Molecular Human Reproduction, Vol. 5, No. 9, 803-808,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of ovarian function |
Cyclic expression of mRNA transcripts for connective tissue components in the mouse ovary
1 Department of Molecular Biology and Medical Biochemistry, and 2 Department of Obstetrics and Gynaecology, Turku University Central Hospital, University of Turku, Kiinamyllynkatu 4–6, FIN-20520 Turku, Finland
Abstract
In the ovary, differentiation of germinal cells into primordial follicles, functional ovulatory follicles and corpus luteum, all take place in a connective tissue matrix. We postulated that extracellular matrix (ECM) of the ovary participates actively in ovarian functions. To test this, the mRNA levels for several ECM components were determined in the mouse ovary at six distinct stages of the 4-day oestrous cycle. Northern analysis revealed statistically significant cyclic expression patterns for the mRNAs coding for type III, IV and VI collagens as well as for the small proteoglycan, biglycan, and for syndecan-1 and osteonectin. The cyclic changes observed in the mRNAs for these structural components exceeded those for matrix metalloproteinases (MMP)-2, -9 and -13, and for tissue inhibitors of matrix metalloproteinases (TIMP)-1, -2 and -3, where the changes were not statistically significant, despite their apparent role in ECM remodelling in the ovary. These observations support the hypothesis that cyclic changes in the production and degradation of ECM are part of normal ovarian function connected with follicular maturation, rupture and corpus luteum formation.
collagen/extracellular matrix/oestrous cycle/ovary/proteoglycan
Introduction
The principal functions of the ovary are to produce mature oocytes and excrete sex hormones. Both of these processes take place within a connective tissue matrix, which provides structural support for the ovary. This property is commonly ascribed to the 19 different types of collagen molecules which undergo self-assembly into various supramolecular structures, fibrils and different kinds of networks (Vuorio and de Crombrugghe, 1990
; Prockop and Kivirikko, 1995) onto which other constituents, such as proteoglycans and glycoproteins, adhere.
In addition to providing structural strength to tissues the extracellular matrix (ECM) also plays a role in differentiation, development and cell migration by providing a permissive or instructive environment (Hay, 1991; Meredith et al., 1993). Several steps of oocyte maturation take place in intimate contact with ovarian connective tissue. These include differentiation of germinal cells into primordial follicles, their further development into functional ovulatory follicles and rupture, as well as formation of corpus luteum and its subsequent atresia. Cell migration as well as displacement, destruction and repair of ECM during these steps brings out the possibility that connective tissue performs non-structural functions also in the ovary (Woessner, 1982, 1991). Several recent studies have demonstrated changes in the production and activity of matrix degrading enzymes, especially matrix metalloproteinases (MMPs), and their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs), during follicular development (Hulboy et al., 1997; Duncan et al., 1998), but surprisingly little is known about possible changes in the synthesis of ovarian ECM.
The present study is based on a working hypothesis, that in addition to providing structural strength to the ovaries, collagens and other ECM components have additional roles connected with different stages of follicular development. As modulation of ECM production during this process can be considered an indication of such a role, we tested our hypothesis by determining the mRNA levels for several matrix components in the mouse ovary at six distinct stages of the 4-day oestrous cycle. The mRNAs selected for these analyses represent those for collagens which have previously been detected in the ovary, for proteoglycans associated with such collagen networks in other tissues, and for MMPs and TIMPs believed to be involved in the proteolytic degradation cascades of ovarian collagens. Furthermore, mRNA levels for two proteins involved in connective tissue activation, syndecan-1 and osteonectin, were studied.
Materials and methods
Ovarian samples
This study is based on the analysis of 30 C57 blxDBA mice (aged 17–25 weeks; mean ± SD 19.7 ± 2) which were housed in a pathogen-free animal facility under controlled lighting conditions with lights on from 05.00 to 19.00. The animals were given water and pelletted food ad libitum.
Staging of the oestrous cycle
Vaginal cytology was used for determination of the oestrous cycle. Smears were taken daily between 12.00 and 14.00. A trimmed tip of a disposable plastic pipette was placed at the vaginal orifice. Two drops of water were instilled into the vagina, aspirated back into the tip twice, and then transferred onto a microscopic slide. Dry smears were fixed in absolute methanol for 30 s, drained and stained with haematoxylin–eosin and a modified Papanicolaou method (Merck, Darmstadt, Germany) to facilitate identification of the cycle phases. The oestrous cycle was classified into six phases (di-oestrus, early pro-oestrus, late pro-oestrus, oestrus, meta-oestrus I, meta-oestrus II; Figure 1) according to vaginal cell morphology (Nelson et al., 1982).
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To obtain representative samples, the oestrous cycle of each animal was followed by analysis of the vaginal smears at 4 consecutive days before the animal was killed by cervical dislocation. Ten mice were discarded from the study as their cycle phase could not be unambiguously determined or they became pseudopregnant. The ovaries were excised, trimmed free from surrounding tissues, and frozen at –80°C. The study protocol was approved by the institutional committee for animal welfare.
RNA extraction and mRNA analyses
For extraction of total RNA, the ovaries were pulverized under liquid nitrogen in a mortar and dissolved in guanidinium isothiocyanate as described previously (Chirgwin et al., 1979). Aliquots (10 µg) of total RNA were denatured with glyoxal and formamide, fractionated on 0.75 % agarose gels, and blotted onto nylon transfer membranes, and hybridized with [32P]-labelled cDNA inserts at 42°C for 20 h. The hybridizations and washes were performed as suggested by the supplier (Pall BioSupport Division, Glen Cove, NY, USA). The bound probes were detected and quantified using a BioRad phosphor imager or by autoradiography and densitometry. One-way analysis of variance (ANOVA) for repeated measure (for n > 3) and the unpaired student's t-test (for n = 2) were used to analyse differences in mRNA levels between cycle phases. When the ANOVA disclosed significant differences, t-tests were used to determine which means were different. P < 0.05 was considered to be statistically significant.
The following collagen cDNA clones were used as probes in Northern hybridization: pMCol1a1-1 for mouse pro
1(I) collagen mRNA, pMCol2a1-1 for mouse pro1(II) collagen mRNA, and pMCol3a1-1 for mouse pro1(III) collagen mRNA (Metsäranta et al., 1991), p1234 and p1572 for mouse 1(IV) and 2(IV) collagen mRNA respectively (Oberbaumer et al., 1985), pMCol6a2-1 for mouse 2(VI) collagen mRNA (A.-M.Säämänen and E.Vuono, unpublished) and pMCol9a1-1 for mouse 1(IX) collagen mRNA (Metsäranta et al., 1991). The mRNAs for core proteins of small proteoglycans decorin, biglycan and fibromodulin were detected with probes pMDcn-1, pMBgn-1 and pMFmn-1 (A.-M.Säämänen, H.Salminen and E.Vuorio, unpublished), those for syndecan-1 with clone PM-4 (Saunders et al., 1989), those for MMP-2, 9 and 13 with clones pK-191 (Huhtala et al., 1990), M92 KD-1 (Reponen et al., 1994) and pMMMP-13 respectively (M.Perälä and E.Vuorio, unpublished), and those for TIMP-1, 2 and 3 with clones pMTIMP-1, pMTIMP-2 and pMTIMP-3 respectively (K.Joronen, V.Glumoff and E.Vuorio, unpublished). Osteonectin mRNA was detected using clone pHon 164 (Young et al., 1990), and the 28 S rRNA with clone 341–1 (Iruela-Arispe et al., 1991). The inserts were liberated from the plasmids by appropriate restriction enzymes for labelling by the random priming method.
Results
Northern analysis revealed cyclic variation in the mRNA levels for the constituent chains of type I, III, IV and VI collagens (Figures 2 and 3). For type I collagen the expression was maximal during late pro-oestrus and oestrus, but the changes in the mRNA levels were not statistically significant (Figure 3A). On the contrary, type III collagen mRNAs were significantly higher during oestrus and meta-oestrus than di- and early pro-oestrus (P< 0.05, Figure 3B). The mRNAs for the 1 and 2 chains of type IV collagen also revealed distinct expression profiles: the differences in 1(IV) collagen mRNAs were not statistically significant, whereas those in 2(IV) mRNAs were (Figure 3F, G). The mRNA for the 2 chain of type VI collagen exhibited higher levels during diestrus, early pro-oestrus and late pro-oestrus than during oestrus and meta-oestrus (P < 0.05, Figure 3H). Transcripts for cartilage specific type II and IX collagens were not detected at any stage of the oestrous cycle (data not shown).
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The gene expression patterns of the small proteoglycans were analysed similarly. While the mRNA levels for decorin remained relatively unaltered (Figure 3C
), those for biglycan varied depending of the stage of the oestrous cycle showing significantly higher levels during late pro-oestrus, oestrus and meta-oestrus I than during di-oestrus, early pro-oestrus and meta-oestrus II (P < 0.05, Figure 3D). The changes in fibromodulin mRNA levels were not quite statistically significant (P = 0.058, late pro-oestrus and oestrus versus di/pro-oestrus, Figure 3E). Syndecan-1 and osteonectin mRNA levels exhibited statistically significant cyclic variation with a peak at late pro-oestrus and oestrus (P < 0.05, Figure 3J, K).
Finally, the mRNA levels of three MMPs and three TIMPs were determined. MMP-2 mRNA exhibited no apparent cyclicity in its gene expression (Figure 4A), whereas the mRNA levels for MMP-9 were very low and those for MMP-13 undetectable (data not shown). TIMP-1 mRNA levels exhibited statistically insignificant cyclic changes with the highest value during oestrus (Figure 4B), whereas those for TIMP-2 and TIMP-3 demonstrated no cyclicity (Figure 4C, D).
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Discussion
Although several recent studies have focused on changes in the production and activity of proteolytic enzymes during ovulation and luteal rescue (Inderdeo et al., 1996; Hulboy et al., 1997; Duncan et al., 1998), possible changes in the production of new connective tissue matrix during these processes have received little attention and no systematic studies are available on the mRNA levels of ECM components during the different stages of normal ovarian function. The presence of fibrillar collagens of type I and III in the human ovary and their production by cultured ovarian cells have been reported previously (Zhu et al., 1993; Auersperg et al., 1994; Kruk et al., 1994). Interestingly, we found the mouse ovary to express mRNAs coding for type I and III collagens with different cyclic patterns. Elevated ratios of type III to type I collagens and their mRNAs, characteristic for connective tissue repair (Gay et al., 1978; Glumoff et al., 1994), were seen during corpus luteum formation (meta-oestrus). These and several earlier studies on cell cultures have demonstrated good correlation between collagen production and cellular mRNA levels of the corresponding -chains (Kähäri et al., 1984), which justifies the use of mRNA levels for estimating protein production rates. Our data thus suggests that formation of corpus luteum resembles a connective tissue repair process as has also been suggested by histological studies (Woessner, 1982).
Type IV collagen, a characteristic component of the basement membranes, is produced by cultured ovarian epithelial cells (Kruk et al., 1994). Accordingly, we found the mouse ovary to express mRNAs coding for the 1 and 2 chains of type IV collagen but with different profiles. Although the 1 and 2 chains are the major constituents of type IV collagen and their genes share a common promoter (Soininen et al., 1988), considerable variation has been observed in their respective mRNA levels also in other tissues (Boot-Handford et al., 1987). The biological significance of this variation, if any, remains unknown.
Type VI collagen is the major component of microfibrils in several tissues (Chu et al., 1990). Therefore, it was not unexpected to detect the mRNA coding for the 2(VI) collagen chain in the ovary.
Little is known about the overall activity and regulation of genes coding for proteoglycans in the ovary. Although the small proteoglycans have been shown to associate with collagen fibrils (Iozzo, 1997), their expression patterns do not demonstrate any clear-cut correlation with those of major fibrillar collagen types (I, II and III). In the present study only the cyclicity of biglycan mRNA was statistically significant and exhibited patterns which suggested co-expression with type I and III collagen mRNAs.
Our observations of increased transcript levels of syndecan-1 and osteonectin at oestrus are also in accordance with earlier studies on tissue remodelling and repair. The expression of syndecan-1 is greatly induced during tooth morphogenesis (Vainio et al., 1989) and in healing wounds (Elenius et al., 1991). Osteonectin (also called SPARC) has also been localized to areas of active morphogenesis, steroid production, and remodelling (Nomura et al., 1988; Porter et al., 1995). The presence of osteonectin in the developing follicle and corpus luteum has also been observed by others (Smith et al., 1996; Bagavandoss et al., 1998) but no earlier reports are available on syndecan production by the ovary.
Activation of ovarian collagenolytic mechanisms must be an important step in the ovulatory response as considerable thinning of the collagenous layers, terminating in their rupture upon ovulation, has been observed during follicular maturation (Espey et al., 1967; Reich et al., 1985). Accordingly, the activity and mRNA levels of MMP-1 and MMP-2 (type I and IV collagenases) in human follicular samples have been shown to increase as ovulation proceeds (Reich et al., 1991; Hulboy et al., 1997), apparently under regulation by oestradiol (Puistola et al., 1995). Surprisingly, in the present study, the mRNA levels of MMP-2 failed to demonstrate any apparent cyclicity when the mouse ovary was studied as an entity. The mRNA levels of the other type IV collagenase (MMP-9) and for MMP-13 (the mouse equivalent of MMP-1) capable of degrading fibrillar collagens, were too low to permit reliable quantification. Different explanations are available for the apparent discrepancies between the present study and earlier reports on MMP production by the ovary. First, earlier data are largely based on the analysis of human follicular samples and cultures of granulosa/luteal cells, whereas in the present study the mouse ovary was studied as an entity. Secondly, earlier data have been obtained largely from in-vitro induction and analysis of samples representing the luteal phase whereas in the present study the mouse ovary was studied in vivo through the entire oestrous cycle. We are aware of only one earlier study where a similar approach has been used, for the analysis of TIMP gene expression in the ovary (Inderdeo et al., 1996). Thirdly, MMPs other than those analysed in the present study could contribute to the collagenolytic activity. Finally, as MMPs are produced and secreted as proenzymes the results of activity measurements cannot be directly compared with mRNA levels since complex activation and inhibition pathways regulate the collagenolytic activity of MMPs in tissues (Hulboy et al., 1997).
Earlier studies have also demonstrated increases in the mRNA levels of TIMP-1 and TIMP-3, but not of TIMP-2, in the mouse and rat ovary during luteolysis (Nothnick et al., 1995; Inderdeo et al., 1996), and after in-vitro administration of human chorionic gonadotrophin (HCG) (Tsafriri et al., 1992; Duncan et al., 1998). Although the changes seen in the mouse ovary in the present study were not statistically significant, they are mostly in accordance with earlier observations.
The cyclic variation in the mRNA levels for several ECM components (which in most biological circumstances reflect similar variation in the production of the corresponding proteins) in the mouse ovary during the oestrous cycle are reminiscent of changes seen during tissue repair. We interpret these results to indicate that cyclic production and degradation of extracellular matrix is an integral part of normal ovarian function. Lack of significant cyclicity in total ovarian MMP and TIMP mRNA levels suggests that their activity is mainly controlled at post-transcriptional level at sites of follicular rupture and luteal rescue. Our observations warrant further studies on spatial and temporal distribution of the mRNAs in the mouse ovary, zymographic analyses of MMP activities as well as studies on human ovaries, not only during the normal menstrual cycle, but also in diseases affecting follicular maturation, e.g. polycystic ovary syndrome. Such studies are currently under way. Further applications of this study may come from recent observations that ECM supports the viability and growth on human primordial and primary follicles in long-term culture (Hovatta et al., 1997). Identification of the ECM components responsible for the maintenance of in-vitro follicular maturation could have important implications in infertility treatments in the future.
Acknowledgments
The authors are grateful to Tuula Oivanen and Anu Kupiainen for expert technical assistance. Drs. Karl Tryggvason, Markku Jalkanen, Yoshi Yamada and F.Marion Young are acknowledged for providing cDNA probes for these studies. This study was financially supported by grants from the Academy of Finland (project no 37311), Sigrid Juselius Foundation and the Turku University Central Hospital (project no 13449).
Notes
3 To whom correspondence should be addressed 
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Submitted on February 8, 1999; accepted on June 8, 1999.
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