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Expression and Regulation of Plasminogen Activators, Plasminogen Activator Inhibitor Type-1, and Steroidogenic Acute Regulatory Protein in the Rhesus Monkey Corpus Luteum

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences (K.L., Q.F., H.-J.G., Z.-Y.H., R.-J.Z., Y.-C.L. Y.-X.L.), Beijing 100080, People’s Republic of China; and Serono Reproductive Biology Institute (K.L.), Rockland, Massachusetts 02370

Address all correspondence and requests for reprints to: Prof. Yi-Xun Liu, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China. E-mail: liuyx{at}panda.ioz.ac.cn.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. Using primate materials obtained from rhesus monkeys, we have in this study investigated the expression and regulation of the plasminogen activators (PAs) and PA inhibitor type 1 (PAI-1) during CL development and regression. Adult (5–7 yr old) female rhesus monkeys were treated with pregnant mare serum gonadotropin/human chorionic gonadotropin to induce ovulation and follicular luteinization. At various luteal developmental stages, CL or whole ovaries were obtained for preparing luteal cells, Northern blot, in situ hybridization, and immunohistochemistry. We demonstrated that luteal cells from the rhesus monkey were able to produce both tissue type PA (tPA) and urokinase type PA, as well as the physiological PAI-1. During luteal development in the monkey, urokinase type PA was the major PA species taking part in the active angiogenesis and tissue remodeling processes in the forming CL. However, the mRNA as well as the enzymatic activity levels of tPA increased dramatically in monkey CL with the advent of luteolysis. This change of tPA levels was in a temporal coordination with the regulation of PAI-1 expression, resulting in an increased tPA activity at the initiation of luteolysis. Therefore, we suggest that tPA might be a luteolytic factor to the monkey CL. A PAI-1 modulated tPA activity might be important for the initiation of luteolysis in the monkey. In addition, we have also demonstrated that the expression of steroidogenic acute regulatory protein in the monkey CL was in accordance with the changes of progesterone production, suggesting that steroidogenic acute regulatory protein expression may be considered as a reliable marker for CL function in primates.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE CORPUS LUTEUM (CL) is a transient endocrine organ that secretes progesterone to prepare the uterine environment for implantation, provided fertilization has occurred (1). A CL is formed from a ruptured follicle in a dynamic process of tissue remodeling and angiogenesis. In both rodents and primates, the development of CL is a rapid process with very high cellular turnover (2, 3). A CL is usually developed within hours in rats and mice and within days in monkeys and human, and mature CL receive the greatest blood supply per unit tissue in the whole body (4). However, if fertilization has not occurred or if the implantation is unsuccessful, the functional phase of the CL will be terminated and luteolysis will be initiated, which involves a rapid loss of progesterone production (the functional luteolysis) followed by degradation of the luteal tissue into small fibrous remnants in days (the structural luteolysis) (1, 5, 6, 7). Therefore, the involvement of proteolytic activities in CL formation and regression has been suggested (8, 9, 10, 11, 12, 13).

Proteolytic activities generated by the plasminogen activator (PA) system has been associated with many physiological and pathological processes that involve extracellular matrix remodeling, such as angiogenesis, wound healing, inflammation, embryo implantation, tumor invasion and metastasis, and ovulation (for papers and reviews, see Refs. 14, 15, 16, 17, 18, 19, 20). The PA system is a versatile, temporally controlled enzymatic system in which plasminogen is activated to the proteolytic enzyme plasmin by either of the two physiological PAs, tissue type PA (tPA) or urokinase type PA (uPA). The PA system is regulated at the level of synthesis where tPA and uPA are released by specific cells in response to hormones, growth factors, and cytokines (15, 16), and also at the level of modulation by two specific PA inhibitors, PA inhibitor type 1 (PAI-1), and PA inhibitor type 2 (PAI-2), which are also released in response to stimulatory signals (15, 19, 21).

In rodents, the regulation and functions of PAs and PAI-1 in the CL have been widely studied for the last decade. For example, in the rat, proteolytic activities mediated by tPA and regulated by PAI-1 are important for the CL formation and regression (9, 10). In primates, our previous study had shown that high levels of uPA and PAI-1 mRNA were simultaneously expressed in the functional monkey CL, indicating that their interplay may participate in the functional maintenance in the rhesus monkey (11). However, what role the PA system may play during monkey CL formation and regression is not totally clear. The interaction between tPA and PAI-1 in the monkey CL is not very well understood either. To address this question, we have performed a series of assays in the present study for PAs and PAI-1 as well as for steroidogenic acute regulatory protein (StAR) in the monkey CL. We suggest that these molecules may play important roles during CL formation, function, and regression in the primate.

	Materials and Methods

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Animals
Female rhesus monkeys (5–7 yr old) were obtained from the Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China), and were kept in standard cages. The use of monkeys for this study was approved by the Academic Committee of the Institute of Zoology, Chinese Academy of Sciences, and the World Health Organization Review Committee. Monkeys with two to three observed successive estrous cycles of 26–28 d were selected. To induce superovulation and luteinization, an initial dose of 950 IU pregnant mare serum gonadotropin (PMSG) was given im on d 1 of menses. From d 2–7, 300 IU PMSG was administered every other day, followed by daily injections of 300 IU PMSG for an additional 5 d. On d 13, a single injection of 4000 IU human chorionic gonadotropin (hCG) was administered and superovulation was usually observed 36–48 h after the injection. At various time points of CL development, monkeys were maintained anesthetized by ketamine, and blood samples were collected. The ovaries were removed for further analysis by abdominal surgery in a standard operation room.

Hormones and chemicals
McCoy’s 5A medium (modified, without serum), penicillin, streptomycin, L-glutamine, HEPES, trypan blue, and thrombin were obtained from Sigma Chemical Co. (St. Louis, MO). Acrylamide, N,N-methylenebisacrylamide, sodium dodecyl sulfate (SDS), TEMED, ammonium persulfate, and Coomassie brilliant blue were purchased from Bio-Rad Laboratories (Richmond, CA). Purified human plasminogen, tPA, uPA, and tPA monoclonal antibody were obtained from Biopool Laboratory (Umea, Sweden). PMSG was obtained from Changchun Institute of Biological Products (Changchun, China). hCG was obtained from the laboratory of Dr. Yushu Xu (Institute of Zoology, Chinese Academy of Sciences, Beijing, China). Collagenase and DNase were purchased from Vector Laboratories (Burlingame, CA). ³H-progesterone was purchased from Shanghai Institute of Nuclear Energy (Shanghai, China). LH (NIADDK oLH-25) was obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

Granulosa cells and luteal cell preparation and culture
To obtain preovulatory granulosa cells, monkey ovaries were obtained by surgery, and ovaries were washed in McCoy’s 5A media before being punctured with 26-gauge needles repeatedly to release the granulosa cells. To obtain luteal cells, individual CL were dissected from monkey ovaries, cut into small pieces, rinsed, and incubated with 0.1% collagenase at 37 C for 15–30 min. Luteal cells were obtained by frequent pipetting of the segments. The cells were washed three times with fresh medium, and 1 x 10⁶ cells were cultured in 1 ml serum-free McCoy’s 5A medium in poly-D-lysine-coated 35-mm Falcon culture dishes in a CO₂ incubator at 37 C for 24–48 h. The media were collected at the end of the culture and stored at -70 C for measurement of PA and PAI-1 activities as well as progesterone levels.

SDS-PAGE and fibrin or reversed fibrin overlay
PA and PAI-1 antigens in the samples were fractionated by SDS-PAGE according to the method of Laemmli (22). Electrophoresis was performed at 50 V overnight until the dye front reached the bottom of the gel. After electrophoresis, the gel was washed twice for 45 min in 2.5% (vol/vol) Triton X-100 solution to remove SDS in the gel and rinsed once with distilled water before being applied to the surface of a fibrin/agar indicator gel. The preparation of fibrin/agar indicator gel for measuring PA or PAI-1 activities was performed according to Loskutoff et al. (23) with modifications as described previously (24). The overlaid gels were incubated at 37 C in a humid chamber for autolysis. The PAI-1 activity was shown by antilysis bands on the indicator gel.

Riboprobe preparation
The riboprobes for rhesus monkey tPA and PAI-1 were prepared as previously described (25). Briefly, fragments of monkey tPA (346 bp) and PAI-1 (388 bp) cDNA were subcloned into pGEM-3Z vectors, and sequencing results revealed that the monkey tPA and PAI-1 cDNA fragments have 95.78% and 96.79% similarities to the corresponding human cDNA sequences, respectively (25). For Northern blot analysis, cDNA templates were linearized so that the antisense cRNA probes can be labeled with ³²P-UTP. The specific activity of the probes ranged between 2 and 5 x 10⁸ cpm/µg RNA.

Northern blot analysis
Total RNA from the CL was extracted as reported previously (10), fractionated on 1% agarose gel in the presence of 2.2 M formaldehyde, and transferred to nylon filters. After cross-linking under a UV light, the filters were prehybridized at 62 C for 2 h in a solution containing 50% formamide, 5x saline sodium citrate (SSC) (1x SSC = 0.15 M NaCl, 0.015 M sodium citrate), 8x Denhardt’s solution (1.6 mg/ml Ficoll, 1.6 mg/ml polyvinylpyrrolidone, and 1.6 mg/ml of BSA), 0.1% SDS, 10 mM EDTA, and 25 mM yeast tRNA. The hybridization was performed in the same solution with ³²P-UTP probes at 62 C overnight, followed by two to three washes in 0.1x SSC containing 0.1% SDS at 65 C for about 1 h. The hybridized filters were exposed to the screen of the PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) cassette, and data were analyzed using programs of the PhosphorImager.

In situ hybridization
Frozen sections (10 µm) were prepared from monkey ovaries at various stages, and in situ hybridization was performed as described previously (10). Slides used for comparison were prepared and hybridized at the same time. To monitor background levels and the specificity of the hybridization, the sense strands of the probes were included in each experiment. Photographs were taken with a Carl Zeiss camera attached to a Carl Zeiss Axioplan microscope (Zeiss, New York, NY) at a magnification of x4–10.

Immunohistochemistry
Immunohistochemistry was performed with a Vectastain ABC (avidin-biotin peroxidase) kit (Vector Laboratories) as described previously (26). Briefly, the deparaffinized sections were incubated with 3% H₂O₂ for 10 min and then incubated with 10% normal goat serum (NGS) in PBS for 30 min. The primary antibody against StAR was diluted in PBS containing 10% NGS and incubated with the sections for 1 h. For negative control, sections were incubated with 10% NGS in PBS only. Sections were then washed in PBS for 3 x 5 min, incubated with biotinylated second antibodies for 1 h, and washed in PBS for another 3 x 5 min. After incubation with avidin-biotin-peroxidase complex for 1 h and washing in PBS for 3 x 5 min, sections were incubated in diaminobenzidine tetrachloride in 0.05 M Tris-HCl (pH 7.2) with 0.01% H₂O₂ for 2–7 min. The sections were then dehydrated through a graded series of ethanol and mounted.

Progesterone assay
Progesterone levels in monkey serum and in luteal cell culture media were assayed by RIA, as previously described (27). The results were processed by RIACALC (Wallac, Redmond, WA) program in the IBM computer.

Statistical analysis
All experiments were repeated at least three times, and a representative photograph as well as mean values ± SD values (mean ± SD) of three experiments (where applicable) were shown. The relative amount of specific tPA and PAI-1 mRNA was determined by densitometric scanning of autoradiographies and normalized against levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples. Differences among groups were calculated by Tukey’s multiple-comparison test, and a significant difference was determined by a value of P < 0.05.

	Results

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The gonadotropin-induced ovulation model in the rhesus monkey
In this study, we have used a PMSG/hCG induced ovulation model to obtain CL at different developmental stages from the rhesus monkeys. As reported previously, ovulation usually takes place 36–48 h after the hCG injection. The newly formed CL (3–5 d after hCG injection) appeared to be filled with plenty of blood contents, indicating an aggressive ongoing angiogenesis. As shown in Table 1, the serum progesterone level accompanying the newly developed functional CL (d 5) was significantly higher than later luteal stages. The CL from this model lasted until 13 d after hCG injection, and serum progesterone levels decreased in a stage-dependent manner. At 13 d after hCG injection, the progesterone level dropped to a very low level when functional luteolysis took place (Table 1).

View larger version (22K): [in this window] [in a new window]   FIG. 1. Gonadotropin regulation of tPA and uPA activities in luteinized granulosa cells and luteal cells during CL formation. Luteinized granulosa cells and luteal cells (1 x 106) at indicated time points after hCG injection were cultured in 1 ml McCoy’s 5A medium for 24 h at 37 C. PA activities in the culture media were measured by fibrin overlay as described in Materials and Methods. For each experiment and time point, luteal or granulosa cells from the ovaries of one female monkey were used. The result shown is a representative photograph from three independent experiments. 24 h and 36 h, 24 or 36 h after hCG injection; D3 and D5, 3 or 5 d after hCG injection.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Relative levels of tPA (A) and PAI-1 (B) mRNAs in monkey CL at various stages. Total RNA from each group (n = 3 monkeys per time point) of monkey CL or luteinized follicles was extracted, and 30 µg of total RNA for each time point was fractionated by agarose gel electrophoresis and transferred to nylon filter for hybridization with 32P-labeled monkey antisense tPA, PAI-1, or human antisense GAPDH RNA probes, as described in Materials and Methods. The relative amounts of tPA or PAI-1 mRNA (mean ± SD) were estimated by densitometric scanning of the autoradiographies and normalized against the corresponding amounts of GAPDH mRNA from three Northern blot analyses. 30 h represents 30 h after hCG injection; D10, D13, and D15 represent 10, 13, or 15 d after hCG injection, respectively.

Localization of tPA, PAI-1, and StAR in the monkey CL
To confirm the expression of tPA and PAI-1 mRNAs in monkey CL measured by Northern blot analysis, we have performed in situ hybridization with digoxigenin-labeled RNA probes. As shown in Fig. 3, in accordance with our Northern blot analyses, the semiquantitative in situ hybridization data showed that tPA mRNA was low in the functional CL (10 d after hCG injection). However, in the luteolytic monkey CL (13 d after hCG injection), tPA mRNA expression was much higher, with a dotted pattern. Also in agreement with our Northern blot analysis results, PAI-1 mRNA was expressed at high levels in the functional CL (10 d after hCG injection) (Fig. 3). However, the expression of PAI-1 mRNA measured by in situ hybridization decreased to undetectable levels in the luteolytic monkey CL (13 d after hCG injection). At this stage of study, we have not been able to provide evidence to identify cell types that express tPA and PAI-1 mRNA.

View larger version (104K): [in this window] [in a new window]   FIG. 3. Localization of tPA and PAI-1 mRNAs in the monkey CL. Semiquantitative in situ hybridization for monkey tPA and PAI-1 mRNAs were performed with digoxigenin-labeled monkey tPA and PAI-1 riboprobes, as described in Materials and Methods. Slides used for comparison were prepared at the same time. Photographs were taken at original magnification of x4–10. Representative pictures from three individual experiments (n = one monkey per time point for each experiment) were shown. D10 and D13, 10 or 13 d after hCG injection, respectively.

View larger version (157K): [in this window] [in a new window]   FIG. 4. Expression of StAR antigen in monkey CL at various stages. Immunohistochemistry of StAR protein was performed with a Vectastain ABC kit as described in Materials and Methods. A representative result was shown from three similar experiments (n = one monkey per time point for each experiment). A, Negative control; B–F: 5, 8, 9, 13, or 18 d after hCG injection, respectively. Original magnification, x10–20.

Regulation of PAI-1 activities in luteal cells before luteolysis
We have suggested that tPA may play a role in luteolysis initiation in the monkey CL and hypothesized that the interaction between tPA and PAI-1 in preluteolytic CL may result in a fine-tuned tPA activity for the advent of luteolysis. To study the interaction of tPA and PAI-1 in monkey luteal cells just before luteolysis, luteal cells (5 x 10⁵ cells/well in a six-well plate) prepared from CL at 10 d after hCG injection were incubated in 0.5 ml serum-free McCoy’s 5A medium for 24–48 h with or without LH, monoclonal activity-neutralizing antibody against tPA, or a combination of both. The PAI-1 activities and progesterone production in the media were measured by reversed fibrin overlay and RIA techniques, respectively.

As shown in Fig. 5A, the progesterone production was significantly enhanced by the addition of LH to the luteal cell culture, indicating that we have a good pool of viable luteal cells. A combination of LH and tPA antibody can further enhance the progesterone production of the luteal cells. Although the mRNA level of PAI-1 in CL at 10 d after hCG injection is considerably high (Figs. 2 and 3), measurable PAI-1 activity in the untreated luteal cell culture media is not very high (Fig. 5B, lane C). LH alone did not have any effect on the secretion of PAI-1 activity by luteal cells (Fig. 5B, lane LH). However, the addition of tPA-neutralizing antibody to the culture media dramatically enhanced the measurable PAI-1 activity (Fig. 5B, lane tPA ab). Nevertheless, as shown in Fig. 5B, a combination of tPA antibody and LH did not result in any synergistic stimulation of PAI-1 activity.

View larger version (41K): [in this window] [in a new window]   FIG. 5. Regulation of progesterone production (A) and PAI-1 activities (B) by LH and exogenous tPA monoclonal antibody in cultured monkey luteal cells. Luteal cells (5 x 105 cells/well) obtained from monkey CL at 10 d after hCG injection were incubated for 24–48 h in 0.5 ml serum-free McCoy’s 5A medium with LH (100 ng/ml), tPA monoclonal antibody (tPA-ab, 100 ng / ml), or a combination of LH and tPA antibody. Progesterone production (A) and PAI-1 activities (B) in the culture media were measured by RIA and reverse fibrin-overlay, respectively. The experiments were repeated three times (n = 2 monkeys per experiment), and a representative photograph for PAI-1 activity was shown. The relative levels of progesterone were shown by the average of three experiments (mean ± SD). Lowercase letters (a–c) indicate significant differences (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There exist species differences in terms of roles of PAs in the CL. In the present study, we reported that uPA is highly expressed in the developing monkey CL; therefore it may be involved in the tissue remodeling and angiogenesis processes during CL formation. As CL undergoes luteolysis (11), tPA was up-regulated in the monkey CL to provide the proteolytic activity needed for luteal regression. However, in the rat, our previous report has shown that tPA plays a role in both CL formation and regression (10). A spatially coordinated expression of tPA and PAI-1 in the newly formed rat CL helped to generate a proteolytic activity only in the outer region of the CL in which active angiogenesis and tissue remodeling took place, whereas a temporally coordinated expression of tPA and PAI-1 led to an increased tPA proteolytic activity in the luteolytic rat CL (10). The interaction between tPA and PAI-1 in the luteolytic monkey CL is very similar to what we observed in the rat CL (10).

We have also studied the expression of the PA system in the CL of pseudopregnant mice (28). No coordinated expression of the PAs and their inhibitor PAI-1 was observed in the mouse CL. Our data showed that uPA is the major PA in the mouse CL and it is constantly expressed in mouse CL at relatively high levels regardless of different luteal developmental stages, implying that uPA plays a role in formation, functional maintenance, and regression of the mouse CL. However, when we compared the luteal functions between the uPA gene-deficient mice and their wild-type control mice, no defect in luteal functions was observed as judged by serum progesterone levels, luteinized ovarian weights, and blood vessel densities in the functional CL. This suggests that in the mouse CL the function of uPA is not essential or other similar molecules compensate for the uPA function in its absence (28).

Our result that tPA mRNA increased in monkey CL at the time of luteolysis was well supported by our previous reports that addition of exogenous tPA to cultured rat or monkey luteal cells significantly decreased their progesterone production in the culture media (8, 9). Our data from both the rat and the monkey suggest that tPA might be not only a molecule that participates in the extracellular matrix degradation during luteal tissue regression but also a molecule that may have other activities to inhibit progesterone synthesis via possible autocrine or paracrine pathways. Experiments using rat and monkey luteal cells are currently under way to address this question.

In this study, we have also demonstrated that treatment with activity-neutralizing tPA monoclonal antibody of cultured monkey luteal cells considerably increased measurable PAI-1 activity and progesterone production in the culture media. Our explanation for this is that when tPA is not needed or excessive tPA activity could be harmful to the integrity of the CL, tPA is suppressed in a complex with PAI-1. Once tPA activity was required for the coming luteolysis, the expression of PAI-1 was quickly decreased from the mRNA level so that active tPA molecule was liberated. The temporally coordinated induction of tPA and PAI-1 may reflect a fine-tuned regulatory mechanism by which PAI-1 activity was used to limit excessive tPA activity by forming an inhibitory PA/PAI-1 complex.

In conclusion, the current study is our first step to investigate the important roles of the PA system in the development and regression of primate CL. We have demonstrated that uPA may participate in the active tissue remodeling and angiogenesis processes during CL formation, whereas the interplay between tPA and its inhibitor PAI-1 may be crucial to initiate luteolysis in rhesus monkeys. Despite the PA system, functions of other extracellular matrix degrading proteases, such as molecules from the matrix metalloproteinase family, have also been investigated in the physiological processes during CL development and regression (19, 28, 29). With the help of gene-deficient mice that lack a protease or a combination of proteases, we have wished that functions of certain proteases in the ovary would be revealed. However, due to possible overlapped or compensated proteases functions, it has been more complicated than anticipated (19, 28). We hope that our current effort using the recently developed small interference RNA technology (30, 31), by which we can quickly knock down the expression of more proteins in cultured cells, can help us identify important proteases for luteal development, function, and regression.

	Acknowledgments

 
We thank Prof. Douglas M. Stocoo for providing StAR cDNA and the antibody; Prof. Tor Ny for providing tPA uPA, and PAI-1 cDNA and tPA antibody; and Prof. A. F. Parlow for providing the NIDDK hormones.

	Footnotes

 
This work was supported by the Rockefeller Foundation, World Health Organization, 973 Program (G1999055901), CAS Chuang-Xin Program (KSCX2-SW-201), and the National Natural Science Foundation of China (39970284, 90208025, 30170452).

Present address for Q.F.: Department of Obstetrics and Gynecology, Research Division, University of British Columbia, British Columbia’s Women’s Hospital & Health Center, 4490 Oak Street, Room 2H30, Vancouver, British Columbia V6H 3N1, Canada.

Present address for H.-J.G.: Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, Maryland 21210.

Abbreviations: CL, Corpus luteum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; NGS, normal goat serum; PA, plasminogen activator; PAI-1, PA inhibitor type 1; PMSG, pregnant mare serum gonadotropin; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; StAR, steroidogenic acute regulatory protein; tPA, tissue type PA; uPA, urokinase type PA.

Received March 10, 2003.

Accepted for publication May 2, 2003.

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