Takahisa Oda

Gonadotropin stimulates ovarian renin-angiotensin system in the rabbit

The present study was undertaken to assess the role of ovarian renin-angiotensin system (RAS) in the preovulatory cascade induced by gonadotropin exposure. In the in vitro perfused rabbit ovaries, exposure to human chorionic gonadotropin (hCG) enhanced the secretion rate of angiotensin II (Ang II) within 1 h. The secretion rate reached maximal levels at 6 h and then declined thereafter. The intrafollicular Ang II content and renin-like activity were also significantly increased at 2 and 4 h after exposure to hCG, compared with control ovaries perfused with medium alone. The level of intrafollicular Ang II after hCG exposure significantly exceeded the concentration of Ang II in an equivalent volume of plasma. The addition of 1 microM captopril to the perfusate significantly inhibited the secretion rate of Ang II stimulated by hCG; however, captopril affected neither the ovulatory efficiency nor prostaglandin production in ovaries treated with hCG. Captopril significantly inhibited the resumption of meiosis in the ovulated ova and follicular oocytes stimulated by hCG. The administration of 100 micrograms Ang II at 2-h intervals to the perfusate reversed the inhibitory effects of captopril on hCG-induced oocyte maturation. In conclusion, these data indicate that gonadotropin stimulates renin-like activity and Ang II production in the rabbit ovary. Ovarian renin-angiotensin system may play an important role in the process of oocyte maturation after exposure to gonadotropin.

Possible Contribution of Prolactin in the Process of Ovulation and Oocyte Maturation

The present study was undertaken to evaluate the effects of PRL in the process of ovulation and oocyte maturation. In the first experiment, using an in vitro perfused rabbit ovary model, the addition of PRL to the perfusate inhibited hCG-induced ovulation in a dose-related fashion, without any reduction in progesterone synthesis. In a subsequent experiment, PRL directly inhibited both the degeneration and decomposition of surface epithelial cells and the disruption of connective tissue at the apex of the follicle wall. Furthermore, PRL inhibited hCG-stimulated plasminogen activator (PA) activity in mature follicles in a dose-related fashion. In the final experiment, we demonstrated conditions in which rabbit oocytes matured in vitro acquire competence for early embryonic development. PRL, as well as gonadotropins and estradiol, was an important constituent in the process of oocyte maturation, promoting embryonic development. These results suggest that the preovulatory environment of PRL within the follicle may influence the process of ovulation and oocyte maturation.

Effects of prolactin on fertilization and cleavage of human oocytes.

The effects of PRL on fertilization and cleavage of human oocytes and subsequent pregnancy were studied. Forty-five patients (47 cycles) with euprolactinemic normal menstrual cycles undergoing in vitro fertilization (IVF) for the treatment of tubal infertility were selected for this study. The patients were divided into three groups dependent upon their mean serum PRL concentrations for the 3 days prior to oocyte retrieval; hypoprolactinemic (less than 10 micrograms/l), euprolactinemic (10-30 micrograms/l) and hyperprolactinemic cycles (greater than or equal to 30 micrograms/l). Multiple follicular development was induced with hMG, and 10 patients were randomized to receive bromocriptine beginning with the previous menstrual cycle. In the hypoprolactinemic cycle group, the fertilization rate was significantly lower than in the hyperprolactinemic cycle group, and the cleavage rate was significantly lower than in the other groups. The fertilization rates and the cleavage rates in the hyperprolactinemic cycle group were higher than those in the euprolactinemic cycle group; however, these differences were not statistically significant. While the pregnancy rates in the euprolactinemic cycle group were higher than in the other two groups, the numbers were too small for meaningful statistical comparison. The present study demonstrates that below normal concentrations of PRL have deleterious effects on IVF outcome. These data suggest that PRL may play a beneficial stimulatory role in oocyte maturation and the acquisition of developmental capacity.

Direct Ovarian Effect of Growth Hormone in the Rabbit

PROBLEM: This study was undertaken to assess whether growth hormone (GH) can stimulate follicle growth and ovarian steroidogenesis via putative GH receptors.

Improved semen qualities after continuous-step density gradient centrifugation, application to artificial insemination and pregnancy outcome

For increasing sperm fertilizability in artificial insemination, it is effective to concentrate progressively motile sperm from whole ejaculate. We developed the continuous-step density gradient for the selective concentration of progressively motile sperm. The present procedure was applied to intrauterine artificial insemination. Infertile couples (n = 152) whose diagnoses involved oligoasthenospermia, cervical factor, and unexplained infertility were selected for artificial insemination with washed and concentrated sperm. Successful pregnancies (47) were obtained, with an overall pregnancy rate of 31% in the program. Sperm processing by this procedure improved pregnancy rates in couples with the sole diagnosis of oligoasthenospermia or cervical factor. In the patients in whom other fertility problems coexisted, however, it was essential to treat female fertility problems. Continuous-step density gradient centrifugation is effective in increasing the pregnancy rate in artificial insemination.

Quantitative Assessment of Human Sperm Acrosome Reaction by Using Fluorescein Isothiocyanate Conjugated Concanavalin a—Comparison Between Highly Purified Acrosome Reacted with Non-Acrosome Reacted Sperm

In the present study, to achieve quantitative assessment of human sperm acrosome reaction by using fluorescein isothiocyanate conjugated Concanavalin A (FITC-Con A), the optimum conditions for microscopic observation were established by comparing the fluorescent images between the purified human sperm with the intact acrosome (non-acrosome reacted sperm; non-AR sperm) and those with exposed inner acrosomal membrane (acrosome reacted sperm; AR sperm). FITC-Con A stained not only the inner acrosomal membrane but also the other portions. These non-specific bindings were excluded by the competitive dissociation with alpha-D-mannose (Man) solution, the specific labeling of the inner acrosomal membrane with FITC-Con A was accomplished in the presence of 0.5 mg/mL Man. The influence of methanol fixation on the acrosome status were examined. The assessment of the acrosome reaction should evaluate whether the inner acrosomal membrane is exposed or masked by the plasma membrane, and methanol fixation is often employed in the histochemical stain. By methanol fixation, the fluorescent profiles of AR sperm were not altered before and after the treatment, bright fluorescence were found in the acrosomal regions. On the other hand, non-AR sperm gave weak fluorescence on the sperm heads before the treatment, the acrosomal regions fluoresced brightly after the treatment similar to those of AR sperm. Methanol fixation caused false positive results, it might denaturate the plasma membranes and facilitate permeability of FITC-Con A. FITC-Con A fluorescent stain and the modified triple stain (rose bengal stain) gave good positive correlation to evaluate AR sperm. This result concluded that the optimum conditions with FITC-Con A stain in the present procedure might be an useful tool for observation of human sperm acrosome reaction.

The effects of gonadotropin-releasing hormone agonist on androstenedione production and follicular development during controlled ovarian hyperstimulation.

Purpose:We performed a prospective randomized study to assess the effects of a GnRH agonist (GnRH-a) onfollicular development and steroidogenesis during controlled ovarian hyperstimulation (COH).Methods:Patients undergoing in vitro fertilization (IVF)for tubal infertility received human menopausal gonadotropin (hMG) stimulation with or without the GnRH-a, buserelin, beginning in the midluteal phase of the prior cycle. We analyzed serum hormone levels, follicular development, and outcome of IVF.Results:The mean number of retrieved oocytes was significantly greater, and the implantation rate per embryo was significantly higher, in the GnRH-a/hMG group (n = 101) than in the hMG-only group (n = 97). The concentration of androstenedione (A) and the A/estradiol ratio in the serum were significantly lower in the GnRH-a treatment group throughout the follicular phase.Conclusions:The concomitant use of GnRH-a during COH prevents atretic change of the follicles and enhances follicular development by reducing androgen accumulation, resulting in a higher developmental competence of the oocytes.

Comparative study of hormonal dynamics in pregnant and nonpregnant cycles during pulsatile subcutaneous administration of human menopausal gonadotropin in anovulatory infertile women.

OBJECTIVE To assess the clinical relevance of daily hormonal changes for achieving a successful pregnancy in anovulatory infertile women. DESIGN A comparative study of hormonal dynamics in pregnant and nonpregnant cycles during the pulsatile subcutaneous administration of hMG. Subjects received subcutaneous injection of either 9.375 IU or 14.0625 IU of hMG diluted in 50-microL physiological saline (total daily dose, 150 or 225 IU) at 90-minute intervals by means of a portable peristaltic pump. SETTING Kyorin University Hospital and Ichikawa General Hospital. PATIENTS We analyzed 18 pregnant and 42 nonpregnant cycles in 17 patients with secondary hypothalamic/pituitary amenorrhea who conceived after receiving pulsatile hMG treatment. Another 14 women with normal spontaneous ovulation, including 14 pregnant and 15 nonpregnant cycles, served as controls. MEASUREMENTS Serum concentrations of LH, FSH, E2, and P were measured, and the P:E2 ratio was determined. RESULTS Serum concentrations of LH and FSH did not differ significantly between the pregnant and nonpregnant cycles. Serum levels of P and E2 were significantly higher during the hMG treatments than those of the spontaneous ovulatory cycles throughout the follicular and luteal phases. Up to the midluteal phase, the P and E2 values in the nonpregnant cycles during the hMG treatments did not differ significantly from those in the pregnant cycles. The P:E2 ratios were comparable between the pulsatile stimulatory cycles and the normal spontaneous ovulatory cycles. However, the P:E2 ratio in the early and midluteal phases was significantly greater in the pregnant cycles than in the nonpregnant cycles. CONCLUSION The P:E2 ratio in the early and midluteal phases is a more important indicator of hormonal function for implantation than the absolute levels of either P or E2.

Possible Involvement of Lipoxygenase Products in Human Corpora lutea

The present study was undertaken to determine the ability of cultured luteal cells from human corpora lutea to secrete progesterone (P4) and prostaglandins (PGs), and to assess the effects of the products of the lipoxygenase pathway on luteal P4 production. Luteal cells responded to human chorionic gonadotropin (hCG) with a significant increase (2- to 7-fold) in P4 production. Arachidonic acid significantly stimulated PGE2 synthesis by luteal cells in a dose-dependent manner. Both basal PGE2 production and the responsiveness to arachidonic acid were maintained for 8 days. In contrast, both PGF2 alpha and 6-keto-PGF1 alpha production abruptly declined as the culture proceeded. However, the addition of hCG did not further stimulate the accumulation of the 3 PGs assayed. In the subsequent experiment, 5-hydroxyeicosatetraenoic acid (5-HETE) and the reaction products of soybean lipoxidase of arachidonic acid (AA-LIP) were utilized for evaluating the involvement of the lipoxygenase pathway in luteolysis. The addition of 5-HETE dose-dependently inhibited P4 production by the cultured luteal cells. Although treatment with either arachidonic acid or lipoxidase alone had no effect on P4 production, AA-LIP significantly reduced P4 production in the presence or absence of hCG. These results suggest that the products of the lipoxygenase as well as of the cyclo-oxygenase pathway may be important in regulating the life span and function of human corpora lutea.

Role of Corpus luteum Function in Embryo Implantation

The present study was undertaken to evaluate the role of corpus luteum function in the implantation process of cryopreserved embryos. Six patients with tubal infertility were studied, with a total of 27 fresh embryos being replaced following controlled ovarian hyperstimulation and in vitro fertilization. These embryo transfers failed to produce pregnancy. From the patients, 22 supernumerary embryos were cryopreserved and 10 viable frozen-thawed embryos were transferred in a subsequent natural cycle, resulting in 2 pregnancies. In 1 of the pregnancies, the serum concentrations of estradiol (E2) and progesterone (P4) in the midluteal phase were 29 pg/ml and 5.6 ng/ml, respectively. These values were considered to be below the physiological range. These data suggest that a pregnancy can be achieved in spite of low circulating E2 and P4 levels during the luteal phase, which are generally indicative of the luteal phase defect.