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.     

Vascular endothelial growth factor production in growing pig antral follicles

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.     

Expression of basigin, an inducer of matrix metalloproteinases, in the rat ovary

The extensive tissue remodeling that occurs during follicular development, ovulatory rupture, and the formation and regression of the corpus luteum (CL) requires local degradation of the extracellular environment by matrix metalloproteinases (MMPs). This report characterizes the expression pattern of basigin (Bsg), a putative regulator of MMP induction, in the rat ovary. An induced superovulation model (eCG/hCG) was used in immature rats to evaluate Bsg expression profiles in ovaries collected during the follicular phase, the preovulatory period, and the luteal lifespan. Levels of Bsg mRNA were unchanged through follicular growth (0-48 h post-eCG) and increased during postovulatory luteinization (24 and 48 h post-hCG; P < 0.01). Bsg expression persisted into pseudopregnancy (4-8 days post-hCG) and after functional luteal regression (12 days post-hCG). The profile of Bsg expression during regression of the CL was examined using a model of induced luteolysis. Both functional and structural regression was associated with a decline in Bsg expression levels. Bsg mRNA and protein localized to the theca of preovulatory follicles (12 h post-hCG) and formative and functional CL (24 h-8 days post-hCG). Bsg expression profiles in the induced ovulation and CL regression models were similar to observations made in naturally cycling mature rats. In the cycling ovary, Bsg signaling localized to newly forming CL, the theca of preovulatory follicles, and appeared to be lower in CL from previous estrous cycles. A putative regulatory mechanism of Bsg expression was identified using an in vitro model; treatment of cultured granulosa cells with hCG significantly augmented Bsg mRNA expression levels. The processes of ovulation and luteogenesis may be facilitated by Bsg expression and its induction or regulation of the MMPs.     

Expression and regulation of ang-2 in murine ovaries during sexual maturation and development of corpus luteum

The aim of this study was to examine the expression and regulation of angiopoietin-2 (Ang-2) in murine ovaries during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and RT-PCR. By in situ hybridization Ang-2 mRNA was mainly localized in granulosa cells, thecal cells and corpus luteum, otherwise in oocytes. Moreover, Ang-2 mRNA was highly expressed in corpus luteum and granulosa cells of atretic follicles. According to RT-PCR data, Ang-2 mRNA was lowly expressed on day 10 after birth, then expression levels gradually increased and reached their highest values on day 25 after birth. In the superovulated model of immature mice, Ang-2 expression was strongly induced by equine chorionic gonadotropin (eCG) 48 h post the eCG injection, and was high from 0.5 to 13 h after hCG treatment. in situ hybridization showed that Ang-2 mRNA was highly expressed in corpus luteum from day 2 to 9 post the hCG injection, then the expression levels gradually declined on days 11 and 13 after hCG treatment. According to RT-PCR data, the levels of Ang-2 mRNA expression showed a decline after the hCG injection, with a nadir on day 3, followed by an increase, reaching the highest level on day 9 post-hCG injection. Then again Ang-2 expression gradually declined from day 11 to 15 after hCG injection. These results suggest that Ang-2 may be involved in follicular development, atresia, ovulation, and corpus luteum formation and regression.     

Effect of Antiprogesterone RU486 on VEGF Expression and Blood Vessel Remodeling on Ovarian Follicles before Ovulation

Background

The success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P₄) remains to be clarified, in particular when its concentration rapidly increases before ovulation.

Aim

This in vivo study was designed to clarify the effect promoted by a P₄ receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration.

Material and Methods

Preovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture.

Results and Conclusions

VEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P₄ antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may affect the functionality of the corpus luteum (CL) and the success of pregnancy.     

The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.     

Ultrasound and endocrine evaluation of the ovarian response to a single dose of 500 IU of eCG following a 12-day treatment with progestogen-releasing intravaginal sponges in the breeding and nonbreeding seasons in ewes

A standard dose of 500 IU of eCG is commonly given to progestogen pre-treated anestrous ewes for induction of estrus. Twelve seasonally anestrous and 12 cyclic Western White Face ewes were treated for 12 days with intravaginal sponges impregnated with medroxyprogesterone acetate (MAP). In trials in both the breeding and nonbreeding seasons, six randomly selected ewes were given 500 IU of eCG at sponge removal to determine the effects of low dose of eCG on ovarian antral follicular dynamics and ovulation. Ultrasound scanning and blood sampling were done daily. Treatment with eCG did not have marked effects on antral follicular growth. All ewes ovulated, except for five of six control anestrous ewes. Luteal structures and progesterone secretion were confirmed in all but the control anestrous ewes. In the breeding season, peak progesterone concentrations were greater (P<0.05) in eCG-treated compared to control ewes. Daily serum estradiol concentrations were greater in the periovulatory period in eCG-treated compared to control ewes (treatment-by-day interaction; P<0.05), particularly in anestrus. Progestogen-treated ewes ovulated follicles from several follicular waves, in contrast to ovulations of follicles from the final wave of the cycle in untreated, cyclic ewes. Anestrous ewes exhibited more frequent follicular waves and FSH peaks compared to cyclic ewes after a progestogen/eCG treatment. In conclusion, 500 IU of eCG given after 12 days of progestogen treatment had limited effects on the dynamics of ovarian follicular waves. However, eCG treatment increased serum concentrations of estradiol during the periovulatory period, particularly in anestrous ewes; this probably resulted in the synchronous estrus and ovulation in anestrous ewes.     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.     

Cyclic guanosine 5'-monophosphate-dependent protein kinase II is induced by luteinizing hormone and progesterone receptor-dependent mechanisms in granulosa cells and cumulus oocyte complexes of ovulating follicles

Cyclic GMP (cGMP)-dependent protein kinase II (Prkg2, cGK II) was identified as a potential target of the progesterone receptor (Nr3c3) in the mouse ovary based on microarray analyses. To document this further, the expression patterns of cGK II and other components of the cGMP signaling pathway were analyzed during follicular development and ovulation using the pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-primed immature mice. Levels of cGK II mRNA were low in ovaries of immature mice, increased 4-fold in response to pregnant mare serum gonadotropin and 5-fold more within 12 h after hCG, the time of ovulation. In situ hybridization localized cGK II mRNA to granulosa cells and cumulus oocyte complexes of periovulatory follicles. In progesterone receptor (PR) null mice, cGK II mRNA was reduced significantly at 12 h after hCG in contrast to heterozygous littermates. In primary granulosa cell cultures, cGK II mRNA was induced by phorbol 12-myristate 13-acetate enhanced by adenoviral expression of PR-A and blocked by RU486 and trilostane. PR-A in the absence of phorbol 12-myristate 13-acetate was insufficient to induce cGK II. Expression of cGK I (Prkg1) was restricted to the residual tissue and not regulated by hormones. Guanylate cyclase-A (Npr1; GC-A) mRNA expression increased 6-fold by 4 h after hCG treatment in contrast to pregnant mare serum gonadotropin alone and was localized to granulosa cells of preovulatory follicles. Collectively, these data show for the first time that cGK II (not cGK I) and GC-A are selectively induced in granulosa cells of preovulatory follicles by LH- and PR-dependent mechanisms, thereby providing a pathway for cGMP function during ovulation.     

Synchronization of estrus and ovulation in sows not conceiving in a scheduled fixed-time insemination program

A field study was conducted to investigate the effectiveness of a treatment with altrenogest, eCG and hCG or the GnRH-analogue D-Phe(6)-LHRH to synchronize estrus and ovulation of sows diagnosed as non-pregnant in order to reintegrate them back into a scheduled fixed-time insemination program. Sows (n=531) diagnosed as non-pregnant by ultrasonography on days 21-35 after insemination were subjected to one of three treatments: (1) 16 mg altrenogest/day/animal orally for 15 days to block follicular growth, followed by injection of 1000 IU eCG intramuscularly (i.m.) 24h after withdrawal of altrenogest to stimulate follicular growth and 500 IU hCG i.m. 78-80 h after eCG to induce ovulation; (2) similar to (1) except that 20mg altrenogest and 800 IU eCG were used and (3) similar to (2) except that 50 microg D-Phe(6)-LHRH was used to induce ovulation. Females were artificially inseminated (AI) twice at 24 and 40 h, respectively, after hCG/D-Phe(6)-LHRH. Success of treatments was checked by ultrasonography of the ovaries. Rates of conception and farrowing (CR, FR), and number of total and live born piglets (TB, LB) were recorded and compared to those of synchronized first served sows. Females had differing ovarian structures prior to treatment. Altrenogest effectively blocked follicular growth in >80% of the females irrespective of dosage, but 16 mg increased the development of polycystic ovarian degeneration. Four to 18% of the females still had corpora lutea after altrenogest. Most females ovulated either between both inseminations or thereafter (P<0.05). Females treated with D-Phe(6)-LHRH tended to ovulate earlier than those injected with hCG. The CR and FR were up to 25% lower for sows diagnosed as non-pregnant than for sows after first service (P<0.05). Among sows diagnosed as non-pregnant the CR was higher in females treated with D-Phe(6)-LHRH (P<0.05). No differences were found in regard to numbers of TB and LB. In conclusion, a treatment with 20mg altrenogest per day per animal, followed by 800 IU eCG and 50 microg the GnRH-analogue D-Phe(6)-LHRH is appropriate to synchronize estrus and ovulation of sows diagnosed as non-pregnant. Whether there might be a need to feed altrenogest for a longer interval of 18 days has to be investigated.     

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.     

Gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific

We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after pregnant mare's serum gonadotropin (PMSG) and after an ovulatory gonadotropin stimulus. Twenty-four hours after PMSG, there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48 h later by 10 IU of hCG. GCLm protein and mRNA levels increased dramatically from 0 to 4 h after hCG and then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48 h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.     

Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.     

Membrane type 1-matrix metalloproteinase (MMP)-associated MMP-2 activation increases in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin

Gonadotropins stimulate ovarian proteolytic enzyme activity that is believed to be important for the remodeling of the follicular extracellular matrix. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified in vitro as an activator of pro-MMP-2 by forming a complex with tissue inhibitors of metalloproteinase-2 (TIMP-2). In the present study, the expression pattern of MT1-MMP mRNA and the role of MT1-MMP were examined in the ovary using the gonadotropin-treated immature rat model. Ovaries were collected at selected times after eCG or hCG. RNase protection assays revealed a transient increase in MT1-MMP mRNA beginning 4 h after hCG. High expression of MT1-MMP mRNA was localized to the theca-interstitial layer of developing and preovulatory follicles, while low expression was observed in the granulosa cell layer of developing follicles by in situ hybridization. The localization pattern of MT1-MMP mRNA was compared with TIMP-2 mRNA. Both MMP-2 and TIMP-2 mRNA were expressed in the theca layer of preovulatory follicles, showing a similarity to MT1-MMP mRNA expression. To further determine whether MT1-MMP activates pro-MMP-2 in the ovary, crude plasma membrane fractions from preovulatory ovaries were analyzed by gelatin zymography. In plasma membrane fractions, pro-MMP-2 increased around the time of ovulation. Upon incubation, pro-MMP-2 was activated with the highest levels of activation at 12 h post-hCG. The addition of MT1-MMP antibody or excess TIMP-2 to membrane fractions inhibited pro-MMP-2 activation. The increase in MT1-MMP mRNA may be an important part of the mechanism necessary for the efficient generation of active MMP-2 during the ovulatory process.