Temporal and spatial expression of MMP-2,-9,-14 and their inhibitors TIMP-1,-2,-3 in the corpus luteum of the cycling rhesus monkey

A CL develops by extensive cellular reorganization and neovascularization of the remnants of the evacu-ated follicle following ovulation. In both rodent and primate, the development of CL is a rapid process with very high cellular turnover[1,2]. A CL is u…     

Expression of gelatinases and their tissue inhibitors in rat corpus luteum during pregnancy and postpartum

Extensive tissue remodeling occurs in the corpus luteum (CL) during both formation and luteolysis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play pivotal roles in these processes. In the present study, to evaluate the potential roles of matrix degrading proteases in luteal development and regression, we examined gelatinases and TIMP-1, -2, -3 mRNA expressions, as well as gelatinase activity in rat CL during pregnancy and postpartum using Northern blot, in situ hybridization, and gelatin zymography, respectively. The results showed that MMP-2 mRNA was only expressed at the early stages of pregnancy; TIMP-2 mRNA was highly expressed at the early and late pregnancy and day 1 postpartum, but could not be detected during the mid-phase of pregnancy; TIMP-3 mRNA expression was abundant during early pregnancy and peaked at day 7, but was absent from other time points examined. MMP-9 and TIMP-1 mRNAs in rat CL were below detectable level in the current study. Furthermore, the active MMP-2 was only present during the early stages of pregnancy, and no MMP-9 activity was observed in the zymogram. Taken together, our results suggest that MMP-2 and TIMP-3 may have functional roles in rat luteal formation, while TIMP-2 may be implicated in both formation and regression of the pregnant CL.     

Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.     

Gonadotropin and steroid regulation of matrix metalloproteinases and their endogenous tissue inhibitors in the developed corpus luteum of the rhesus monkey during the menstrual cycle

The factors regulating the dynamic expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the primate corpus luteum (CL) during the menstrual cycle are unknown. We hypothesized that LH or progesterone (P) regulate interstitial-collagenase (MMP-1), the gelatinases (MMP-2 and -9), TIMP-1, and TIMP-2 in the CL. Hormone ablation/replacement was performed in rhesus monkeys on Days 9-11 of the luteal phase in five treatment groups (n = 4/group): control (no treatment), antide (GnRH antagonist), antide + LH; antide + LH + trilostane (TRL; 3beta-hydroxysteroid dehydrogenase inhibitor), and antide + LH + TRL + R5020 (nonmetabolizable progestin). On Day 12, the CL was removed and the RNA and protein isolated for real-time polymerase chain reaction and immunoassays, respectively. The MMP-1 mRNA increased 20-fold with antide, whereas LH replacement maintained MMP-1 mRNA at control levels. Likewise, TRL increased MMP-1 mRNA 54-fold, and R5020 prevented this effect. Immunodetectable MMP-1 protein also increased with antide or TRL; these increases were abated with LH or R5020. Gelatinase mRNA and/or protein levels increased with antide (e.g., 3-fold, MMP-2 mRNA), and LH replacement reduced protein levels (e.g., 11-fold, MMP-2). The TRL increased MMP-9, but not MMP-2, expression; however, R5020 replacement had no effect on mRNA or protein levels. The LH treatment increased TIMP-1 and -2 mRNA and TIMP-1 protein expression compared to controls and antide groups, whereas R5020 enhanced only immunodetectable TIMP-1. These data strongly suggest that LH suppresses MMP-1 in the primate CL via P and that it also suppresses gelatinases, either at the mRNA (MMP-2) or protein (MMP-2 and -9) levels, perhaps in part via steroids, including P. In contrast, LH promotes TIMP expression, perhaps via steroids, including P.     

Expression and regulation of tumor necrosis factor (TNF) and TNF-receptor family members in the macaque corpus luteum during the menstrual cycle

Members of the tumor necrosis factor (TNF)-receptor (R) family may be involved in the tissue remodeling that occurs in the primate corpus luteum (CL) during development and regression. As a first step towards addressing this issue, studies assessed TNF ligand-R expression and regulation in CL collected from monkeys during the early (ECL, Days 3-5), mid (MCL, Days 7-8), mid-late (MLCL, Days 10-11), late (LCL, Days 14-16), and very late (VLCL, menses) luteal phase of the menstrual cycle. CL were also collected after gonadotropin and/or steroid ablation and replacement (with hLH and the progestin R5020) for 3 days at mid-late luteal phase. TNF-alpha, -beta, FAS ligand (FASL), and TNF-R1 mRNA levels were two- to sixfold greater (P < 0.05) at the MLCL or LCL phase as compared to earlier (ECL, MCL). In contrast, TNF-R2 and FAS mRNA levels did not change during the luteal phase. Immunohistochemical staining for TNF-beta, TNF-R1, TNF-R2, FAS, and FASL was observed in luteal cells, whereas only TNF-beta staining was observed in endothelial cells. Several TNF-R components were influenced by LH and/or steroid ablation; notably, steroid ablation reduced (P < 0.05) luteal TNF-alpha, but not TNF-beta, mRNA levels, which was prevented by progestin treatment. In contrast, steroid ablation increased (P < 0.05) luteal cell immunostaining for FAS and FASL, which was reduced by progestin treatment. Thus, several members of the TNF R-ligand family are expressed in the primate CL in an LH- and/or progestin-dependent manner. Peak expression in the late luteal phase may signify a role for the TNF-R system in death receptor-mediated apoptosis during luteolysis.     

Cellular localization of gelatinases and tissue inhibitors of metalloproteinases during follicular growth, ovulation, and early luteal formation in the rat

The matrix metalloproteinase (MMP) system consists of a proteolytic component, the metalloproteinases, and an associated class of tissue inhibitors of metalloproteinases (TIMPs). We investigated the cellular localization of the TIMPs and the gelatinase family of MMPs throughout the latter stages of follicular growth and during the periovulatory period. Immature female rats were injected with eCG, and ovaries were collected at the time of eCG administration (0 h) and at 6, 12, 24, or 36 h after eCG injection (i.e., follicular development group). A second group of animals (periovulatory) was injected with eCG followed by hCG 48 h later, and ovaries were collected at 0, 12, and 24 h after hCG. Ovaries were processed for the cellular localization of gelatinase or TIMP mRNA or gelatinolytic activity. Gelatinase mRNA (MMP-2 and MMP-9) was localized to the theca of developing follicles and to the stroma. Following a hCG stimulus, MMP-2 mRNA increased as the granulosa cells of preovulatory follicles underwent luteinization during formation of the corpus luteum (CL). MMP-9 mRNA remained predominantly in the theca during this period. In situ zymography for gelatinolytic activity demonstrated a pattern of activity that corresponded with the localization of MMP-2 and MMP-9 mRNA around developing follicles. Gelatinolytic activity was observed at the apex of preovulatory follicles and throughout the forming CL. The mRNA for TIMP-1, -2, and -3 was localized to the stroma and theca of developing follicles. TIMP-3 mRNA was present in the granulosa cells of certain follicles but was absent in granulosa cells of adjacent follicles. At 12 h after hCG, luteinizing granulosa cells expressed TIMP-1 and TIMP-3 mRNA, but TIMP-2 mRNA was at levels equivalent to the background. In the newly forming CL at 24 h after hCG administration, the luteal cells expressed TIMP-1, -2, and -3 mRNA, although the pattern of cellular expression was unique for each of the TIMPs. These findings demonstrate that the MMPs and TIMPs are in the cellular compartments appropriate for impacting the remodeling of the extracellular matrix as the follicle grows, ovulates, and forms the CL.     

Expression of apoptosis-related genes Fas/FasL, Bax/Bcl-2 and Caspase-3 in rat corpus luteum during luteal regression

Corpus luteum (CL) is a transient endocrine organ formed from the ovulated follicle. CL produces progesterone and estrogen that are important in preparing the uterine environment for implantation and maintaining gestation. Pregnancy maintains the CL function; otherwise, CL re-gresses rapidly. Cycling formation and regression of CL is essential for initiation of new follicular growth and differentiation, and subsequently ovulation and luteinization[1]. Luteal regression could be divided int…     

Expression of apoptosis-related genes Fas/FasL,Bax/Bcl-2 and Caspase-3 in rat corpus luteum during luteal regression

Using immunohistochemistry, in situ hybridization, Western blot and TUNEL methods, we have studied the expression of Fas/FasL, Bcl-2/Bax and caspase-3 in the corpora lutea (CL) at various stages of pseudopregnant rat induced by injection of PMSF/hCG. The results showed that no apoptotic signal could be observed until Day 14 after hCG injection. Fas weakly expressed in the CL at all the stages increased when luteolysis took place. FasL signal increased dramatically on Day 14 and reached the maximum level on Day 21. The expression of Bcl-2 and Bax was detected in a time-dependent manner. At the early stage of CL development, Bcl-2 expression was stronger, while Bax was low. The expression of Bcl-2 and Bax in the CL was completely reversed. Caspase-3 antigen could be detected throughout all the phases of CL development in a time-dependent fashion, low on Day 2 and reaching the maximum on Day 21. These results suggest that luteal regression at the late phases may be related to cell apoptosis.     

Expression of mRNA encoding insulin-like growth factor binding protein-2 (IGFBP-2) during induced and natural regression of equine corpora lutea

The insulin-like growth factors, IGF-I and -II, have been shown to play a key role in luteal function in some species. The IGF binding proteins, IGFBP-2 and -3, have been shown to inhibit binding of IGF-I and -II to bovine luteal cells and decrease progesterone production. We have recently shown that equine follicles have the genetic capacity to produce IGFBP-2, and that levels decrease in healthy preovulatory follicles. In the present study expression of mRNAs encoding IGFBP-2, as well as the rate-limiting steroidogenic enzyme, P450scc, were studied in equine corpora lutea to investigate whether IGFBP-2 might be involved in luteolysis. Corpora lutea were collected from mares in mid-luteal phase (day 10), at early regression (day 14), late regression (day 17), and 12 and 36 h after intramuscular administration of the PGF(2alpha) analogue, cloprostenol (0.5 microg/kg). During early natural regression, and 12 h after administration of cloprostenol on day 10, steady state levels of mRNAs encoding P450scc had decreased significantly compared with day 10 of dioestrus (P < 0.001). Levels of mRNA encoding IGFBP-2 increased significantly between mid-diestrus and early (P < 0.01) and late (P < 0.001) regression, and 36 h after cloprostenol administration (P < 0.001). We conclude that the genetic capacity for increased IGFBP-2 production in the early stages of natural luteolysis in the mare may act to sequester IGF-I in the CL, assisting in inhibition of progesterone production. However the delay in increase in mRNA encoding IGFBP-2 after cloprostenol administration, combined with the sharp fall in expression of P450scc mRNA, suggests that the luteolytic action of a pharmacological dose of cloprostenol may not be mediated via IGFBP-2 in the mare.     

Expression of mRNA and proteins for GnRH I and II and their receptors in primate corpus luteum during menstrual cycle

The differential expression of mRNA and protein of GnRH I, II and their receptors (RI and RII) in the monkey corpus luteum (CL) were measured during different stages of the luteal phase of the menstrual cycle as an initial step towards considering the role and regulation of GnRH (I and II) system during luteinization and luteolysis in primates. RT-PCR confirmed the sequence identity of PCR products and real time PCR quantified specific mRNA expressions. Proteins were localized by immunohistochemistry (IHC). Changes in mRNA expression patterns of GnRH I and II (increased) and GnRH RII (decreased) were maximal at mid-late to late stages, that is, at CL regression, where as GnRH RI was low during the entire luteal phase. However, RT-PCR and IHC studies confirmed the presence of GnRH RI at both mRNA and protein levels, respectively. IHC results showed the presence of GnRH I, II and their receptors in steroidogenic cells (granulose-luteal cells and thecal-luteal cells) across the luteal phase. Hence, GnRH I and II systems may have a role on both luteinization (from early to mid stages of CL) and luteolysis (from mid-late to very-late stages of CL). These novel findings suggest that monkey luteal GnRH system may have a role in fertility regulation in paracrine and/or autocrine manner.     

Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.     

Effect of the preovulatory gonadotropin surge on matrix metalloproteinase (MMP)-14, MMP-2, and tissue inhibitor of metalloproteinases-2 expression within bovine periovulatory follicular and luteal tissue

The matrix metalloproteinases (MMPs) have been implicated in the ovulatory process, but the specific roles of individual MMPs are unclear. This study examined the effect of the preovulatory gonadotropin surge on localization and regulation of MMP-2, MMP-14, and tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA and MMP-2 and TIMP-2 activity in bovine preovulatory follicles and new corpora lutea (CL). Ovaries containing ovulatory follicles or new CL were collected at approximately 0, 6, 12, 18, 24, and 48 h (CL) after a GnRH-induced gonadotropin surge. Messenger RNA for TIMP-2 and MMP-14 increased within 6 and 24 h of the gonadotropin surge, respectively, whereas MMP-2 mRNA was constitutively expressed. Activity for MMP-2 in follicular fluid and follicle homogenates was not changed, but follicular fluid TIMP-2 activity increased in response to the gonadotropin surge. Messenger RNA for MMP-2 was localized to the thecal layer of bovine preovulatory follicles, whereas MMP-14 mRNA was localized primarily to the thecal layer and adjacent ovarian stroma. Expression of MMP-14 was also observed in the granulosal layer after the gonadotropin surge. In contrast, TIMP-2 mRNA was localized predominantly to the granulosal layer with intense expression in the antral portion of the granulosal layer in response to the gonadotropin surge. These data support the hypothesis that increased expression of MMP-14 and TIMP-2 may help regulate follicle rupture and/or the ovulatory follicle-CL transition in cattle.     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.     

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.