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Endocrinology, doi:10.1210/en.2007-1736
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Endocrinology Vol. 149, No. 10 5070-5077
Copyright © 2008 by The Endocrine Society

Expression and Localization of the Serine Proteases High-Temperature Requirement Factor A1, Serine Protease 23, and Serine Protease 35 in the Mouse Ovary

Patrik Wahlberg, Åsa Nylander, Nina Ahlskog, Kui Liu and Tor Ny

Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden

Address all correspondence and requests for reprints to: Professor Tor Ny, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. E-mail: tor.ny{at}medchem.umu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RELEASE OF the mature ovum during the process of ovulation is essential for mammalian reproduction. This process is induced by a preovulatory surge of LH from the pituitary gland and ends with the rupture of the ovarian follicle. Extracellular matrix (ECM)-degrading proteases have been suggested to play essential roles for ovulation to occur. In particular, the plasminogen activator (PA) and matrix metalloproteinase (MMP) systems have repeatedly been implicated as important mediators of follicular rupture (1, 2). Furthermore, the PA and MMP systems have been shown to cooperate in several other tissue remodeling processes such as wound healing and placentation. In these cases, suppressing either the PA or the MMP system had a moderate effect on the tissue remodeling processes, however, when both protease systems were suppressed, a dramatic effect was recorded (3, 4).

We have recently studied the ovulation efficiency in plasminogen-deficient mice treated with a broad-spectrum MMP inhibitor, and found that the combined suppression of both the PA and MMP systems had only a modest effect on the ovulation efficiency (5). This suggests that these protease classes may be of less importance in this particular process than previously anticipated, and that proteases from other classes may play crucial roles in ovulation.

Because the mammalian genome contains hundreds of proteases from several different classes (6), we have in this study used a microarray approach to identify proteases that are expressed during ovulation and that may be regulated by gonadotropins. We found three extracellular serine proteases, and explored their expression patterns and levels during different stages of mouse ovary development and maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
C57BL/6 wild-type mice were obtained from Taconic (Ry, Denmark). The mice were kept on a 12-h light, 12-h dark cycle with the light cycle initiated at 0600 h, and were fed chow and water ad libitum. Experimental protocols were approved by the regional ethical committee of Umeå University.

Twenty-five-day-old female mice were injected ip with 4 IU pregnant mare serum gonadotropin (PMSG) to stimulate follicular development and 48 h later with 5 IU human chorionic gonadotropin (hCG) to induce ovulation. Ovulation normally takes place at 10–12 h after hCG injection (7). Mice were killed at different time points, and the ovaries were collected.

For PMSG withdrawal, 23-d-old female mice were injected ip with 4 IU PMSG, killed 96 h later, and the ovaries were collected and frozen in optimal cutting temperature compound (Histolab, Gothenburg, Sweden) (8).

To induce pseudopregnancy in mice for obtaining physiological corpora lutea (CLs), mature 8- to 12-wk-old female mice were housed with vasectomized males until a vaginal mating plug was detected, which defined d 1 pseudopregnancy. Mice were killed at different time points, and the ovaries were collected.

Induced CL regression was achieved by injecting pseudopregnant females on d 6 of pseudopregnancy with a single sc dose of 2 µg of the stable prostaglandin (PG)-F2{alpha} analog cloprostenol (Sigma-Aldrich, Stockholm, Sweden) in 100 µl 0.9% sodium chloride, as adapted from Liu et al. (9). The mice were killed after 72 h, and the ovaries were collected and frozen in optimal cutting temperature compound.

Ovaries used for microarray or real-time PCR experiments were used directly, whereas ovaries used for in situ hybridization experiments were stored at –80 C.

Microarray analysis of gene expression
Total ovarian RNA was extracted with the Ultraspec solution from Biotecx (Houston, TX) according to the protocol supplied by the manufacturer. Total RNA was further purified with RNeasy spin columns from QIAGEN (Hilden, Germany), and RNA integrity was confirmed with agarose/formaldehyde gels. The pure total RNA was then amplified, labeled, and hybridized to the Mouse Expression Array 430A from Affymetrix, Inc. (Santa Clara, CA), according to the manufacturer’s instructions. Quality was confirmed for each step in the procedure. For each time point, two to three arrays were used, and for each array, a pool of high-quality RNA from five mice was used.

Real-time PCR
Total ovarian RNA was extracted with Ultraspec solution, and RT-PCR was performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Sundbyberg, Sweden) according to the manufacturer’s instructions. Real-time PCR was performed using the iCycler Multicolor PCR Detection System (Bio-Rad Laboratories). For fluorescent quantization, iQ SYBR Green Supermix (Bio-Rad Laboratories) was used according to the accompanying protocol. The primers (DNATechnology, Copenhagen, Denmark) were selected using Beacon Designer 6 (Premier Biosoft International, Palo Alto, CA) and submitted to Basic Local Alignment Search Tool search to verify the correct gene target sequence (Table 1Go). The method was validated at each step according to User Bulletin no. 2 (Applied Biosystems, Foster City, CA). The program chosen used an initial denaturation at 96 C for 3 min, followed by 40 cycles of denaturation (30 sec at 95 C), annealing (30 sec at 60 C), and extension (30 sec at 72 C). Ovaries from four to five mice were used for each RNA preparation and each time point. All measurements are the average of five measurements performed on two occasions and on two different RNA preparations (a total of 20 measurements per time point). The relative expression of the target mRNA was analyzed using the iCycler iQ program and the comparative {Delta}{Delta}CT method (10) according to User Bulletin no. 2. In short, the expression level of each mRNA was normalized against the expression of ribosomal L19 mRNA and then normalized against the respective expression in the untreated control sample. The resulting relative expression level was then expressed as 2{Delta}{Delta}CT. The data points were analyzed by one-way ANOVA, followed by the Bonferroni post hoc test using SPSS software (SPSS, Inc., Chicago, IL). A P value of less than 0.05 was considered statistically significant.


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  		TABLE 1. Primers for quantitative real-time PCR

 
Cloning of riboprobes
RT-PCR on ovarian RNA was performed for high-temperature requirement factor A1 (HtrA1) [National Center for Biotechnology Information (NCBI) accession no. NM_019564; nucleotides 468–892], serine protease 23 (PRSS23) (NCBI accession no. NM_029614; nucleotides 328–733), and serine protease 35 (PRSS35) (NCBI accession no. NM_178738; nucleotides 2791–3200) (Table 1Go). PCR fragments were separated and purified from agarose gels, ligated into pGEMT vectors (Promega, Madison, WI), and sequenced to confirm the correct identities. Plasmids were linearized with appropriate restriction enzymes, and then transcribed in vitro to generate both sense and antisense riboprobes. The probes were labeled with digoxigenin using the DIG RNA Labeling Kit from Roche Molecular Biochemicals (Mannheim, Germany).

In situ hybridization
In situ hybridization was performed on 10-µm cryostat sections using digoxigenin-labeled antisense riboprobes as previously described (11, 12). Images were taken with a Leica DC300F digital camera attached to a Leica DM LB microscope (Leica, Wetzlar, Germany).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining
TUNEL staining was performed on 10-µm cryosections with the In Situ Cell Death Detection Kit, POD from Roche Diagnostics (Mannheim, Germany). The slides were mounted with Mount Quick solution (Daido Sangyo Co., Ltd., Tokyo, Japan), and images were taken as described previously.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of ovarian proteases with microarray analysis
Follicular development and ovulation involve extensive tissue remodeling and degradation of ECM components (1, 2). To identify proteases that are expressed in these processes, we extracted RNA from periovulatory ovaries obtained from gonadotropin-primed immature mice at different time points, and analyzed the RNA expression with Affymetrix microarrays. As shown in Fig. 1Go, we found several proteases and protease inhibitors that were regulated by gonadotropins, including some that were already known to be expressed in the ovary. Most of the genes regulated by PMSG or hCG treatment were up-regulated (Fig. 1Go, A and B, respectively). There were also a few proteases and inhibitors that were down-regulated by PMSG or hCG treatment (Fig. 1Go, C and D, respectively). However, most of the proteases and inhibitors that were highly expressed did not appear to be regulated during ovulation (Fig. 1EGo). Of the proteases detected this way, we selected three relatively highly expressed extracellular serine proteases for further analysis: HtrA1, PRSS23, and PRSS35. HtrA1 has been able to degrade some matrix components (13). PRSS23 and PRSS35 proteins belong to the trypsin class of serine proteases, but their proteolytic activities have not yet been characterized. According to the microarray data, HtrA1 was highly expressed but did not appear to be regulated much during ovulation (Fig. 1EGo). PRSS35 was up-regulated and PRSS23 was down-regulated during ovulation (Fig. 1Go, A and D, respectively).


Figure 1
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  		FIG. 1. Expression of proteases and protease inhibitors during ovulation. Microarray analysis of total ovarian RNA was performed as described in Materials and Methods. Protease and protease inhibitor transcripts that were deemed "present" by the Affymetrix software were sorted by their regulation by gonadotropins and visualized using the TIGR MultiExperiment Viewer (The Institute for Genomic Research, La Jolla, CA). A, Genes that were up-regulated by PMSG treatment. B, Genes that were up-regulated by hCG treatment. C, Genes that were down-regulated by PMSG treatment. D, Genes that were down-regulated by hCG treatment. E, Genes that were regulated less than 2-fold by gonadotropin treatment. The 20 genes with the highest expression level at 8 h after hCG treatment are shown. For clarity, proteasome subunits were omitted in E. The color scale represents the log2 expression levels normalized to the mean value of all probes in the respective sample. Green color represents low-expression levels, and red color represents high-expression levels. ADAMTS-1, A disintegrin and metalloproteinase with thrombospondin motifs-1; ctrl, control; TIMP-3, tissue inhibitor of metalloproteinase-3; tPA, tissue plasminogen activator; ADAM8, a disintegrin and metalloproteinase 8; ATPase, adenosyl triphosphatase; CNDP, cytosolic nonspecific dipeptidase; IMP2, inner membrane peptidase 2; MT1-MMP, membrane-type matrix metalloproteinase 1; PAI-3, plasminogen activator inhibitor 3; SUMO, small ubiquitin-like modifier.

 
To validate the microarray data, we performed real-time PCR to follow the relative expression level of these three proteases during ovulation (Fig. 2Go).


Figure 2
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  		FIG. 2. Real-time PCR quantification of HtrA1, PRSS23, and PRSS35 mRNA levels in mouse ovaries at different developmental stages. Total ovarian mRNA was collected from untreated immature mice (control), mice treated with PMSG (48 h) followed by hCG (4 or 8 h), after gonadotropin withdrawal (96 h after PMSG treatment), and from pseudopregnant mice at d (D) 1, 6, and 16 of pseudopregnancy, as well as after cloprostenol treatment (Clo). A–C, The relative expression of HtrA1 mRNA during follicular development, ovulation, and in the CL, respectively. D–F, The relative expression of PRSS23 mRNA during follicular development, ovulation, and in the CL, respectively. G–I, The relative expression of PRSS35 mRNA during follicular development, ovulation, and in the CL, respectively. The values are normalized to the ribosomal L19 mRNA levels and to the respective expression levels in ovaries from untreated control mice. The values are averages of 20 measurements and presented as mean ± SEM. Data points indicated by different letters are significantly different (P < 0.05).

 
Quantitative analysis of the HtrA1, PRSS23, and PRSS35 expression in the ovary
To obtain quantitative expression data and validate the microarray data, we performed real-time PCR on total ovarian RNA from different time points during gonadotropin-induced ovulation and in pseudopregnant mice. As shown in Fig. 2BGo, the expression of HtrA1 mRNA was slightly up-regulated after ovulation was induced by hCG treatment. Throughout pseudopregnancy, HtrA1 expression remained on a level comparable to that in control mice but tended to increase during CL regression (Fig. 2CGo). Gonadotropin withdrawal (96 h after PMSG treatment) and induction of premature CL regression with the stable PGF2{alpha} analog cloprostenol (9) had no major effect on the expression of HtrA1 mRNA (Fig. 2Go, A and C, respectively). The expression of PRSS23 mRNA was down-regulated by hCG treatment (Fig. 2EGo) and remained at a low level during pseudopregnancy (Fig. 2FGo). Gonadotropin withdrawal resulted in a slight up-regulation of PRSS23 mRNA expression (Fig. 2DGo). The expression of PRSS35 mRNA was dramatically induced after gonadotropin treatment (Fig. 2Go, G and H). The up-regulation was somewhat higher than that observed on microarrays; however, the general expression pattern was the same. PRSS35 mRNA was also highly expressed during CL formation and regression, and down-regulated during the functional CL stage (Fig. 2IGo). Gonadotropin withdrawal reverted the expression of PRSS35 mRNA to a level similar to that of untreated control mice (Fig. 2GGo). Similarly, when premature CL regression was induced by cloprostenol treatment, the expression of PRSS35 mRNA was up-regulated and reached a level comparable to that during physiological CL regression (Fig. 2IGo).

Expression of HtrA1, PRSS23, and PRSS35 during gonadotropin-induced follicular development and ovulation
To investigate the expression pattern of these proteases during follicular development and ovulation, immature mice were superovulated with PMSG and hCG. The localization of protease transcripts was then determined with in situ hybridization. As shown in Fig. 3AGo, we found that in ovaries from untreated control mice, HtrA1 mRNA was expressed at high levels in granulosa cells of tertiary follicles and at lower levels in smaller follicles. PRSS23 mRNA was highly expressed in granulosa cells of a subset of follicles at this time point (Fig. 3BGo). PRSS35 mRNA was expressed in the theca tissues of all kinds of follicles (Fig. 3CGo).


Figure 3
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  		FIG. 3. Expression of HtrA1, PRSS23, and PRSS35 during follicular development and ovulation. In situ hybridization was performed on tissue sections from different time points during gonadotropin-induced ovulation. Positive signal appears as blue-black. A–C, Ovaries from untreated control mice. D–F, Ovaries 48 h after PMSG treatment. G–I, Ovaries 8 h after hCG treatment. J–L, Ovaries 24 h after hCG treatment. Sections were hybridized with antisense RNA probes for HtrA1 (A, D, G, and J), PRSS23 (B, E, H, and K), and PRSS35 (C, F, I, and L). Magnification bar, 200 µm.

 
After stimulating follicular development with PMSG for 48 h, the expression of HtrA1 mRNA was similar to that in ovaries from untreated mice (Fig. 3DGo). At this time point, some expression of this protease was also observed in the theca layers of large antral follicles. The expression of PRSS23 mRNA was similar to that in ovaries from untreated control mice; however, its expression appeared to be down-regulated in the more developed follicles (Fig. 3EGo). PRSS35 was still expressed in the theca layers of all types of follicles, but the expression appeared to be slightly up-regulated (Fig. 3FGo). It was also expressed in the ovarian stroma and the granulosa cells of large antral follicles at low levels.

Eight hours after inducing ovulation with hCG, which is a time point that is just before ovulation, HtrA1 mRNA was highly expressed in granulosa cells of preovulatory follicles (Fig. 3GGo). PRSS23 mRNA was expressed at very low levels in granulosa cells of preovulatory follicles, and it was also expressed at a low level in the theca layers of the same follicles (Fig. 3HGo). PRSS23 was also expressed in the ovarian stroma in a dotted fashion. PRSS35 mRNA was expressed in the theca layers of the preovulatory follicles; however, it was also induced in the granulosa cells in these follicles (Fig. 3IGo).

Twenty-four hours after hCG administration, newly formed CLs were seen in the ovaries. The CLs are formed through rapid angiogenesis and tissue remodeling processes (14). At this time point, HtrA1 and PRSS35 mRNAs were both highly expressed in the forming CL (Fig. 3Go, J and L, respectively). PRSS23 mRNA was expressed at a very low level, in a dotted pattern (Fig. 3KGo).

PRSS23 is expressed in atretic follicles
PRSS23 mRNA expression was found to be elevated in a subset of developing follicles. To assess whether this expression correlated with follicular atresia, we performed in situ hybridization for PRSS23 mRNA and TUNEL staining on consecutive ovary sections from untreated control mice and from PMSG-treated mice at 48 or 96 h after PMSG treatment. As shown in Fig. 4Go, the expression of PRSS23 mRNA (Fig. 4Go, A–C) was specifically elevated in the TUNEL-positive atretic follicles (Fig. 4Go, D–F) at all time points investigated.


Figure 4
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  		FIG. 4. PRSS23 is expressed in atretic follicles. TUNEL staining and in situ hybridization were performed on tissue sections from untreated control mice (A and D) and PMSG-treated mice at 48 h (B and E) and 96 h (C and F) after PMSG treatment. In situ hybridization signal appears as blue-black. TUNEL signal appears as red. Magnification bar, 200 µm.

 
Expression of HtrA1, PRSS23, and PRSS35 in the CL of pseudopregnancy
CL formation and regression are characterized by extensive tissue remodeling. By using in situ hybridization on ovary sections from pseudopregnant mice, we investigated the expression pattern of HtrA1, PRSS23, and PRSS35 in the CL (Fig. 5Go).


Figure 5
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  		FIG. 5. Expression of HtrA1, PRSS23, and PRSS35 in the CL. In situ hybridization was performed on tissue sections from pseudopregnant mice at different stages of CL development. Positive signal appears as blue-black. A–C, CL at d (D) 1. D–F, CL at d 6. G–I, CL at d 16. J–L, CL 72 h after cloprostenol treatment (clo). Tissue sections were hybridized with antisense RNA probes for HtrA1 (A, D, G, and J), PRSS23 (B, E, H, and K), and PRSS35 (C, F, I, and L). Magnification bar, 200 µm.

 
We found that in the forming CL (d 1), the expression of HtrA1 (Fig. 5AGo) and PRSS35 (Fig. 5CGo) mRNAs were both high and evenly distributed throughout the CL. At this time point, PRSS23 mRNA was expressed at a very low level in the CL (Fig. 5BGo). At d 6 of pseudopregnancy, when the CL is functional and little tissue remodeling takes place, the expression of HtrA1, PRSS23, and PRSS35 mRNAs was low (Fig. 5Go, D–F, respectively). At d 16 of pseudopregnancy, expression of HtrA1 mRNA was induced in the regressing CL (Fig. 5GGo). PRSS23 mRNA was expressed at a low level in a dotted fashion (Fig. 5HGo), whereas PRSS35 mRNA expression was induced in the regressing CL in a dotted fashion (Fig. 5IGo).

To investigate further the relationship between expression of HtrA1 and PRSS35 and CL regression, we induced premature CL regression in pseudopregnant mice by treatment with the PGF2{alpha} analog cloprostenol (Fig. 5Go, J–L). Seventy-two hours after cloprostenol treatment, expression of HtrA1 (Fig. 5JGo) and PRSS35 (Fig. 5LGo) mRNAs was induced in the regressing CL, whereas expression of PRSS23 mRNA remained low (Fig. 5KGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies from our group and other laboratories have implicated the PA and MMP systems as crucial mediators of the follicular wall breakdown during ovulation (1, 2). However, recent ovulation experiments in which plasminogen-deficient mice were treated with the broad-spectrum MMP inhibitor galardin have shown that the functional importance of these protease systems may be less than previously believed (5). In the current study, we have used a microarray approach to identify proteases involved in ovulation. We found three extracellular serine proteases that were expressed in the mouse ovary. The proteases HtrA1 and PRSS35 appeared to be involved in follicular development, ovulation, and CL formation and regression. The protease PRSS23 appeared to be involved in follicular atresia.

Because there are several hundred proteases in the mammalian genome (6), the microarray approach has enabled us to study a large number of proteases at the same time. By this way, we detected three serine proteases, HtrA1, PRSS23, and PRSS35, and studied their expression and regulation in the mouse ovary.

During follicular development and ovulation, we found that the HtrA1 was expressed in many types of follicles and that it was not regulated much. This suggests that this protease may have either a housekeeping function or that its activity is mainly regulated on a posttranslational level. HtrA1 has previously been shown to be able to degrade different matrix components (13), some of which are present in the ovary (15, 16). It can also stimulate IGF signaling by degrading IGF-binding protein 5 (17), and inhibit signaling by proteins from the TGF β-family (18). Moreover, this protease has also been shown to be involved in apoptosis (19). Altogether, these findings indicate that besides matrix degradation, there are many other potential roles that HtrA1 may play in ovarian physiology.

In contrast with HtrA1, PRSS35 was found to be dynamically regulated during the ovulatory process. It was highly induced in granulosa cells of preovulatory follicles, suggesting a role in the breakdown of the follicular wall during ovulation. This is in agreement with the recent study by Miyakoshi et al. (20), who used a suppression subtractive hybridization technique (21) and identified PRSS35 as an ovary specific protease. By using dot blot hybridization, these researchers could not detect any expression of PRSS35 in tissues other than the ovary (20). Likewise, in this study we used in situ hybridization to investigate PRSS35 expression in several other tissues, including testis, liver, kidney, and intestine, but failed to detect any major site of expression (data not shown). Therefore, the highly specific expression of PRSS35 suggests that this protease plays important roles in the ovary. Miyakoshi et al. (20) also demonstrated that PRSS35 is regulated by progesterone. Because mice deficient for the progesterone receptor fail to ovulate (22), this finding provides further evidence for a role for this protease in ovulation.

Less than 1% of the follicles in mammalian ovaries reach ovulation. The other follicles are eliminated by atresia (23). Extracellular proteases are expressed in atretic follicles, and are likely to play central roles in their destruction and the concomitant reorganization of the ECM (24). Miyakoshi et al. (20) noted that the serine protease PRSS23 appeared to be expressed differentially in follicles at different stages of development. In this study we used TUNEL staining as a marker for atretic follicles and found that PRSS23 was specifically up-regulated in these follicles. Because the physiological substrates for this protease are unknown, we can only speculate about what roles PRSS23 may play in this process. One possibility is that it degrades ECM in the follicles destined for atresia, thereby promoting apoptosis through deficient cell-matrix adhesion (25). Alternatively, it may facilitate atresia by acting upon local paracrine factors or their receptors to stimulate or inhibit their actions (26).

The formation and regression of the CL are both accompanied by extensive tissue remodeling and matrix turnover. Previous studies have shown that the matrix-degrading proteases from the PA and MMP systems are involved in these processes (1, 2). However, our recent data have shown that these protease systems may not be required for normal CL formation (27). The high expression of HtrA1 and PRSS35 in the forming CL suggests that these proteases may play important roles for CL formation. It also supports our hypothesis that many proteases from different classes act in parallel to ensure proper reproductive function.

HtrA1 and PRSS35 were also expressed during CL regression, but not in the functional CL, indicating involvement mainly in tissue remodeling and degradation. However, it remains to be determined whether these proteases act directly upon matrix components or indirectly by modulating the actions of paracrine factors.

In summary, we have found three serine proteases that are expressed in the mouse ovary. Their dynamic expression pattern during ovulation and CL formation and regression suggests that they are involved in different tissue remodeling processes in the ovary.


   Acknowledgments
 
We thank Dr. Henrik Andersson for help with the microarray analysis.


   Footnotes
 
This work was supported by the Swedish Research Council (521-2005-6701) and the Medical Faculty of Umeå University.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2008

Abbreviations: CL, Corpus luteum; ECM, extracellular matrix; hCG, human chorionic gonadotropin; Htr-A1, high-temperature requirement factor A1; MMP, matrix metalloproteinase; NCBI, National Center for Biotechnology Information; PA, plasminogen activator; PG, prostaglandin; PMSG, pregnant mare serum gonadotropin; PRSS23, serine protease 23; PRSS35, serine protease 35; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Received December 14, 2007.

Accepted for publication June 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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