Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

Abstract

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Expression and regulative function of tissue inhibitor of metalloproteinase 3 in the goat ovary and its role in cultured granulosa cells

“…In the human and rat ovary, the high dose of TIMP3 has been found to promote apoptosis of ovarian granulosa cells (Li and Curry, 2009;Rosewell et al, 2013). Our results are consistent with involvement of TIMP3 activation in granulosa cell apoptosis in human and rat.…”

“…Additionally, the presence of TIMP3 has been reported in ovaries from mouse (Inderdeo et al, 1996), rat (Curry and Wheeler, 2002), pig (Shores and Hunter, 2000) and human (Rosewell et al, 2013). Although we found that the expression level of TIMP3 in goat ovary is lower, its function on ovary is significant.…”

“…Earlier studies have demonstrated that hCG can induce the expression of TIMP3 mRNA in the ovary (Curry and Wheeler, 2002;Li and Curry, 2009;Rosewell et al, 2013). Li and Curry (2009) reported a biphasic increase of TIMP3 expression at 2 and 16 h after hCG treatment, and TIMP3 protein level increased at 8 h after hCG treatment and remained high through 24 h of hCG treatment.…”

“…TIMP3-induced apoptosis has been shown to be involved in stabilization of cell surface death receptors (tumor necrosis factor-α receptor) and activation of caspase-8 in a variety of cancer cell lines (Ahonen et al, 2003;Bond et al, 2002;Smith et al, 1997). Recently, it was demonstrated that recombinant TIMP3 could increase the apoptosis of human granulosa lutein cells as well as cultured rat granulosa cells (Li and Curry, 2009;Rosewell et al, 2013). In addition, the downregulation of TIMP3 inhibited progesterone secretion from rat granulosa cells (Li and Curry, 2009).…”

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

“…Follicles were fixed in 4% formaldehyde, embedded in paraffin, sectioned (7 m), and processed for immunostaining as previously described (16). The sections were incubated in a humidified chamber at 4°C overnight with CCL20 primary antibodies (AF360; R&D Systems) as shown in Table 2 at a concentration of 10 g/mL.…”