Dynamic Expression of Mrnas and Proteins for Matrix Metalloproteinases and Their Tissue Inhibitors in the Primate Corpus Luteum During the Menstrual Cycle

Molecular Human Reproduction Vol.8, No.9 pp. 833–840, 2002

Dynamic expression of mRNAs and proteins for matrix metalloproteinases and their tissue inhibitors in the primate corpus luteum during the menstrual cycle

K.A.Young1,3, J.D.Hennebold1 and R.L.Stouffer1,2

1Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Ave, Beaverton, Oregon 97006 and 2Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97201, USA 3To whom correspondence should be addressed. E-mail: [email protected]

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) may be involved in tissue remodelling in the primate corpus luteum (CL). MMP/TIMP mRNA and protein patterns were examined using real-time PCR and immunohistochemistry in the early, mid-, mid-late, late and very late CL of rhesus monkeys. MMP-1 (interstitial collagenase) mRNA expression peaked (by >7-fold) in the early CL. MMP-9 (gelatinase B) mRNA expression was low in the early CL, but increased 41-fold by the very late stage. MMP-2 (gelatinase A) mRNA expression tended to increase in late CL. TIMP-1 mRNA was highly expressed in the CL, until declining 21-fold by the very late stage. TIMP-2 mRNA expression was high through the mid-luteal phase. MMP-1 protein was detected by immunocytochemistry in early steroidogenic cells. MMP-2 protein was prominent in late, but not early CL microvasculature. MMP-9 protein was noted in early CL and labelling increased in later stage steroidogenic cells. TIMP-1 and -2 proteins were detected in steroidogenic cells at all stages. Thus, MMPs and TIMPs are dynamically expressed in a cell-specific manner in the primate CL. Early expression of MMP-1 is suggestive of a role in tissue remodelling associated with luteinization, whereas MMP-2 and -9 may contribute to later stage luteolysis. TIMP expression may control MMP activity, until declining at luteolysis.

Key words: corpus luteum/MMP/ovary/primate/TIMP

Introduction as latent zymogens and can be locally inhibited in both latent and Following ovulation, the corpus luteum (CL) forms from the wall of active forms by their specific tissue inhibitors (TIMPs). the ruptured follicle and functions, through secretion of progesterone, MMPs have been implicated in the substantial ECM turnover and to permit both the initiation and maintenance of intrauterine tissue remodelling associated with the structural and functional pregnancy. Substantial tissue remodelling is required for the cellular changes occurring in the ovary throughout the reproductive cycle et al. changes characterizing both luteinization (or CL formation) and (Smith , 1999). Many members of the MMP family are differentially expressed in ovarian tissue, and recent experiments in luteolysis (or CL regression) during the ovarian cycle (Knobil, 1973; this laboratory and others have elucidated discrete expression patterns Stouffer and Brannian, 1993). The molecular mechanisms that regulate of MMP family members regulated by gonadotrophins and steroids the extensive tissue remodelling occurring in the primate CL during in the peri-ovulatory follicle (Nothnick et al., 1996; Chaffin and the menstrual cycle are, for the most part, unknown. Stouffer, 1999). However, while expression patterns of certain MMP Matrix metalloproteinases (MMPs) are a family of structurally and tissue inhibitor of metalloproteinase (TIMP) mRNAs and proteins related Znϩ- dependent endopeptidases that cleave both extracellular have been characterized in the CL of various species (Endo et al., matrix (ECM) components and non-matrix proteins. MMPs can 1993; Tsang et al., 1995; Nothnick et al., 1996; Smith et al., 1996; Ͼ proteolytically activate other MMP enzymes, and together the 25 Duncan, 2000; Pitzel et al., 2000), very little is known about their members of the MMP family can cleave all constituents of the ECM. regulation in the ovary (Fata et al., 2000). Through their protease activity, MMPs are essential for regulating As a first step towards characterizing the role and regulation of the interactions between cells and the ECM (Salamonsen, 1996). In MMPs in luteal formation and regression in primates, we sought addition to mediating general remodelling in a variety of tissues, to establish a time course for simultaneous expression and cell MMPs also regulate the release of signals from matrix-embedded localization of interstitial collagenase (MMP-1), gelatinase A proteins that can influence cell proliferation, differentiation and death (MMP-2), gelatinase B (MMP-9) and TIMP-1 and -2 in the CL of (Massova et al., 1998). Activity of the MMPs is regulated on multiple the non-human primate. CL were removed from rhesus macaques at levels, including transcriptional and translational regulation, by specific stages of the luteal phase of spontaneous menstrual cycles. cytokines, hormones, chemokines, oncogenes and other cell–matrix This protocol allows a detailed investigation of MMP/TIMP mRNA interactions (Hulboy et al., 1997). In addition, MMPs are produced and protein expression throughout the natural luteal lifespan, including

© European Society of Human Reproduction and Embryology 833 K.A.Young, J.D.Hennebold and R.L.Stouffer

Table I. Sets of TaqMan Probes and forward (FOR) and reverse (REV) primers designed for real-time amplification of luteal MMP and TIMP mRNAs

Gene Sequence type Sequence Tm (°C)

MMP-1 FOR primer 5Ј-CACGCCAGATTTGCCAAGA 59 REV primer 5Ј-CTTGGTGAATGTCAGAGGTGTGA 59 TaqMan probe 5Ј-GGCTTTCTCAATGGCCTGGTCCACA 70 MMP-2 FOR primer 5Ј-CCTCCCTGTGCCCAAGAAT 59 REV primer 5Ј-GAGGGTTGGTGGGATTGGA 59 TaqMan probe 5Ј-CTGATTGTATTCCTCCCAGGTGCCCC 69 MMP-9 FOR primer 5Ј-CACCTTCACTCGCGTCGAA 70 REV primer 5Ј-CCGTCCTTCCCGTCGAA 59 TaqMan probe 5Ј-TCCGTGCTCCGCGACACCAA 59 TIMP-1 FOR primer 5Ј-TAAACAGGGAAACACTGTGCATTC 59 REV primer 5Ј-GTTTCGTGGCTCCCTGGAA 60 TaqMan probe 5Ј-TGAACGGGGGGCGCTGAGCTA 70 TIMP-2 FOR primer 5Ј-CAGGGCACGATGAAGTCACA 59 REV primer 5Ј-GCTGGACGTTGGAGGAAAGA 59 TaqMan probe 5Ј-CCCCCTCGGCCTTTCCTGCAAT 70

Tm ϭ melting temperature.

CL formation (early luteal phase; ECL), peak CL function (mid- and probe sets for the real-time assay (Primer Express software; Perkin-Elmer phase; MCL) and CL on the verge of (mid-late luteal phase; MLCL), Applied Biosystems, Foster City, CA, USA). Perkin-Elmer parameters were or undergoing, functional and structural regression (late luteal phase; adhered to during probe design: sequences with clusters of identical nucleotides LCL and very late luteal phase, menstruation; VLCL). were avoided to prevent non-specific interactions, selected probes were Ͻ27mer, contained less than three Gs or Cs at the 5Ј end, and had a melting temperature at least 10°C higher than both forward and reverse primers to Materials and methods ensure sufficient hybridization stability of probes during primer extension (Table I). Oligonucleotide primer sequences were synthesized by Gibco and Animals TaqMan probes were synthesized by Perkin-Elmer. Probes were labelled The general care and housing of rhesus monkeys (Macaca mulatta) at the with the 5Ј reporter dye 6-carboxyfluorescein and the 3Ј quencher dye 6- Oregon National Primate Research Center (ONPRC) has been described carboxytetramethylrhodamine. A matrix of varying primer concentrations was previously (Wolf et al., 1990). Menstrual cycles of adult female rhesus employed to determine optimal concentrations of assay components. monkeys were monitored daily. Six days after the onset of menses, daily MMP and TIMP mRNA expression were analysed using TaqMan PCR blood samples were collected by saphenous venipuncture. Serum was separated Core Reagent Kit with the ABI PRISM 7700 Sequence Detection System (PE and stored at –20°C until assayed for hormone concentrations. The first day Applied Biosystems). To control for the amount of total RNA added to each of low serum estradiol following the mid-cycle estradiol peak has been RT reaction and to normalize the target signal, 18S mRNA was used as an demonstrated to correspond with the day after the LH surge, and was therefore active endogenous control in each well. Amplifications were conducted in a termed day 1 of the luteal phase (Duffy et al., 1999). All protocols were 10 µl final volume containing: 250 nmol/l TaqMan MMP/TIMP probe, approved by the ONPRC Animal Care and Use Committee, and conducted in 300–900 nmol/l MMP/TIMP forward and reverse primers (Table I), 250 nmol/ accordance with NIH Guidelines for the Care and Use of Laboratory Animals. l TaqMan 18S probe (labelled with the 5Ј reporter dye VIC), 80 nmol/l forward and reverse 18S primers, 20 ng (MMPs) or 8 ng (TIMPs) cDNA, Hormone assays and 5 µl TaqMan Universal PCR master mix containing ROX dye as a passive Plasma concentrations of estradiol and progesterone were determined by reference (PE Biosystems). The PCR reactions were conducted in sealed 96- specific electrochemoluminescent assay using a Roche Elecsys 2010 Analyzer well optical plates with thermal cycler conditions of: 2 min at 50°C, 10 min by the Endocrine Services Laboratory, ORPRC. Hormone concentration values at 95°C and 40 cycles of 15 s at 95°C (DNA melting) and 1 min at 60°C were validated against previous radioimmunoassays in this laboratory (Hess (primer annealing/extension). If the cDNA sample contained the target et al., 1981; Zelinski-Wooten et al., 1995). sequence, the TaqMan probe was cleaved with every PCR amplification cycle, resulting in increased fluorescent emission of the reporter dye. During the Tissues amplification cycles, the ABI Prism sequence detector monitored real-time PCR amplification by quantitatively analysing changes in fluorescence emissions in CL (n ϭ 3–4/stage) were isolated and dissected from anaesthetized monkeys each well. The number of amplification cycles for the fluorescence to reach during aseptic ventral mid-line laparotomy surgery as described previously a determined threshold level (C ) was recorded for every unknown and an (Duffy et al., 2000). CL were collected during the early (luteal days 3–5), T mid- (days 6–8), mid-late (days 10–12), late (days 14–16) and very late (days internal standard curve. The internal standard curve, used for relative mRNA quantification, was generated from five 10-fold dilutions of pooled early CL 17–19; menses) luteal phase. A portion of the CL was frozen in liquid nitrogen and stored at –80°C for isolation of total RNA using Trizol (BRL, Gaithersburg, samples. CT values for unknown samples were used to extrapolate the amount of RNA equivalents from the internal standard curve. The RNA equivalent MD, USA) according to BRL standard protocol. A second portion was immersion-fixed for immunohistochemical analysis of protein expression. values were then divided by complimentary 18S RNA equivalent values also derived from the same internal standard curve. Real-time PCR analysis of MMP and TIMP mRNAs RT was carried out on 1 µg DNase (Gibco)-treated RNA using Molony Immunohistochemistry Murine Leukemia Virus reverse transcriptase (Gibco) for 2 h at 37°Cas Portions of the CL were fixed in 10% neutral buffered formalin (Richard- described previously (Chaffin and Stouffer, 1999). Allen Scientific, Kalamazoo, MI, USA) for 1 week. Tissue was then dehydrated For each primer set analysed, PCR was performed on pooled luteal cDNA in a series of ethanol solutions (50, 70 and 100%) and paraffin-embedded. generated from the RT reaction, using primers previously optimized in this For MMP/TIMP immunohistochemistry, 6 µm sections were deparaffinized laboratory for other experiments (Chaffin and Stouffer, 1999). Sequence and hydrated through xylenes and a graded series of ethanol, as reported analysis was performed on the resulting PCR products to obtain the rhesus previously (Hazzard et al., 2000). Sections were incubated in phosphate- macaque sequence. This sequence was then used to design TaqMan primer buffered saline (PBS) prior to pressure cooker-antigen retrieval in citrate buffer 834 MMP expression in primate corpus luteum

Figure 1. Mean (SEM) corpus luteum (CL) mass (left panel) and serum progesterone concentrations (right panel) in adult rhesus macaques during the early (ECL, day 3–5), mid- (MCL, day 7–8), mid-late (MLCL, day 10–12), late (LCL, day 14–16) and very late (VLCL, menstruation day 17–19) luteal phase. Values were obtained on day of lutectomy. Left panel, *significant difference (P Ͻ 0.05) between mid- or mid-late masses and those from all other groups. Right panel, *significant difference (P Ͻ 0.05) in progesterone concentrations at mid-CL as compared with early, mid-late, late and very late CL. H depicts a difference in progesterone concentrations from samples taken during the mid-late luteal phase and those taken during the very late luteal phase.

(Citra; BioGenex Laboratories, Inc., San Ramon, CA, USA). Endogenous peroxidases were then quenched with a 10 min incubation in 3% H2O2. Sections were placed in a blocking buffer [1.5% normal horse serum (NHS) in PBS], then incubated with primary antibody in NHS PBS buffer for 1 h at room temperature and overnight at 4°C. Concentrations for MMP-1 (M35L Ab-1; Oncogene Cambridge, MA, USA), MMP-2 (MS 806; Neomarkers, Freemont, CA, USA), and MMP-9 (IM37; Calbiochem, San Diego, CA, USA) antibodies were 1:100. TIMP-1 (MS 608; Neomarkers) and TIMP-2 (IM82; Calbiochem) antibodies were diluted to 1:200 and 1:75 respectively. Primary antibody was detected using a biotinylated anti-rabbit IgG secondary antibody (1:1000; Vector Laboratories, Burlingame, CA, USA) and the Vector ABC- Elite Kit, visualized with Nickel-enhanced Sigma Fast diaminobenzidine substrate (Sigma, St Louis, MO, USA) and counter-stained with haematoxylin. Figure 2. Mean (SEM) interstitial collagenase (MMP-1) mRNA levels in For each MMP or TIMP examined, negative controls lacking primary antibody the macaque CL during the early (ECL, day 3–5), mid- (MCL, day 7–8), were processed on adjacent tissue sections. mid-late (MLCL, day 10–12), late (LCL, day 14–16) and very late (VLCL, menstruation day 17–19) luteal phase. Values were standardized to Statistical analysis respective 18S control values. *Significant difference (P Ͻ 0.05) between Statistical evaluation of mean differences among experimental groups was early luteal phase and all other groups. performed by analysis of variance with the significance level set at 0.05 using the StatView software package (SAS Institute Inc., Cary, NC, USA). To isolate the cycle (P Ͻ 0.05; Figure 2). MMP-1 mRNA levels declined Ͼ7- significant differences between groups, the Student–Newman–Keuls method fold from early to mid-luteal phase, and remained low in mid-late, was used for pairwise multiple comparisons. late and very late luteal tissue. MMP-2, MMP-9 (gelatinases) Results No significant changes in gelatinase A (MMP-2) mRNA levels were noted during the luteal phase (Figure 3, left panel); however, Hormone assays and CL mass expression tended to be 2-fold higher (P ϭ 0.07) at the late luteal Both CL mass and serum concentrations of progesterone followed phase as compared with earlier stages of the cycle. The expression patterns typically observed in adult macaques during the luteal phase of gelatinase B (MMP-9) (Figure 3, right panel) also peaked near the of the menstrual cycle. Mean CL mass increased significantly from end of the luteal phase. However, MMP-9 mRNA levels peaked in Ͻ the early stage to peak at the mid-luteal phase (Figure 1, left panel; the very late stage (P 0.05), increasing ~41-fold in the VLCL as P Ͻ 0.05); the significant increase was maintained through the mid- compared with all other stages. late stage until declining in the late and very late CL. Concomitant TIMPs with the increase then decrease in CL size was the related increase TIMP-1 mRNA was highly expressed in the CL (Figure 4, left panel) then decline in luteal function. Progesterone concentrations increased through to the late luteal phase without significant changes in significantly from the early stage, peaking at the mid-stage (Figure expression levels. However, TIMP-1 expression declined by the very 1, right panel; P Ͻ 0.05). Serum progesterone remained elevated late luteal phase as compared with all other stages (P Ͻ 0.01), through the mid-late stage, but then declined at the late and very late decreasing ~21-fold during luteal regression. Like TIMP-1, TIMP-2 stages of the CL. mRNA levels tended (P ϭ 0.09) to be highest (Figure 4, right panel) in the mid-late luteal phase. However, unlike TIMP-1, expression of Real-time PCR analyses of MMP and TIMP RNAs TIMP-2 did not vary significantly across the luteal stages. MMP-1 (collagenase-1) Immunohistochemistry Whereas 18S mRNA expression remained constant (P Ͼ 0.05; data not shown), individual MMP and TIMP mRNA levels in CL varied MMP-1 (collagenase-1) significantly between stages of the luteal phase. Interstitial collagenase Intense staining for MMP-1 protein was detected in the cytoplasm of (MMP-1) mRNA levels peaked in the early luteal phase; ECL MMP-1 granulosa luteal and theca luteal cells in the developing CL (Figure expression values were higher as compared with all other stages of 5A). In contrast, endothelial cells and the surrounding stroma were 835 K.A.Young, J.D.Hennebold and R.L.Stouffer

Figure 3. Mean (SEM) gelatinase A (MMP-2; left panel) and gelatinase B (MMP-9; right panel) mRNA expression in the macaque CL during the early (ECL, day 3–5), mid- (MCL, day 7–8), mid-late (MLCL, day 10–12), late (LCL, day 14–16) and very late (VLCL, menstruation day 17–19) luteal phase. Values were standardized to respective 18S control values. *Significant difference (P Ͻ 0.05) in MMP-9 mRNA expression between the very late luteal phase and all other stages.

Figure 4. Mean (SEM) TIMP-1 (left panel) and TIMP-2 (right panel) mRNA expression in macaque CL during the early (ECL, day 3–5), mid- (MCL, day 7–8), mid-late (MLCL, day 10–12), late (LCL, day 14–16) and very late (VLCL, menstruation day 17–19) luteal phase. Values were standardized to respective 18S control values. *Significant difference (P Ͻ 0.05) between TIMP-1 mRNA expression at the very late luteal phase compared with all other groups. devoid of staining (Figure 5A), as were adjacent control sections stages (Figure 6A). Staining was punctate and appeared to be processed without primary antibody (Figure 5A, inset). At later stages, perinuclear (Figure 6A, inset). TIMP-1 immunostaining appeared to MMP-1 staining in the luteal cells was greatly reduced; low to diminish in the very late CL (Figure 6B). No staining was noted in negligible staining was observed in the mid, mid-late, late and very theca luteal or endothelial cells in any section, nor did non-specific late luteal tissues (Figure 5B). immunolabelling occur in adjacent control sections processed without MMP-2, MMP-9 (gelatinases) primary antibody (not shown). Low to negligible gelatinase A (MMP-2) immunolabelling was noted Immunolabelling for TIMP-2 protein was low in early luteal tissue in tissues from early to mid-late luteal phase (Figure 5C). In contrast, (Figure 6C), but staining was more prominent in mid, mid-late and cells associated with the ovarian microvasculature (presumably endo- late CL tissue sections (Figure 6D). TIMP-2 was characterized by thelial cells, pericytes) were immunolabelled for MMP-2 in luteal tissue diffusely staining cytoplasm of granulosa luteal cells at all CL stages, at the late and very late luteal phase (Figure 5D). Granulosa luteal, and was not noted in endothelial cells (Figure 6D). Adjacent sections theca luteal and stromal cells did not positively label for processed without primary antibody did not label (Figure 6D, inset). MMP-2 antibody at any stage, nor was staining observed in control sections processed without primaryMMP-2 antibody (Figure 5D, inset). Unlike staining for MMP-2, gelatinase B (MMP-9) immuno- Discussion labelling was noted at all stages of the luteal phase. In tissues from The present study provides the first characterization of mRNA and the early to mid-luteal phase, MMP-9 protein was only expressed in protein expression for several MMPs and TIMPs in the non-human a subpopulation of cells that appeared to be endothelial cells or primate CL throughout its lifespan in the natural menstrual cycle. pericytes (Figure 5E and inset); this labelling became more prominent The dynamic and differential expression of these proteases and in the mid-late phase. By the late to very late phase, MMP-9 staining inhibitors in specific cell types is suggestive of multiple functions in was observed in the cytoplasm of both luteal cells and cells that the CL. appeared to be endothelial cells and/or pericytes (Figure 5F). No MMP-1 (interstitial collagenase) mRNA expression peaked and staining was noted in surrounding stromal cells (Figure 5F) or in protein immunolabelling was most prominent in the macaque CL adjacent control sections processed without primary antibody (Figure during the early (days 3–5) luteal phase of the cycle. Maximal MMP-1 5F, inset). expression in the developing CL is consistent with our earlier TIMPs observation of markedly elevated MMP-1 mRNA levels in granulosa TIMP-1 immunolabelling was noted in the cytoplasm of granulosa cells of the ovulatory, luteinizing follicle (day 1–2 of the luteal phase) luteal cells at every stage of the CL lifespan, though labelling was (Chaffin and Stouffer, 1999). After ovulation, the follicular tissue is more prominent in tissues from the early, mid, mid-late and late CL reorganized as granulosa cells hypertrophy, theca and granulosa cells 836 MMP expression in primate corpus luteum

Figure 5. Immunohistochemistry for MMP-1 (A,B), MMP-2 (C,D) and MMP-9 (E,F) in the macaque CL at specific stages of the menstrual cycle. (A) Tissue section from early CL demonstrating intense MMP-1 immunolabelling localized to the cytoplasm of granulosa-luteal (G) and theca luteal (T) cells, but not to cells in the surrounding stroma (S). Inset depicts adjacent control section processed without primary MMP-1 antibody. (B) Section from the mid-late CL, with negligible MMP-1 staining, also representative of results in the mid-, late and very late phases. (C) Section from the mid-late CL depicting scant MMP-2 staining typical of early, mid- and mid-late stages. (D) Section from the late CL, with MMP-2 immunolabelling localized to the cytoplasm of cells in the microvasculature, representative of tissue from the late and very late stages. Inset depicts adjacent control section processed without primary MMP-2 antibody. (E) MMP-9 protein expression in a subpopulation of microvasculature cells (presumably endothelial cells, pericytes) in the mid-CL, inset depicts higher magnification of positively-stained cells. (F) Intense cytoplasmic staining for MMP-9 in the granulosa luteal and putative endothelial cells and pericytes of the late CL. The MMP-9 labelling is typical of late and very late CL. Inset depicts adjacent control section processed without primary MMP-9 antibody. differentiate into luteal cells, and vasculature forms in the granulosa are sequestered in the ECM and necessary for luteal development. luteal compartment. The increased expression of MMP-1 suggests Since MMP-1 expression is low from the mid-luteal phase onward, that this collagenase is involved in the extensive and rapid tissue it is less likely that this collagenase is important in later events in reorganization and angiogenesis occurring in the early CL. Indeed, the luteal lifespan. MMP-1 has been implicated in facilitating the in-vitro migration of In contrast to MMP-1 expression, MMP-2 (gelatinase A) and microvascular endothelial cells, and is therefore associated with MMP-9 (gelatinase B) mRNA expression peaked and protein immuno- advancing angiogenesis (Partridge et al., 2000). In addition to ECM labelling was most prominent near the end of the luteal phase. breakdown, MMP-1 may promote the release of growth factors that Maximal gelatinase expression correlated positively with functional 837 K.A.Young, J.D.Hennebold and R.L.Stouffer

Figure 6. TIMP-1 (A,B), and TIMP-2 (C,D) immunolabelling in the macaque CL at specific stages of the corpus luteum. (A) Tissue section from the mid-late CL demonstrating TIMP-1 immunolabelling localized to the cytoplasm of granulosa luteal (G) cells, but not to thecal luteal cells or cells in the surrounding stroma (S). This TIMP-1 staining was typical of labelling in early, mid-, mid-late and late tissues. Inset depicts a section detail showing punctate and perinuclear labelling. (B) Section from the very late CL depicting reduced TIMP-1 labelling. (C) Section from the early CL depicting scant TIMP-2 immunolabelling. (D) Section from the late CL demonstrating positive TIMP-2 immunolabelling localized to the cytoplasm of granulosa luteal cells, but not in microvascular cells or surrounding stroma (S). Inset depicts adjacent cross section processed without primary TIMP-2 antibody. and structural regression of the CL (days 14–19 of the luteal phase; the late luteal phase suggest that the removal of inhibitor activity menses). Luteolysis is characterized by targeted regression (cell facilitates gelatinase action during luteolysis. In addition, a range of detachment and apoptosis) of steroidogenic cells and the vascular functions, independent of protease inhibition, have been attributed to network and supporting connective tissue, leading to tissue involution TIMPs, including TIMP-1 serving as an autocrine and paracrine (Modlich et al., 1996; Fraser et al., 1999). Increases in gelatinase survival factor in Hodgkin/Reed-Sternberg cells, and both TIMP-1 expression are associated with extensive tissue degradation common and -2 as cell growth factors in serum (Hayakawa et al., 1992, 1994; in such clinical cases as abdominal aortic aneurysms and muscle Oelmann et al., 2002). Therefore, TIMP expression in the ovary may denervation atrophy (Schoser and Blottner, 1999; Yamashita et al., be critical for a variety of functions other than protease control. 2001). Interestingly, mRNA expression for MMP-2 (gelatinase A) In addition to the divergent patterns of expression for individual peaked in cells associated with ovarian microvasculature just prior to MMPs/TIMPs, the cellular localization of each MMP and TIMP the peak in MMP-9 (gelatinase B) in steroidogenic and endothelial protein was distinct. For example, immunoreactive MMP-1 was cells. This consecutive cell-specific pattern may be important, and is prominent in the cytoplasm of granulosa luteal and theca luteal cells, suggestive of primary degeneration of ovarian microvasculature (and but not endothelial cells, of the developing CL. In contrast MMP-2 associated cells) followed by general degradation of the remaining was detected in the cells associated with the microvasculature luteal tissue in later luteolysis. Thus, increased gelatinase expression (presumably endothelial cells, pericytes), but not luteal cells, of the in the primate CL may contribute to the tissue degradation occurring regressing CL. The cell-specific patterns of localization suggest that during luteolysis at the end of the menstrual cycle. there are differences in the site(s) of cellular sources of synthesis The MMP inhibitors TIMP-1 and TIMP-2 were highly expressed and/or action of these proteins. As such, MMP-1 could be important in macaque CL; however, TIMP-1 was significantly reduced by the for ECM remodelling, allowing hypertrophy of luteal cells and end of the luteal phase. TIMP-1 binds to and inhibits both the latent development of microvessels in the luteinizing tissue, whereas MMP-2 and activated forms of MMP-1 and -9, whereas TIMP-2 locally may be required for vessel degeneration during luteal regression. inhibits both latent and active forms of MMP-2 and -9 (Salamonsen, However, since MMPs are synthesized as latent enzymes and their 1996). High expression levels during the early and mid-luteal stages site of synthesis may differ from the site(s) of action, further studies suggest that TIMPs may serve to control collagenase or gelatinase are required to determine if immunolocalization reflects MMP/TIMP activity in the developing/functional CL. However, the inverse patterns origins or activity. In general, expression patterns for MMP/TIMP of TIMP (declining) and MMP-2 and -9 (increasing) expression in mRNAs reflected the immunolabelling profiles for their respective 838 MMP expression in primate corpus luteum proteins. For instance, MMP-1 and TIMP-1 immunostaining closely (R.L.S.) through co-operative agreement (U54-HD18185) as part of the mirrored changes in mRNA levels during the luteal phase. However, Specialized Cooperative Centers Program in Reproductive Research, NCRR RR00163 (R.L.S.) and NICHD Training Grant (T32-HD-07133). MMP-9 staining of a cohort of putative microvascular cells in the early luteal phase, a time when mRNA expression was relatively low, illustrates the need for additional studies at the cellular level. The observed changes in MMP and TIMP expression in the References macaque CL during its lifespan in the ovary cycle are similar, yet Chaffin, C.L. and Stouffer R.L. (1999) Expression of matrix metalloproteinases distinct from, those observed in other species. Interestingly, our and their tissue inhibitor messenger ribonucleic acids in macaque periovulatory granulosa cells: time course and steroid regulation. Biol. observation of elevated MMP-1 levels during CL formation are Reprod., 61,14–21. consistent with reports from domestic animals and rodents (Goldberg Duffy, D.M., Abdelgadir, S.E., Stott, K.R., Resko, J.A., Stouffer, R.L. and et al., 1996; Nothnick et al., 1996), but contrast with the report that Zelinski-Wooten, M.B. (1999) Androgen receptor mRNA expression in the MMP-1 expression does not change throughout the CL lifespan in rhesus monkey ovary. Endocrine, 11,23–30. humans (Duncan et al., 1998). However, there is evidence for Duffy, D.M., Chaffin, C.L. and Stouffer, R.L. (2000) Expression of estrogen increased gelatinase (MMP-2 and -9) expression in the later stages receptor alpha and beta in the rhesus monkey corpus luteum during the menstrual cycle: regulation by luteinizing hormone and progesterone. of the luteal lifespan of women and pigs (Duncan et al., 1998; Pitzel Endocrinology, 141, 1711–1717. et al., 2000) as well as monkeys (current study), but not in cattle Duncan, W.C. (2000) The human corpus luteum: remodelling during luteolysis (Goldberg et al., 1996). Different TIMP expression patterns have also and maternal recognition of pregnancy. Rev. Reprod., 5,12–17. been reported, with sharp declines noted at luteolysis in pigs (Tanaka Duncan, W.C., McNeilly, A.S. and Illingworth, P.J. (1996) Expression of et al., 1992), Old World (current study) and New World (Duncan tissue inhibitor of metalloproteinases-1 in the human corpus luteum after et al., 1996) monkeys, but not in humans (Duncan et al., 1998). luteal rescue. J. Endocrinol., 148,59–67. Many factors may contribute to the observed differences, including Duncan, W.C., McNeilly, A.S. and Illingworth P.J. (1998) The effect of luteal rescue on the expression and localization of matrix metalloproteinases and species variation, differences in methods or accuracy in defining their tissue inhibitors in the human corpus luteum. J. Clin. Endocrinol. stages in the CL lifespan, or lack of testing of other MMPs or Metab., 83, 2470–2478. proteases that may serve to control luteal formation, function or Endo, T., Aden, R.F., Wang, F. and Barman, H.R. (1993) Coordinate induction regression. Indeed, the protease activity of the plasminogen activator and activation of metalloproteinase and ascorbate depletion in structural system has been implicated in primate luteal tissue remodelling (Liu luteolysis. Endocrinology, 133, 690–698. Fata, J.E., Ho, A.T., Leco, K.J., Moorehead, R.A. and Khokha, R. (2000) et al., 1997). It is likely that these and other proteases, including Cellular turnover and extracellular matrix remodeling in female reproductive MMPs not examined in this study, are involved with ECM breakdown tissues: functions of metalloproteinases and their inhibitors. Cell. Mol. Life during the luteal lifespan. Sci., 57,77–95. The specific temporal and spatial expression patterns of MMPs Fraser, H.M., Lunn, S.F., Harrison, D.J. and Kerr, J.B. (1999) Luteal regression and TIMPs in the present study suggest that these proteins are under in the primate: different forms of cell death during natural and gonadotropin- precise, probably endocrine, regulation. Previous studies (Iwamasa releasing hormone antagonist or prostaglandin analogue-induced luteolysis. Biol. Reprod., 61, 1468–1479. et al., 1992; Chaffin and Stouffer, 1999) have demonstrated a Goldberg, M.J., Moses, M.A. and Tsang, P.C. (1996) Identification of matrix regulatory role for both gonadotropins and ovarian steroids of metalloproteinases and metalloproteinase inhibitors in bovine corpora lutea collagenase 1 and TIMP-1 expression in the ovulatory, luteinizing and their variation during the estrous cycle. J. Anim. Sci., 74, 849–857. follicle (i.e. day 1 and 2 of the luteal phase). It is therefore possible Hayakawa, T., Yamashita, K., Tanzawa, K., Uchijima, E. and Iwata, K. that gonadotrophins and steroids, notably progesterone, continue (1992) Growth-promoting activity of tissue inhibitor of metalloproteinases- regulation of MMPs and TIMPs in the primate CL during the luteal 1 (TIMP-1) for a wide range of cells. A possible new growth factor in serum. FEBS Lett., 298,29–32. phase of the menstrual cycle. In addition, it is likely that other factors Hayakawa, T., Yamashita, K., Ohuchi, E. and Shinagawa, A. (1994) Cell regulate MMPs in the CL. Indeed, when late luteal cells from pigs growth-promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP- are cultured with either tumour necrosis factor α or prostaglandin 2). J. Cell Sci., 107, 2373–2379. F2α (agents that are associated with luteolysis in domestic ruminants), Hazzard, T.M., Christenson, L.K. and Stouffer, R.L. (2000) Changes in MMP-1, -2 and -9 are significantly up-regulated (Pitzel et al., expression of vascular endothelial growth factor and angiopoietin-1 and -2 in the macaque corpus luteum during the menstrual cycle. Mol. Hum. 2000). Future studies will address the potential regulatory role of Reprod., 6, 993–998. gondadotrophins and local factors in the regulation of MMPs and Hess, D.L., Spies, H.G. and Hendrickx, A.G. (1981) Diurnal steroid patterns TIMPs throughout the luteal lifespan. during gestation in the rhesus macaque: onset, daily variation, and the In conclusion, the cell-specific expression of both MMPs and effects of dexamethasone treatment. Biol. Reprod., 24, 609–616. TIMPs appears to be regulated in a temporal manner, potentially Hulboy, D.L., Rudolph, L.A. and Matrisian, L.M. (1997) Matrix mediating structural and functional changes that occur in the primate metalloproteinases as mediators of reproductive function. Mol. Hum. Reprod., 3,27–45. CL during the spontaneous menstrual cycle. CL formation, a time of Iwamasa, J., Shibata, S., Tanaka, N., Matsuura, K. and Okamura, H. (1992) luteal cell hypertrophy and angiogenesis, is associated with high The relationship between ovarian progesterone and proteolytic enzyme levels of TIMP-1, TIMP-2 and MMP-1. In contrast, regression of activity during ovulation in the gonadotropin-treated immature rat. Biol. the CL is characterized by increases in the gelatinases MMP-2 and Reprod., 46, 309–313. MMP-9, and with declines in TIMP expression. The current data Knobil, E. (1973) On the regulation of the corpus luteum. Biol. Reprod., 8, provide the groundwork for further research into the regulation of 246–258. cell-specific MMP/TIMP expression and action in the primate CL. Liu, K., Liu, Y.X., Hu, Z.Y., Zou, R.Y., Chen, Y.J., Mu, X.M. and Ny, T. (1997) Temporal expression of urokinase type plasminogen activator, tissue type plasminogen activator, plasminogen activator inhibitor type 1 in rhesus monkey corpus luteum during the luteal maintenance and regression. Mol. Acknowledgements Cell. Endocrinol., 133, 109–116. The authors are grateful for the expert contributions of the Division of Animal Massova, I., Kotra, L.P., Fridman, R. and Mobashery, S. 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