Relaxin Modulates the Ovulatory Process and Increases Secretion of Different Gelatinases from Granulosa and Theca-Interstitial Cells in Rats1

Abstract

There were two related objectives in this study. The first was to determine the influence of endogenous relaxin on ovulation in rats. The second was to investigate the effect of relaxin on the secretion of gelatinases involved in extracellular matrix remodeling from rat ovarian cells. Immature rats were primed s.c. with 10 IU eCG; 51 to 52 h later, a monoclonal antibody specific for rat relaxin (MCAR), a control antibody against fluorescein (MCAF), or PBS vehicle was administered via intraovarian bursal injection under anesthesia, and 15 IU hCG was injected i.p. immediately thereafter. Rats were killed 26 h later, and oviducts were isolated and examined under the microscope to determine the number of ovulated oocytes. MCAR (0.25 and 2.5 micrograms/ovary) partially suppressed gonadotropin-induced ovulation as compared to the value for PBS controls. There was no significant difference in the number of ovulated oocytes between animals treated with MCAF and PBS controls. Also, porcine relaxin, given s.c. immediately after MCAR treatment, could reverse the inhibitory effect of MCAR on ovulation. To examine a possible mechanism for the effect of relaxin on ovulation, granulosa cells and theca-interstitial cells were obtained from ovaries of eCG-primed immature rats. The gelatinases secreted from cultured cells were analyzed using gelatin zymography and scanning densitometry. In the granulosa cell culture, relaxin increased the secretion of two major gelatinases of about 92 and 63 kDa in a dose-and time-dependent manner within 24 h of treatment. In the theca-interstitial cell culture, relaxin induced dose- and time-dependent increases in the secretion of two other major gelatinases of about 76 and 71 kDa. These gelatinases were characterized as metalloproteinases but not serine/cysteine proteinases. Furthermore, an immunoblot study demonstrated that relaxin stimulated the secretion of a 72-kDa type IV collagenase-like substance from cultured theca-interstitial cells but not from granulosa cells. This study demonstrates several original findings. First, endogenous relaxin may facilitate the ovulatory process in rats. Second, exogenous relaxin exhibits a biological effect on cultured rat theca-interstitial cells in addition to granulosa cells. Third, exogenous relaxin regulates the secretion of different major forms of gelantinases from cultured rat granulosa cells and theca-interstitial cells. The study supports the idea that relaxin may play an autocrine/paracrine role that is involved in modulating ovarian function.

Downregulation of progesterone biosynthesis in rat granulosa cells by adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) bran extracts

“…The preparation of granulosa cells was modified from the method described elsewhere. 20,21 The immature female rats (22-25 days of age) were injected subcutaneously with PMSG (15 IU/rat). After 48 h, rats were killed by cervical dislocation.…”

Pituitary adenylate cyclase-activating polypeptide acts synergistically with relaxin in modulating ovarian cell function in rats

“…Cell culture and treatment Isolation of ovarian granulosa cells and theca-interstitial cells from eCG-treated immature rats was performed as previously described (Too et al 1984, Liu & Hsueh 1986 with slight modifications (Hwang et al 1996). Ovarian cells were plated in 24-well plates (Falcon Labware, Lincoln Park, NJ, USA) at approximately 5 10 5 viable cells per well in 500 µl DMEM/F-12 medium containing 10% fetal bovine serum, 2 µg/ml bovine insulin, 100 U/ml penicillin and 100 µg/ml streptomycin.…”

“…In granulosa cell cultures, relaxin (5 µg in 10 µl) was given three times a day at 4-h intervals as previously described (Hwang et al 1996). The final concentration of relaxin was expressed as 10 µg/ml.…”

“…Human relaxin can induce ovulation in the in vitro perfused rat ovary (Brannstrom & MacLennan 1993). Furthermore, administration of monoclonal antibody specific for rat relaxin partially suppressed gonadotropininduced ovulation in rats, and this was reversed by concomitant treatment with porcine relaxin (Hwang et al 1996). Limited studies suggest that this effect of relaxin may be mediated through regulating the remodeling of extracellular matrix.…”

“…Relaxin increases the secreted activity of plasminogen activator, collagenase (MMP1) and proteoglycanase in rat ovarian granulosa cells (Too et al 1982(Too et al , 1984. In addition, relaxin increases the secretion of different major gelatinases from rat granulosa cells and theca-interstitial cells including a type IV collagenase (MMP2) (Hwang et al 1996).…”