of progesterone in the plasma in Group I increased after ovulation, reached a maximum level around Day 12, and subsequently returned to the basal level on Day 17. The concentrations of progesterone in Group II rose after ovulation, but reached a plateau around Day 6 and maintained the level up to Day 9, then rapidly decreased from Day 9 to 10 to the basal level. The concentrations of progesterone in Group II were lower on Days 7 to 15 than those in Group I (P<0.01). The concentrations of progesterone in Group III increased after ovulation, reached a maximum level around Day 8, then dropped from Day 10 to 13 to the basal level. The concentrations of progesterone in Group III on Days 11 to 15 were lower than those in Group I (P<0.05 on Day 11, P<0.01 on Days 12 to 15). These results demonstrate that endogenous LH is essential for normal development and maintenance of the CL function during the estrus cycle in the goat. Further, this study suggests that while the functional maintenance of the caprine CL depends entirely on LH support, such functional dependence during early CL development is only partial.

Increased lh pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.

A quantitative comparison in the bovine of steroids and gonadotropin receptors in normally developing follicles and in follicular and luteinized cysts.

Abstract Total numbers of FSH and LH receptors and concentrations of steroids were examined in ovarian cysts, as compared with those of normally developing follicles. The ovarian cysts ( diameter ≧ 23 mm ) were divided into luteinized cysts (plasma concentration of progesterone ≧ 1 ng/ml ) and follicular cysts (plasma concentration of progesterone These results suggest that the luteinized cysts contain many LH receptors in the theca interna and the functional luteal tissue, whereas the follicular cysts possess few LH receptors in the theca interna and have the capacity to secrete androstenedione to some extent. They also indicate that the total numbers of FSH and LH receptors in granulosa cells are reduced and the secretion of estradiol-17β is inhibited in both the luteinized cysts and the follicular cysts.