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BOR - Papers in Press, published online ahead of print May 5, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029702
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BIOLOGY OF REPRODUCTION 71, 887–893 (2004)
DOI: 10.1095/biolreprod.104.029702
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Plasminogen Activator and Serine Protease Inhibitor-E2 (Protease Nexin-1) Expression by Bovine Granulosa Cells In Vitro1

Mingju Cao, Malha Sahmi, Jacques G. Lussier, and Christopher A. Price2

Centre de recherche en reproduction animale (CRRA), Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Remodeling of the extracellular matrix (ECM) occurs during antral follicle growth, and the plasminogen activators (PA) have been implicated in this process in rodents. In the present study, we measured the expression and secretion of PA and the PA inhibitor protease nexin-1 (SerpinE2) in antral and basal bovine granulosa cells from small (<6 mm), medium (6–8 mm), and large follicles (>8 mm) during 6 days of culture in serum-free medium. Casein zymography revealed that the cells secreted predominantly tissue-type PA (tPA) with urokinase (uPA) being associated mainly with cell lysates, and Western blot demonstrated that the cells secreted SerpinE2. Overall, secreted tPA activity was higher in cultures of cells from small follicles compared with large follicles, and secreted SerpinE2 levels were higher in cultures of cells from large follicles. In cultures of cells from small follicles, secreted tPA levels increased with time of culture for antral but not basal cells, and SerpinE2 levels increased with time for basal but not antral cells. In cultures of granulosa cells from large follicles, tPA activity increased significantly with time of culture, whereas SerpinE2 levels decreased. Cell-associated uPA activity decreased with time in cells from medium and large follicles. Reverse-transcription polymerase chain reaction and Northern blot analysis showed that SerpinE2 secretion was regulated largely at the transcriptional level, whereas tPA secretion was not. The data suggest stage-dependent regulation of granulosa cell PA and SerpinE2 production, consistent with a role in ECM remodeling during follicle growth.

follicle, granulosa cells, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian follicular growth and development involve extensive tissue remodeling, cell proliferation, and differentiation [1]. Tissue remodeling involves a number of protease enzyme cascades, including the matrix metalloproteinases (MMP) and plasminogen activators (PA). Plasminogen activators are serine proteases that convert the abundant extracellular zymogen plasminogen into plasmin, an active protease that degrades components of the extracellular matrix (ECM) [2]. Two forms of PA, tissue type (tPA) and urokinase (uPA), have been described in mammals [3] and are products of the Plat and Plau genes, respectively. The type of PA secreted is species- and cell-specific. Although rat [4–6] and pig [7] granulosa cells secrete predominantly tPA, uPA secretion is predominant in mice and chicken granulosa cells [8–10]. In bovine preovulatory follicles, Plat mRNA was localized primarily in granulosa cells, whereas Plau mRNA was detected in granulosa and in theca cells [11, 12].

One mechanism for the regulation of plasminogen activation is through the production of PA inhibitors (PAI). The three major inhibitors are PAI-1, PAI-2, and protease nexin-1 (PN-1) [13, 14], encoded by the serine protease inhibitor (Serpin) family of genes [15]. In rodents and cattle, Serpine1 (encoding PAI-1) is expressed predominantly by theca-interstitial cells [16–18]. Serpinb2 encodes the weak inhibitor, PAI-2, and is expressed at low levels in the theca layer of hCG-treated rats and in cumulus and granulosa-lutein cells of hCG-stimulated human ovaries [19, 20]. In contrast to Serpine1 and Serpinb2, the gene encoding PN-1, Serpine2, is strongly expressed in granulosa cells of rats and cattle [9, 21, 22].

The PAs and Serpins are widely considered to be important during the process of ovulation when proteolytic degradation of the follicle wall occurs. During final preovulatory growth in rats and monkeys, there are concomitant increases in granulosa cell Plat expression/tPA secretion and thecal Serpine1 expression/secretion. However, a few hours prior to ovulation there is a significant decrease in Serpine1 mRNA and protein levels, presumably resulting in an increase in net tPA activity that initiates the proteolytic cascade necessary for the degradation of the follicle wall [23, 24]. Periovulatory increases in PA activity/mRNA have also been described for pigs, sheep, and cattle [12, 25, 26].

Tissue remodeling is also important for the growth and development of small follicles, as bovine follicles typically increase in size several hundred-fold between preantral and preovulatory stages. Studies in rats have shown that uPA is the predominant PA in small growing follicles, whereas tPA is predominant in preovulatory follicles [27, 28], suggesting a role for uPA in early follicle growth. It is not clear if or how PA activity is regulated by inhibitors at this stage of follicle development, although available evidence suggests that SerpinE1 and SerpinB2 are not involved. In rats, Serpine1 expression was low in small growing follicles and increased as follicles differentiated [28], consistent with the role in ovulation described above. Serpinb2 expression was not readily detected in bovine preovulatory follicles before the induction of ovulation by GnRH [18]. Interestingly, Serpine2 is highly expressed in small growing follicles in rats [9, 21] and in preantral and growing antral follicles in cattle [22].

As SerpinE2 would appear to be the major PA inhibitor expressed in granulosa cells and in small growing follicles, we hypothesize that this member of the Serpin family plays a role in the remodeling of the membrana granulosa during follicle growth by regulating PA activity, mainly uPA. The objective of the present study was to measure Serpine2, Plau, and Plat expression and protein secretion from bovine granulosa cells at different stages of development. To do so, we employed an established cell culture system that permits long-term estradiol secretion in vitro and maintains the follicular phenotype of the cells [29, 30].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

The cell culture system was based on that described by Gutiérrez et al. [29], with slight modifications [30]. All materials were obtained from Invitrogen Life Technologies (Burlington, ON, Canada) except where otherwise stated. Briefly, bovine ovaries were collected from adult cows, irrespective of stage of the estrous cycle, at a local abattoir and were transported to the laboratory in PBS at 35°C containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (1 µg/ml). Follicles were dissected from the ovaries, and those with obvious signs of atresia (avascular theca, debris in antrum) were discarded. Lightly adherent ‘antral’ granulosa cells from small (2–5 mm in diameter), medium (6–8 mm), and large (>8 mm) follicles were released by dissection or aspiration; the adherent ‘basal’ granulosa cells were subsequently collected by repeatedly passing the follicle wall through a pipette. Cells were washed twice by centrifugation at 980 x g for 20 min each and suspended in {alpha}-minimal essential medium containing Hepes (20 mM); sodium bicarbonate (10 mM); sodium selenite (4 ng/ml); BSA (0.1%; Sigma-Aldrich Canada, Oakville, ON, Canada); penicillin (100 IU/ml); streptomycin (100 µg/ml); transferrin (2.5 µg/ml); nonessential amino acid mix (1.1 mM); androstenedione (10–7 M); insulin (10 ng/ml); 1 ng/ml FSH (AFP-5332B; National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD); and human recombinant insulin-like growth factor-I (IGF-I, 10 ng/ml). Cell viability was estimated with 0.4% trypan blue stain. Cells were seeded into 24-well tissue culture plates (Corning Glass Works, Corning, NY) at a density of 106/well in 1 ml medium. Cultures were maintained at 37°C in 5% CO2 in air, with 700 µl medium being replaced every 2 days. Medium and cells were recovered on Days 2, 4, or 6 of culture. Medium samples were stored at –20°C until assay, whereas cells were collected in Trizol and stored at –70°C until RNA, DNA, and protein extraction.

RNA, DNA, and Protein Extraction and Quantification

Total RNA, DNA, and protein were extracted using Trizol according to the manufacturer's instructions. Total RNA was quantified by absorbance at 260 nm. Total DNA was quantified in duplicate by measuring fluorescence in the presence of bisbenzimide (Hoechst 33258) and compared with a calf thymus DNA standard (Sigma-Aldrich) curve. Total protein was measured by the Lowry assay [31] using BSA (Sigma-Aldrich) as standard.

Casein Zymography

Casein zymography was used to measure tPA and uPA activity in culture medium and granulosa cell extracts [12]. Briefly, samples were subjected to electrophoresis at 120 V for 90 min in 10% nondenaturing polyacrylamide gels containing 0.2% casein (Sigma-Aldrich), 0.1% SDS, and 3.75 mU/ml bovine plasminogen (Sigma-Aldrich). After electrophoresis, gels were washed once in 2.5% Triton X-100 for 45 min to remove SDS and placed in incubation buffer (50 mM Tris, 0.1 M NaCl, pH 7.6) at 37°C for 16 h with gentle shaking. The gels were then stained using 0.05% Coomassie blue in 10% acetic acid, 40% methanol for 2 h; destained in 10% acetic acid, 40% methanol; and then fixed in 10% glycerol. The identity of the enzymatic activities was investigated by comparing molecular size with human tPA (Calbiochem, Darmstadt, Germany) and uPA (NIBSC, Hertfordshire, UK) standards. Amiloride (1 mM), a specific inhibitor of uPA, was included in some gels. Plasminogen-free gels were used to confirm that the activity detected was plasminogen dependent. Bands of activity were visualized against a dark (blue) background as clear zones where casein degradation occurred. The volume of medium analyzed was corrected for cell number (total DNA). To correct for gel-to-gel variation, all samples were expressed relative to a control sample (spent medium from a culture of cells from small follicles) that was included in every gel.

Western Blot

SerpinE2 protein abundance was analyzed by Western blot. Media samples were concentrated by lyophilization (Dura-Dry MP Corrosion Resistant Freeze-Dryer, Stone Ridge, NY), and the volume analyzed was adjusted to correct for cell number. Samples were subjected to electrophoresis at 120 V for 90 min in 10% denaturing polyacrylamide gels. Proteins were then electrotransferred onto nitrocellulose membrane (0.45 µm; Bio-Rad, Hercules, CA) at 22 V overnight at 4°C in transfer buffer (39 mM glycine, 48 mM Tris-base, 0.037% SDS and 20% methanol, pH 8.3). After blocking for 1 h in TTBS (0.2% Tween 20, 10 mM Tris-HCl, 150 mM NaCl), blots were incubated with 1:5000 rabbit anti-bovine SerpinE2 [22] for 4 h with agitation, followed by three washes (10 min each) with 0.2% TTBS. The blots were then incubated with 1:2500 alkaline phosphatase-linked anti-rabbit IgG (Sigma-Aldrich) for 1.5 h with agitation, followed by three washes (10 min each) with 0.2% TTBS. Finally, the blots were incubated with NBT/BCIP solution (Roche Diagnostics, Indianapolis, IN). Rainbow Coloured Protein Molecular Weight Marker (Pharmacia, Piscataway, NJ) was used to estimate molecular size of the target protein, and bovine follicular fluid (2 µl) was used as positive control.

Semi-Quantitative Reverse-Transcription Polymerase Chain Reaction

Plau, Plat, and Serpine2 mRNA were assayed by reverse-transcription polymerase chain reaction (RT-PCR). Total RNA (1 µg) was reverse transcribed in the presence of 0.2 mM oligo(dT) primer and 200 U SuperScript II (Invitrogen Life Technologies), 2.5 mM MgCl2, 0.5 mM dideoxy-nucleotide triphosphate (dNTPs) mix, and 10 mM dithiothreitol (DTT) in a volume of 50 µl. The RNA samples were heated to 70°C for 10 min and added to the prewarmed (42°C) reaction mixture. The reaction was incubated for 50 min at 50°C, then for 15 min at 70°C. Residual RNA was removed by incubating 20 min at 37°C with 1 µl of RNase H.

A duplex reaction was performed for Serpine2 in which both Serpine2 primers and primers for glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were amplified together for each sample, whereas Plau and Plat primers and Gapdh primers were amplified separately for each sample. The primers used were the following: Plau sense, 5'-GTCTGGTGAATCGAACTGTGGC-3'; antisense, 5'-GGCTGCAAACCAAGGCTG-3' [32]; Plat sense, 5'-AAGGTTGCAGAAGAAGATGG-3'; antisense, 5'-GTGAGGCGGGTACCTCTCCTGGAA-3' [3]; Serpine2 sense, 5'-TCCGTGACGTTGCCCTCTGTG-3'; antisense, 5'-CCGTGATCTCCACAAACCCTT-3' [22]; Gapdh sense, 5'-TGTTCCAGTATGATTCCACC-3'; antisense, 5'-TCCACCACCCTGTTGCTG-3' [33].

An aliquot (0.4 µl) of the reverse transcription reaction was amplified by PCR using 0.2 µl (2.5 U) Taq Polymerase (Amersham Pharmacia Biotech Inc., Oakville, ON, Canada) in a 20-µl PCR buffer (Amersham) containing 0.1 mM dNTP mix and 0.2 µM specific primers. Target cDNA was amplified under the following conditions: 1) an initial denaturation step for 3 min (Plat) and 5 min (Serpine2, Plau) at 94°C; 2) amplification cycles with denaturation at 94°C for 30 sec, annealing for 45 sec at 65°C (Plau), 55°C (Plat), and 62°C (Serpine2), and elongation at 72°C for 1 min; and 3) final elongation at 72°C for 5 min.

Semiquantitative RT-PCR was validated for each gene product. Preliminary experiments verified that the PCR product increased with amount of RNA in the RT reaction. Reactions were performed for 30 cycles for Plau, 26 cycles for Plat and Gapdh, and for 24 cycles for Serpine2. The PCR products were separated on 1% agarose gels with 0.001% ethidium bromide and visualized under UV. Quantification of band intensity was performed with NIH Image software. Target gene mRNA abundance was expressed relative to Gapdh mRNA abundance.

Northern Blot

To verify the RT-PCR results, we performed Northern hybridizations on a subset of samples where the amount of RNA available permitted. The complete Serpine2 cDNA [22] was subcloned into pBK-CMV phagemid and digested by EcoRI and XhoI restriction endonuclease to generate radioactive probes. To prepare Plat and Gapdh cDNA probes, PCR products (see above) were cloned into pGEM-T Easy Vector (Promega, Madison, WI) and digested by EcoRI enzyme for Plat and by PvuII enzyme for Gapdh, respectively. The cDNA probes were labeled with [32P]dCTP (DuPont NEN Research Products, Boston, MA) using the Random Primed DNA Labeling Kit (Roche), and purified by centrifugation through a Microspan S-200 HR column (Pharmacia).

Electrophoresis of 15 µg total RNA, performed through a 1% denaturing formaldehyde-agarose gel, was followed by overnight capillary transfer onto a nylon membrane (Hybond-N; Amersham). Membranes were UV cross-linked in a commercial UV chamber (Bio-Rad, Mississauga, ON, Canada) and incubated for 2 h at 65°C in prehybridization solution containing 5x saline-sodium phosphate-EDTA buffer (SSPE), 5x Denhardt solution, 0.5% SDS, 10% dextran sulfate, and 1% herring sperm DNA (10 mg/ml). Upon adding the purified probe, hybridization was carried out in hybridization buffer at 65°C overnight. After hybridization, membranes were washed in 2x SSPE-0.1% SDS twice at room temperature and twice at 65°C (15 min each). The labeled membranes were exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) at –70°C in the presence of an intensifying screen.

Statistical Analyses

Each experiment was carried out at least three times. Data are presented as mean ± standard error of the mean (SEM). The data were analyzed by ANOVA with follicle size, day of culture, cell subpopulation, and culture replicate as main effects. Where main effects and/or interactions involving follicle size were found, effects of cell subpopulation and/ or day of culture on antral and basal cells were analyzed separately. Data were transformed to logarithms when not normally distributed. Means comparisons were performed with the Tukey-Kramer HSD test. All analyses were performed with JMP software (SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To confirm the identity of PA activities observed, activities were examined in bovine granulosa cell lysates by zymography. Bovine tPA migrated slightly less (approximately 70 kDa) than the human standard (66 kDa) and was not inhibited by amiloride (Fig. 1). Bovine uPA activity migrated at approximately 45 kDa (compared with 53 kDa for the human standard) and was attenuated by amiloride. No proteolytic activity was observed in plasminogen-free gels (Fig. 1). Granulosa cells secreted predominantly tPA, with low and variable amounts of uPA being detected (Fig. 2). Although it appeared that cells from small follicles secreted low amounts of uPA throughout the culture period and that cells from medium and large follicles secreted uPA mainly during the first 2 days of culture (see zymograph in Fig. 2C), the proteolytic bands were too weak or too often absent for accurate quantification and analysis.



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  		FIG. 1. Zymographic demonstration of PA activity in bovine granulosa cells cultured for 6 days in serum-free medium. PA activity in granulosa cell lysate (bGC) was compared with human (hum) tPA and uPA standards. These samples were run in a 10% nondenaturing PAGE gel. Specificity of PA activity was tested by running a cell lysate sample in a 10% gel in the absence (–AM) or presence of 1 mM amiloride (+AM) and in a plasminogen-free gel (–PL). Molecular weight markers (MW x 10–3) are shown



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  		FIG. 2. Secreted PA activity from cultured bovine granulosa cells from (A) small (2–5 mm diameter), (B) medium (6–8 mm), and (C) large (>8 mm) follicles. Antral (open bars) and basal (solid bars) granulosa cells were cultured with 1 ng/ml bFSH, 10 ng/ml insulin, and 10 ng/ml IGF-1 for 2, 4, or 6 days. A control medium sample was used to normalize the variation between gels. The loading volume was adjusted to correct for cell number. Inserts show representative zymographs. Data are least-squares means (relative units) ± SEM. Bars with different letters within follicle size group are significantly different. M, Molecular weight markers

There were significant effects (P < 0.001) of follicle size and day of culture on secreted tPA activity. Overall, secreted tPA activity was higher from cells of small follicles compared with cells of medium and large follicles, and was higher on Day 6 of culture compared with Day 2. When tPA secretion from cells of small, medium, and large follicles were analyzed separately, there was a significant effect of cell subpopulation and an interaction between cell subpopulation and day of culture (P < 0.05) for cells from small follicles. Secreted tPA activity increased with time for antral cells, but there was no significant increase with time for basal cells (Fig. 2A). On Day 2 of culture, basal cells secreted significantly more tPA than did antral cells, whereas on Day 6 antral cells secreted slightly (P = 0.06) more tPA compared with basal cells. There were significant effects of time in culture (P < 0.05) but not of cell subpopulation in cultures of cells from medium and large follicles (Fig. 2, B and C).

There were no main effects of culture or follicle size on cellular tPA activity, but there were significant effects of time in culture and of follicle size for cellular uPA activity (Fig. 3). Cellular uPA was significantly lower on Day 6 of culture than on Day 2 for cells from medium and large follicles (P < 0.01), but not from small follicles. There was no effect of cell subpopulation.



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  		FIG. 3. Cellular PA activity from cultured bovine granulosa cells from (A) small (2–5 mm diameter), (B) medium (6–8 mm), and (C) large (>8 mm) follicles. Antral (open bars) and basal (solid bars) granulosa cells were cultured with 1 ng/ml bFSH, 10 ng/ml insulin, and 10 ng/ml IGF-1 for 2, 4, or 6 days. A control medium sample was used to normalize the variation between gels. Inserts show representative zymographs. Data are least-squares means (relative units) ± SEM. Asterisks indicate means significantly different from Day 2

Abundance of Plat expression was measured by semiquantitative RT-PCR. There were no significant effects of follicle size, cell population, or time in culture (data not shown). RT-PCR results were verified by performing Northern analysis on two replicates of cultures from medium follicles (as there was sufficient RNA); there was a significant correlation between Northern and PCR data (r = 0.6, P < 0.05, n = 12). We could not detect Plau mRNA in samples from cultured granulosa cells after 30 cycles of PCR, although the positive control (uterus) provided a strong band at the expected size.

Granulosa cells from all follicle size groups secreted SerpinE2 as detected by Western blotting. There were significant main effects of cell population and follicle size, and an interaction between day of culture and follicle size (P < 0.01). Overall, cells from large follicles secreted more SerpinE2 compared with cells from small and medium follicles. SerpinE2 secretion from basal but not antral cells of small and of medium follicles increased with time in culture (Fig. 4, A and B; P < 0.05), whereas SerpinE2 secretion from antral (and not basal) cells of large follicles decreased with time in culture (Fig. 4C; P < 0.01).



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  		FIG. 4. Western analysis of secreted protease nexin-1 (SerpinE2) from cultured bovine antral (open bars) and basal (solid bars) granulosa cells from (A) small (2–5 mm diameter), (B) medium (6–8 mm), and (C) large (>8 mm) follicles. Granulosa cells were cultured with 1 ng/ml bFSH, 10 ng/ml insulin, and 10 ng/ml IGF-1 for 2, 4, or 6 days. A control sample (follicular fluid) was used to normalize the variation between blots. The loading volume was adjusted to correct for cell number. Inserts show representative blots. Data are least-squares means (relative units) ± SEM. Asterisks indicate differences between means (P < 0.05)

There were significant effects of time in culture and follicle size on Serpine2 mRNA levels, and an interaction between time and follicle size (P < 0.001). When data from different follicle sizes were analyzed separately, there were no main effects of time or cell population on Serpine2 mRNA levels in cells from small or medium follicles, but there was a significant decrease (P < 0.01) in Serpine2 mRNA with time in culture in cells of large follicles (Fig. 5). RT-PCR results were verified by performing Northern analysis on two replicates of cultures from medium follicles (as there was sufficient RNA); there was a significant correlation between Northern and PCR data (r = 0.65, P < 0.05, n = 12). Overall, there was a significant correlation between SerpinE2 protein and mRNA levels (r = 0.5; P < 0.01); when the data from different follicle sizes were analyzed separately there was a correlation between protein and mRNA for large (r = 0.6, P < 0.01) but not small or medium follicles.



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  		FIG. 5. Serpine2 mRNA levels in cultured bovine antral (open bars) and basal (solid bars) granulosa cells from (A) small (2–5 mm diameter), (B) medium (6–8 mm), and (C) large (>8 mm) follicles. Granulosa cells were cultured with 1 ng/ml bFSH, 10 ng/ml insulin, and 10 ng/ml IGF-1 for 2, 4, or 6 days. Serpine2 mRNA was expressed relative to Gapdh. Inserts show representative agarose gels. Data are least-squares means ± SEM. Bars with different letters are significantly different (P < 0.05). Note the difference in scale of the y-axis for large follicles

Estradiol secretion was significantly affected by follicle size, with interactions between follicle size and time and between follicle size and cell population. For cells of small follicles, estradiol secretion increased with time in culture for antral but not basal cells, whereas for medium follicles estradiol secretion increased with time for basal and not for antral cells (Fig. 6). Estradiol secretion from cells of large follicles decreased significantly with time in culture (Fig. 6; P < 0.01). There was a main effect of time in culture on progesterone secretion, and no main effects of follicle size or cell population; progesterone concentrations were consistently higher on Days 4 and 6 of culture compared with Day 2 (Fig. 6; P < 0.05).



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  		FIG. 6. Culture medium estradiol and progesterone concentrations after culture of bovine antral (open bars) and basal (solid bars) granulosa cells from (A) small (2–5 mm diameter), (B) medium (6–8 mm), and (C) large (>8 mm) follicles. Granulosa cells were cultured with 1 ng/ml bFSH, 10 ng/ml insulin, and 10 ng/ml IGF-1 for 2, 4, or 6 days. Data are least-squares means ± SEM. Within follicle size, bars with different letters are significantly different (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is, to our knowledge, the first study to describe the secretion of members of the PA system from granulosa cells at different stages of development in a nonrodent mammal. Bovine granulosa cells secreted predominantly tPA in culture, with very low amounts of uPA secreted. This is consistent with measurements of PA activity in bovine follicular fluid [12]. In rats, uPA has been described as a major secreted PA in some studies [3], whereas it is low or absent in other studies [6, 34]. The present data show that, overall, secreted tPA activity was higher from cells of small and less differentiated follicles compared with that of the more differentiated medium and large follicles. This is in contrast to the lower level of tPA activity in undifferentiated versus differentiated rat follicles [27] and presents an important species difference in PA secretion at these stages of follicle growth. A species difference in tissue uPA and tPA activity between rodents and cattle has been described for the periovulatory period [12].

Also novel is the measurement of secreted SerpinE2 from granulosa cells. Serpine2 mRNA and protein has previously been localized in rat [9, 21] and bovine [22] granulosa cells by in situ techniques, and has been detected by Western blotting in bovine follicular fluid [22]. During the first 2 days of culture particularly, granulosa cells of large follicles secreted more SerpinE2 than did cells from small follicles, in agreement with the relative intensity of immunostaining described in rat and cow follicles [21, 22]. As Serpine1 or Serpinb2 expression would not be expected in bovine granulosa cells until after the LH surge [18], SerpinE2 is a candidate for the regulation of PA activity within the membrana granulosa of growing follicles.

The pattern of changes of secreted tPA and SerpinE2 differed. Secretion of tPA from antral cells of small follicles increased with time of culture, whereas SerpinE2 secretion did not change. Conversely, tPA secretion from basal cells did not change but SerpinE2 secretion increased. This difference may reflect the needs of the sublayers of granulosa cells. Although the separation of ‘antral’ and ‘basal’ may be simplistic, the basal cells are nearer the basal lamina and more firmly attached to ECM components than are antral cells. In sheep, there are immunocytochemical differences in the intensity of staining for fibronectin and collagen between antral and basal granulosa cells [35]. A difference between cell subpopulations was not seen in cells of medium and large follicles in the present study, potentially reflecting the changes in the basal lamina ECM that occur with follicle growth (reviewed in [36]).

The most striking divergence between tPA and SerpinE2 secretion occurred with cells of large follicles, for which tPA secretion increased with time of culture and SerpinE2 secretion decreased. This would result in a net increase in extracellular PA activity. This is consistent with increases in Plat expression and protein secretion observed with rat granulosa cells [6]. The relationship between tPA and SerpinE2 is related to stage of follicle development, as tPA secretion from cells of medium follicles increased with time of culture in a manner very similar to that of large follicles, but SerpinE2 secretion did not decrease from cells of medium follicles. The most likely explanation is that the cells of large follicles undergo at least partial luteinization in culture, whereas those of medium follicles do not. This is indicated by the steroid data, which show that estradiol secretion decreases with time of culture from cells of large but not medium follicles, as previously observed with this cell model [29]. This is supported in part by data from studies of rats, which show increased Plat expression and enzyme activity during early development of the corpus luteum, and in granulosa cells following the ovulatory LH surge [3, 37]; it is possible that some of the large follicles may have been periovulatory follicles, and thus exposed to elevated concentrations of LH in vivo. Further, there was increased expression of genes encoding collagen and the MMP inhibitor TIMP-1 during in vitro luteinization of bovine granulosa cells [38], demonstrating that changes to the ECM occur during luteinization.

Another explanation is a change in the ECM that occurs during follicle growth, which may in consequence alter the amount and/or type of protease activity secreted and granulosa steroidogenesis. It has been shown that collagen type 1 levels within the granulosa cell layer increase significantly during follicle development in sheep [35] and that estradiol secretion from ovine granulosa cells of large follicles is inhibited when cultured in the absence of collagen [39]. Thus, the cells of large follicles in the present study may have an increased requirement for collagen type 1 that was not met by the culture conditions, resulting in reduced estradiol (but not progesterone) secretion ([39], present study). The cellular response to this collagen deprivation may therefore be a reduction in the normally high secretion of SerpinE2 in order to increase extracellular protease activity and alter the local ECM structure.

Steady-state levels of Serpine2 mRNA largely reflected secreted protein levels. Over the first 2 days of culture, Serpine2 mRNA levels were higher in cells of large follicles compared with those of small and medium follicles, consistent with data from Northern analysis in cattle [22, 40]. Similarly, the decrease in SerpinE2 secretion observed during culture of cells from large follicles was tightly coordinated with a decline in Serpine2 mRNA levels. Thus we conclude that SerpinE2 secretion is controlled at the transcriptional level, at least in vitro. This does not appear to be the case for PA, however, as consistent increases in secreted tPA activity were not correlated with Plat mRNA levels. Although the measurement of tPA activity may be confounded by inhibitor activity, we do not believe this to be the case in the present study for the following reasons. First, when SerpinE1 inhibits tPA activity, a reversible protein-protein complex is formed that is visible by zymography as an additional high molecular mass lytic zone [34]. Second, potential complexes between SerpinE2 and PA should also be visible on Western blots as an additional high molecular mass band. As we did not observe lytic or protein bands corresponding to a PA-inhibitor complex, we conclude that the majority of the PA activity and SerpinE2 protein measured in culture medium occurs in a ‘free’ noncomplexed state. Collectively, these data suggest that secreted tPA concentrations are controlled at the posttranslational level, possibly involving regulation of secretory mechanisms.

We were unable to detect Plau gene expression in bovine granulosa cells in vitro, and uPA was a minor secretory product. Most of the uPA activity was detected in cell lysates, most likely bound to the cell surface [3]. This is in agreement with the readily detectable uPA activity in cell lysates of the bovine follicle wall [12]. The decrease in secreted and cell-associated uPA in cells of medium and large bovine follicles in vitro is consistent with the developmental decrease seen in rats [27, 28, 41]. In cultures of cells from medium and large follicles, cell-associated uPA activity decreased with time of culture, whereas secreted tPA activity increased. It is not clear what effect this change would have on net PA activity in the ECM immediately surrounding the cell, nor the impact of altered SerpinE2 concentrations on local tPA and uPA activity.

In conclusion, we have demonstrated that secretion of tPA and SerpinE2 from granulosa cells and cell-associated uPA activity are regulated in a follicular stage-dependent manner in cattle. There appear to be several differences between rodents and cattle in terms of PA secretion, which makes generalization difficult, but a common theme is a decrease in granulosa cell uPA content as follicles develop. During the first 2 days of culture, this decrease in cell-associated uPA activity occurred as SerpinE2 secretion increased, suggesting a functional link between these two proteins during follicle development. As SerpinE2 is the only known PA inhibitor secreted by the granulosa cell layer, it may play an important role in tissue remodeling of this follicular compartment during early follicle growth.


   ACKNOWLEDGMENTS
 
We thank Drs. A.K. Goff and A. Bélanger for steroid antibodies, and Dr. A.F. Parlow and the NIDDK National Hormone and Peptide Program for providing bovine FSH. We are grateful to Dr. Mark Dow for advice on zymography.


   FOOTNOTES
 
1 This work was supported by NSERC (Canada) and FQRNT (Québec). Back

2 Correspondence: C.A. Price, CRRA, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000 St-Hyacinthe, Québec Canada J2S 7C6. FAX: 450 778 8103; christopher.price{at}umontreal.ca Back

Received: 12 March 2004.

First decision: 16 April 2004.

Accepted: 21 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Smith MF, McIntush EW, Ricke WA, Kojima FN, Smith GW. Regulation of ovarian extracellular matrix remodelling by metalloproteinases and their tissue inhibitors: effects on follicular development, ovulation and luteal function. J Reprod Fertil Suppl 1999 54:367-381[Medline]
  2. Blasi F, Vassalli JD, Dano K. Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors. J Cell Biol 1987 104:801-804[Free Full Text]
  3. Macchione E, Epifano O, Stefanini M, Belin D, Canipari R. Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles. Biol Reprod 2000 62:895-903[Abstract/Free Full Text]
  4. Canipari R, Strickland S. Plasminogen activator in the rat ovary. Production and gonadotropin regulation of the enzyme in granulosa and thecal cells. J Biol Chem 1985 260:5121-5125[Abstract/Free Full Text]
  5. Canipari R, Strickland S. Studies on the hormonal regulation of plasminogen activator production in the rat ovary. Endocrinology 1986 118:1652-1659[Abstract]
  6. Galway AB, Oikawa M, Ny T, Hsueh AJ. Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C. Endocrinology 1989 125:126-135[Abstract]
  7. Politis I, Srikandakumar A, Turner JD, Tsang BK, Ainsworth L, Downey BR. Changes in and partial identification of the plasminogen activator and plasminogen activator inhibitor systems during ovarian follicular maturation in the pig. Biol Reprod 1990 43:636-642[Abstract]
  8. Canipari R, O'Connell ML, Meyer G, Strickland S. Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator. J Cell Biol 1987 105:977-981[Abstract/Free Full Text]
  9. Hägglund AC, Ny A, Liu K, Ny T. Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation. Endocrinology 1996 137:5671-5677[Abstract]
  10. Lafrance M, Croze F, Tsang BK. Influence of growth factors on the plasminogen activator activity of avian granulosa cells from follicles at different maturational stages of preovulatory development. J Mol Endocrinol 1993 11:291-304[Abstract]
  11. Yamada M, Horiuchi T, Oribe T, Yamamoto S, Matsushita H, Gentry PA. Plasminogen activator activity in the bovine oocyte-cumulus complex and early embryo. J Vet Med Sci 1996 58:317-322[Medline]
  12. Dow MP, Bakke LJ, Cassar CA, Peters MW, Pursley JR, Smith GW. Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue. Biol Reprod 2002 66:1413-1421[Abstract/Free Full Text]
  13. Kruithof EK. Plasminogen activator inhibitors—a review. Enzyme 1988 40:113-121[Medline]
  14. Roberts RM, Mathialagan N, Duffy JY, Smith GW. Regulation and regulatory role of proteinase inhibitors. Crit Rev Eukaryot Gene Expr 1995 5:385-436[Medline]
  15. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW, Pemberton PA, Remold-O'Donnell E, Salvesen GS, Travis J, Whisstock JC. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 2001 276:33293-33296[Free Full Text]
  16. Liu YX, Cajander SB, Ny T, Kristensen P, Hsueh AJ. Gonadotropin regulation of tissue-type and urokinase-type plasminogen activators in rat granulosa and theca-interstitial cells during the periovulatory period. Mol Cell Endocrinol 1987 54:221-229[CrossRef][Medline]
  17. Chun SY, Popliker M, Reich R, Tsafriri A. Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary. Biol Reprod 1992 47:245-253[Abstract]
  18. Dow MP, Bakke LJ, Cassar CA, Peters MW, Pursley JR, Smith GW. Gonadotrophin surge-induced upregulation of mRNA for plasminogen activator inhibitors 1 and 2 within bovine periovulatory follicular and luteal tissue. Reproduction 2002 123:711-719
  19. Piquette GN, Crabtree ME, el-Danasouri I, Milki A, Polan ML. Regulation of plasminogen activator inhibitor-1 and -2 messenger ribonucleic acid levels in human cumulus and granulosa-luteal cells. J Clin Endocrinol Metab 1993 76:518-523[Abstract]
  20. Leonardsson G, Peng XR, Liu K, Nordstrom L, Carmeliet P, Mulligan R, Collen D, Ny T. Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators. Proc Natl Acad Sci U S A 1995 92:12446-12450[Abstract/Free Full Text]
  21. Hasan S, Hosseini G, Princivalle M, Dong JC, Birsan D, Cagide C, de Agostini AI. Coordinate expression of anticoagulant heparan sulfate proteoglycans and serine protease inhibitors in the rat ovary: a potent system of proteolysis control. Biol Reprod 2002 66:144-158[Abstract/Free Full Text]
  22. Bédard J, Brûlé S, Price CA, Silversides DW, Lussier JG. Serine protease inhibitor-E2 (SERPINE2) is differentially expressed in granulosa cells of dominant follicle in cattle. Mol Reprod Dev 2003 64:152-165[CrossRef][Medline]
  23. Peng XR, Hsueh AJ, Ny T. Transient and cell-specific expression of tissue-type plasminogen activator and plasminogen-activator-inhibitor type 1 results in controlled and directed proteolysis during gonadotropin-induced ovulation. Eur J Biochem 1993 214:147-156[Medline]
  24. Liu YX, Liu K, Feng Q, Hu ZY, Liu HZ, Fu GQ, Li YC, Zou RJ, Ny T. Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey. Endocrinology 2004 145:1767-1775[Abstract/Free Full Text]
  25. Smokovitis A, Kokolis N, Ploumis T. Great variation in the response of tissue plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition to endotoxin, aspirin and endotoxin after administration of aspirin. Thromb Res 1988 50:495-505[CrossRef][Medline]
  26. Colgin DC, Murdoch WJ. Evidence for a role of the ovarian surface epithelium in the ovulatory mechanism of the sheep: secretion of urokinase-type plasminogen activator. Anim Reprod Sci 1997 47:197-204[CrossRef][Medline]
  27. Karakji EG, Tsang BK. Follicular stage-dependent regulation of rat granulosa cell plasminogen activator system by transforming growth factor-alpha in vitro. Biol Reprod 1995 52:411-418[Abstract]
  28. Li M, Karakji EG, Xing R, Fryer JN, Carnegie JA, Rabbani SA, Tsang BK. Expression of urokinase-type plasminogen activator and its receptor during ovarian follicular development. Endocrinology 1997 138:2790-2799[Abstract/Free Full Text]
  29. Gutiérrez CG, Campbell BK, Webb R. Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics. Biol Reprod 1997 56:608-616[Abstract]
  30. Silva JM, Price CA. Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro. Biol Reprod 2000 62:186-191[Abstract/Free Full Text]
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin Phenol reagent. J Biol Chem 1951 193:265-275[Free Full Text]
  32. Balcerzak D, Querengesser L, Dixon WT, Baracos VE. Coordinate expression of matrix-degrading proteinases and their activators and inhibitors in bovine skeletal muscle. J Anim Sci 2001 79:94-107[Abstract/Free Full Text]
  33. Tsai JS, Curran BH, Sternick ES, Engler MJ. The measurement of linear accelerator isocenter motion using a three-micrometer device and an adjustable pointer. Int J Radiat Oncol Biol Phys 1996 34:189-195[CrossRef][Medline]
  34. Liu YX, Peng XR, Ny T. Tissue-specific and time-coordinated hormone regulation of plasminogen-activator-inhibitor type I and tissue-type plasminogen activator in the rat ovary during gonadotropin-induced ovulation. Eur J Biochem 1991 195:549-555[Medline]
  35. Huet C, Monget P, Pisselet C, Monniaux D. Changes in extracellular matrix components and steroidogenic enzymes during growth and atresia of antral ovarian follicles in the sheep. Biol Reprod 1997 56:1025-1034[Abstract]
  36. Rodgers RJ, Irving-Rodgers HF, Russell DL. Extracellular matrix of the developing ovarian follicle. Reproduction 2003 126:415-424[Abstract]
  37. Liu K, Brandstrom A, Liu YX, Ny T, Selstam G. Coordinated expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 during corpus luteum formation and luteolysis in the adult pseudopregnant rat. Endocrinology 1996 137:2126-2132[Abstract]
  38. Zhao Y, Luck MR. Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture. Reprod Fertil Dev 1996 8:259-266[CrossRef][Medline]
  39. Huet C, Pisselet C, Mandon-Pepin B, Monget P, Monniaux D. Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function. J Endocrinol 2001 169:347-360[Abstract]
  40. Fayad T, Levesque V, Sirois J, Silversides DW, Lussier JG. Gene expression profiling of differentially expressed genes in granulosa cells of bovine dominant follicles using suppression subtractive hybridization. Biol Reprod 2004 70:523-533[Abstract/Free Full Text]
  41. Ny T, Bjersing L, Hsueh AJ, Loskutoff DJ. Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins. Endocrinology 1985 116:1666-1668[Abstract]



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