Tetsuro Honda

Physiological Roles of Integrin α6β1 in Ovarian Functions

We previously reported that human ovarian cells express integrin β1 families. The physiological role(s) of integrins was investigated using in vitro human granulosa cell culture and in vivo mouse ovulation model. In human luteinizing granulosa cell culture obtained from the patients undergoing in vitro fertilization treatment, laminin, which is a ligand for integrin α6β1, suppressed the production of progesterone by granulosa cells. On the other hand, the anti-α6 monoclonal antibody (mAb) GoH3, which partially inhibits the interaction between integrin α6β1 and laminin, enhanced production of progesterone by 2-fold of the control under the culture with laminin, indicating that integrin α6β1 regulates the luteinization of human granulosa cell during the periovulatory phase. In an immature superovulated 13-day-old ICR (CD-1) mice model, intraperitoneal administration of GoH3 induced successful ovulation, whereas no ovulation was observed in the GoH3-nontreated groups, showing that integrin α6β1 is related to gonadotropin-induced follicular growth. These findings suggest that the interaction between integrin α6β1 and laminin plays an important role in the corpus luteum formation and follicular growth.

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on cultured porcine granulosa cells by employing the aminopeptidase assay using alanine-p-nitroanilide and histochemical staining using L-leucyl-beta-naphthylamide. Porcine granulosa cells obtained from follicles 4-5 mm in diameter were cultured for 7 days. The aminopeptidase assay showed that the porcine granulosa cell culture had aminopeptidase activity and that this activity was inhibited in a dose-dependent manner by bestatin which binds to cell surfaces and inhibits cell surface aminopeptidases. Histochemical staining also indicated that cultured granulosa cells had aminopeptidase activity. Porcine granulosa cells were cultured in the presence or absence of porcine follicle stimulating hormone (FSH, 3.125 nmol/l) and/or bestatin (0.4, 4.0 and 40.0 micrograms/ml) for 7 days, and the production of progesterone and oestradiol was measured. In the presence of porcine FSH, the production of progesterone and oestradiol by granulosa cells was increased significantly by approximately 5- and 2-fold respectively. These increases were enhanced further by bestatin (40.0 micrograms/ml). In the absence of porcine FSH, progesterone production was enhanced by bestatin (40.0 micrograms/ml), whereas no significant effect of bestatin on oestradiol secretion was observed. These findings indicate that the inhibition of membrane-bound aminopeptidase(s) on the cell surfaces affects the steroidogenesis of granulosa cells, and that these aminopeptidase(s) are important regulators of granulosa cell differentiation.

Granulosa Cells Express Integrin α6: Possible Involvement of Integrin α6 in Folliculogenesis

To identify differentiation antigens of granulosa cells, we have raised two monoclonal antibodies against porcine granulosa cells (POG-2 antibody) and human granulosa cells (OG-1 antibody). Analysis of N-terminal amino acid sequences of purified OG-1 and POG-2 antigens revealed that they were identical to human integrin α6 and its porcine homolog, respectively. An immunohistochemical study showed that integrin α6 was highly expressed on granulosa cells in preovulatory follicles in the human ovary, whereas it was highly expressed on granulosa cells in small follicles in the porcine ovary. As a common characteristic of integrin α6 expression in both ovaries, granulosa cells located in inner layers, which are not in contact with the basal lamina, expressed integrin α6 on the cell surface. This expression profile suggests that the interaction between integrin and extracellular matrices occurs in the granulosa cells located in inner layers. This cell surface interaction is reported to modulate soluble signals to the cells. Since the expression of integrin α6 on inner-layer granulosa cells is related to stages of differentiation, this interaction may be involved in folliculogenesis.

Laminin and fibronectin concentrations of the follicular fluid correlate with granulosa cell luteinization and oocyte quality

Background and AimsProgesterone production of human cultured luteinizing granulosa cells was reported to be modified by extracellular matrix, suggesting that extracellular matrix regulates luteinization of granulosa cells after ovulation. In the present study, the relationship among laminin, fibronectin, progesterone and estradiol in follicular fluid along with oocyte quality was analyzed to estimate the physiological role of extracellular matrix in follicular luteinization and oocyte quality during ovulation.Methods and ResultsFollicular fluid was collected at oocyte pick-up from the patients undergoingin vitro fertilization treatment and intracytoplasmic sperm injection. The concentrations of laminin, fibronectin, progesterone and estradiol in the follicular fluid were measured by enzyme immunoassay and radioimmunoassay. The morphology of oocytes were also assessed during the procedure of intracytoplasmic sperm injection and was classified into normal and abnormal groups. The fibronectin concentration was higher in the normal ooplasm group than in the abnormal group, but it did not correlate with estradiol or progesterone concentration. However, laminin concentration significantly correlated with that of progesterone, but not with cytoplasm morphology of oocytes. There was no difference in estradiol or progesterone concentration between the normal and abnormal groups.ConclusionThese findings suggest that extracellular matrix plays some roles in regulating human granulosa cell luteinization and oocyte quality during ovulation.

Response to Wang: Perinatal management of cervicoisthmic pregnancy

The authors reported their experience in managing a 39-year-old nulliparous woman with a cervicoisthmic pregnancy from early pregnancy (6 weeks’ gestation) to delivery (32 weeks’ gestation). I applaud their excellent teamwork in dealing with such a high-risk patient, but I am greatly concerned with the decision making in this case. Even though the physicians had informed the couple about the diagnosis of cervicoisthmic pregnancy as early as 6 weeks following gestation, I personally feel that early termination would have been a better choice, because it could have been easily managed with conservative treatment, including a systemic or local methotrexate injection, although this is still highly debated. As the authors mentioned, most physicians consider that a true cervical pregnancy cannot be conservatively managed after 20 weeks’ gestation. In addition, making a diagnosis of cervicoisthmic pregnancy without consideration of possible cervical pregnancy is dangerous, since a missed diagnosis can occur, 2 with resultant catastrophic events, including hemorrhage, hysterectomy, or a life-threatening condition, even though the authors stated that they clearly demonstrated that this case was a cervicoisthmic pregnancy. However, the diagnosis of cervicoisthmic pregnancy cannot be confirmed without the histologic proof that trophoblastic tissue is present in the uterine isthmus, which further supports the concept that it is difficult to clearly diagnose cervicoisthmic pregnancy before surgery, that the diagnosis of cervicoisthmic pregnancy has always been retrospective, and that only a surgical approach can identify it correctly.