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ISSN : 2508-755X(Print)
ISSN : 2288-0178(Online)
Journal of Embryo Transfer Vol.33 No.2 pp.61-68
DOI : https://doi.org/10.12750/JET.2018.33.2.61

Detection of Matrix Metalloproteinases Patterns in Bovine Luteum cell during Pregnancy

Sang-Hwan Kim1, Kyong-Lae Kim1, Ji-Hye Lee2, Da-Hye Shin3, Na-Hyeon Jung3, Ho-Jun Lee1, Jong-Taek Yoon1,4
1Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Korea.
2Major in the Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University, Anseong, Gyeonggi-do, 456-749, Korea.
3Department of Animal Life and Environment science, Graduate School of Future Convergence Technology, Hankyong National University, Anseong, Gyeonggi-do, 456-749, Korea.
4Department of Animal Life Science, Hankyong National University, Ansung 456-749, Korea.
*

The authors contributed equally to this work


Correspondence: Jong Taek Yoon Phone: +82-31-670-5255, +82-31-670-5094, Fax: +82-31-675-8265 E-mail: jtyoon@hknu.ac.kr
09/06/2018 15/06/2018 21/06/2018

Abstract


The major focus of this study is to analyze the expression of bovine MMPs and to monitor their activity during the estrus cycle and pregnancy. During pregnancy, MMP-2 expression was detectable around 30 days but became insignificant by 60 days, then started to increase again around 90 days and reached the maximum at 250 days. The activity of MMP-2 protein changed in accordance with its expression level. As expected, the level of TIMP-2 exhibited a reverse pattern. About MMP-9, high level expression was observed as early as 30 days and gradually increase until 90 days. Then started to decrease after 250 days. Again, the sites of MMP-9 expression were similar to those of MMP-2. On the other hand, expression of TIMP-3 remained low until 90 days but showed a small and temporal increase around 250 days. In summary, expression of different MMPs were differentially regulated during estrus cycle and pregnancy. While the expression of MMP-2 was high in estrus cycle, MMP-9 slowly takes over with the progression of pregnancy. These results indicated that the luteal tissue perform distinct functions during pregnancy and estrus. Perhaps the activity of MMP-2 is required for the structural remodeling of luteum, resulting the suppression of P4 inflow from blood. On the other hand, steady maintenance of MMP-9 throughout luteal development is important for the activation of cell proliferation, maturation and angiogenesis.



초록


    INTRODUCTION

    Maintaining pregnancy is known to maintain pregnancy by the action of progesterone formed in the pregnant corpus luteum. In particular, the composition of corpus luteum is present in two kinds of steroidogenic cell including large luteum cell which is mature luteal cells, forming cellular functional change in luteum cell derived from cumulus cell after ovulation, steroids, protein hormones, growth factors, eicosanoids and cytokines and progesterone directly to the uterine environment and to maintain normal pregnancy (Lei et al., 1991, Reynolds 1999).

    The execution of the normal function of corpus luteum is performed by the action of luteinizing hormone (LH) applied from the pituitary gland, and the granulosa-lutein cell derived from granulosa cells and theca lutein cell derived from the capsular cells. It will bring about the rapid development of luteal cells to promote the formation of blood vessels by the stimulation of vascular endothelial growth factor (VEGF), increasing the production of progesterone using low density lipoprotein (LDL) as an acceptor through newborn blood vessel as an acceptor (Richard et al 2006). In other words, such a process is characterized in that the pre-granulosa cells, which are converted into luteal cells, differ from the surface on which androstenedione is passed from the capsular cells. The two cell bodies of the follicles constitute the corpus luteum (Theca-lutein cells and granulosa-lutein cells). To the destruction of the basement membrane to be affected directly from theca-lutein cell, the rapid appearance of angiogenic factors and the increase of androstenedione the cell cycle of large lutein cell progressed very rapidly, with small lutein cell role exchange will be done (Smith et al., 1999, Curry and Osteen 2003). In particular, the corpus luteum during pregnancy where maturation has been completed can produce high levels of progesterone also in the action of low LH and responds to embryonic human chorionic gonadotropin (hCG) hormone to generate. At the same time increasing the effect of implantation and pregnancy in the uterus can be considered as simultaneous formation of progesterone in corpus luteum and placenta (Berisha and Schams 2005). These results suggest that the function of the corpus luteum at the pregnancy begins to be transferred to placental hormone, and the deterioration of corpus luteum and the reconstitution between cells are formed. However, studies on the reconstitution of luteal cells by pregnancy time are weak. In particular, reports on the action of matrix metalloproteinases (MMPs) that can promote differentiation between cells and act on lutein cell regression it is very rare. Therefore, in this study, we analyzed the expression of MMPs and 20α-hydroxysteroid dehydrogenase (20α-HSD) in order to illuminate changes in corpus luteum from the early stage of gestation to the latter half and examined the reconstitution and change of the cells of the luteal cells of gestation period.

    MATERIALS AND METHODS

    Corpus luteum collection

    Ovarian tissue during 30, 60, 90 and 250 pregnant days in bovine (The day of artificial insemination was designated as day 1) were collected from a local slaughterhouse at Pyeong-Nong, Pyeongtaek, Korea. After samples were placed into an LN2 freezer box, and were transported to the laboratory within 2 h. And corpus luteum at each pregnancy stages were separated and classified as collected, corpus luteum according to the method described by Kim et al., (2011). This study was carried out in strict accordance with the recommendations specified in the guide for the care and use of laboratory animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Hankyong National University (Permit Number: 2016-1).

    Zymography

    To analyze the MMP enzyme reaction from bovine luteal tissues, 20 mg of total protein were added to 10㎕ FOZ loading buffer (5% Bromo phenol blue, 10% SDS and 2% Glycerol) were mixed and allowed to react on ice for 5 minutes and then electrophoresed for 1 h 30 minutes at 150 V with gelatin SDS-PAGE gel containing 100 mg /㎖ gelatin. After electrophoresis, the gel was induced twice in protein renaturation with renaturation buffer (2.5% Triton X-100, 1 × PBS) for 20 minutes and then washed with sterilized water for 20 minutes. The cells were placed in a zymography reaction buffer (1M Tris-HCL pH 7.5, 5M NaCl, 1M CaCl2, 0.2 mM ZnCl2, 0.2% Triton X-100, 0.02% NaN3) and then allowed to react at 37 ℃ for 18 h it was. After completion of the reaction, the zymography gel induced protein staining with Coomassie Brilliant Blue (Bio-rad, USA) for 1 h, after which color faded and discolored parts were analyzed (Balasingam and Yong. 1996).

    Hematoxylin & Eosin stein

    In order to remove paraffin of tissue slide for immunological analysis made of bovine corpus luteum tissues, after dipping in Xylene, 100%, 95%, 70%, 50% Ethanol order twice at 10 minute intervals, hematoxylin and dyeing with eosin and immersing at 10 minute intervals twice in order of 50%, 70%, 95%, 100%, Xylene to remove moisture, use Immunomount to encapsulate 48h after the elapse of time, it was analyzed with an optical microscope (X40).

    in-situ Zymography

    In order to measure the activity of MMPs at each specific stage of corpus luteum, 30 days, 90 days, 250 days of pregnant, paraffin slide of corpus luteum tissue were utilized. In order to perform in-situ Zymography experiment, Deparaffinize / hydrate was firstly repeated twice in Xylene, 100% Ethanol, 95% Ethanol for 10 minutes, washing in ddW for 5 minutes and boiling in 10 mM sodium citrate for 10 minutes. Thereafter, it was repeated 20 times for ice and 3 times for ddW for 5 minutes, then emulsion (ddW, 10% SDS, 2% Glycerol) and zymography reaction buffer were mixed at a ratio of 1: 2 and raised to slide, followed by enzymatic reaction at 37℃ for 48 h in a slide box filled with 1 M Tris. After the reaction was completed, the slide was dried at 37℃, stained with Hematoxylin and Eosin. And Xylene, 95% Ethanol, 100% Ethanol, Permount 50㎕ after twenty minutes for the slide upper. And then overwritten with cover glass and analyzed with an optical microscope (X 40).

    Immunohistochemistry (IHC)

    In order to conduct IHC experiments, paraffin slide of each corpus luteum tissue was used. Deparaffin / hydrate was repeated twice on Xylene, 100% Ethanol, 95% Ethanol for 10 minutes, washing in ddW for 5 minutes and boiling in 10 mM sodium citrate for 10 minutes. Antigen retrieval was performed by heating at 95℃ in 10 mM sodium citrate (pH 6.0). Endogenous peroxidases were quenched with 0.3% hydrogen peroxide in methanol for 5 min at room temperature. After 3 washes in 1× PBS buffer, the slides were blocked in 1% goat serum containing 3% horse serum for 1 h at room temperature. First Antibody (MMPs, TIMPs, PAPP-A) detection was overnight at 4℃, then washing was carried out with 1×PBS for 5 minutes, secondary antibody detection was carried out for 1 h at 4℃, washing was carried out 3 times for 5 minutes in 1×PBS, ABC detection was carried out at room temperature for 30 minutes. After washing, washing was carried out 5 times with 1×PBS for 3 minutes. DAB staining to 300㎕ DAB solution for 1 to 10 minutes, ddW for 5 minutes. Sections were counterstained with periodic Acid-Schiff (PAS) reagent and Harris hematoxylin solution containing 4% acetic acid. Tissues were dehydrated, cleared, and covered with Permount solution (Fisher, NJ, USA), and observed under a microscope (Nikon Corp., Tokyo, Japan) at 200× and 400× magnification.

    RESULTS

    Enzyme activity analysis of MMPs from luteum tissues

    The results of analyzing the activity of MMPs from the corpus luteum tissues at 30, 90 and 250 days of pregnancy are shown in figure 1. Analysis results of MMPs from pregnant luteal tissues, the enzyme activity response of MMPs at 250 days appeared very high compared with 30 days and 90 days, and the corpus luteum on 30 days was mainly from the basal membrane between granulosa-lutein and theca-lutein. In the case of 90 days, it was supposed to be mainly active in the vascular endothelial zone, and it was low activity in other zones. In the case of 250 days, the MMPs activity in the tissue was highly active as a whole. Especially the activity of the granulosa-lutein position was markedly formed.

    Expression pattern of MMPs, TIMPs and PAPP-A in the luteal body of the pregnant days

    Gestation period, the results of analyzing the activity of MMPs from total corpus luteum protein and the expression of TIMPs and PAPP-A protein as inhibitor are the same as in figure 2. In the case of MMP-2, some enzymatic activity was confirmed on 30th day of pregnancy, and the activity of 90 days and 60 days was lowly activity, increased from 250 days thereafter. However, the aspect of active-MMP-2 was very low overall, and in the case of MMP-9 overall, it showed similar activity, but it increased from 90 days, it was high levels. The analysis results of TIMPs were very high overall in the case of TIMP-2, an inhibitor of MMP-2, however showed the decreased at 250 days of pregnancy. The showing that TIMP-3 the expression was overall low level, but expression at 250 days showed very lowliest to one thing relatively to others pregnancy days. As a result of analyzing the expression of PAPP-A (Pregnancy-associated plasma protein A), the higher expression level of PAPP-A protein was increased from the around 30 days to 60 days in the corpus luteum. However, on 90 and 250 days showed that the PAPP-A protein was lowly expressed.

    Expression position analysis of MMPs, TIMPs and PAPP-A form pregnant luteal tissues

    The results of analyzing the expression positions of MMPs and TIMPs are the same as in figure 3. Expression of MMPs was gradually increased from 30 days to 250 days as shown in figure 1, and these results showed the same aspect of western blot results. Expression of each MMP-2 and MMP-9 was expressed in the large lutein cell section in granulose-lutein cell, and its expression in the small lutein cell was very low. Expression of TIMPs, an inhibitor of MMPs, was also very high in the large lutein cell section. However, the expression of TIMP-2 a suppressor of MMP-2 was confirmed that expression was very high in the large lutein cells of 30 days. MMP-9 showed similar expression on the whole, it was expressed in the lutein cytoplasm in general distributed at 30 days, but only on the 90th day high expression was observed in some large lutein cells. The detected pattern of 250 days was similar to the localization expressed of MMP-2. In the case of TIMP-3 known as an inhibitor of MMP-9, the expression was over all low detected. However, in the case of 30 days 60 days, 90 days and 250 days of pregnancy, it was expressed throughout the cytoplasm in the tissue, but it was confirmed that intracellular expression predominated at the 250 days. In addition, the expressed patterns of PAPP-A, which controls progesterone in the corpus luteum based on the results of expression of MMPs, the overall expression increased from 30 days to 250 days. Also PAPP-A protein was mainly high in granulose-lutein cells compared to other sections of the corpus luteum (figure 4).

    DISCUSSION

    Analysis of MMPs expressed in the gutter corpus luteum is very important for analyzing the altered function of pregnant corpus luteum or the effect of reconstitution. In particular, it is known that MMP-9 acts as a basement membrane-degrading enzyme in cellular constituents which can be usefully used for discriminating maintenance of normal pregnancy (Pustovrh et al., 2002). The corpus luteum retained during pregnancy is to continuously produce steroids, protein hormones, growth factors, eicosanoids, cytokines and progesterone, to interact with hormones derived from placenta at the end of pregnancy and to induce normal pregnancy (McCracken et al., 1999; Reynolds and Redmer 1999; Berisha and Schams 2005). And the expressing of HSD was rapidly reducing progesterone from the corpus luteum in late pregnancy (Nakajin et al., 1989).

    In this study that the expression of MMPs was analyzed to illuminate the structural change of luteal body during pregnancy, and it was confirmed that the expression of MMP-2 and 9 were different between the early and late stages of pregnancy. The confirmed that changes in the enzymatic activity of MMPs are mainly formed in the basal membrane between granulosa-lutein and theca-lutein from about 30 days to 250 days after pregnancy. Also, our results can be suggested that changes in structural and functional constitution are activated from about 250th day of pregnancy, which is related to the functional organization of the corpus luteum revealed in Kliem (2007). The MMPs expressed in our results in 250 days were like the reports in Michaluk and Kaczmarek (2007) that MMPs affect the reconstruction of the corpus luteum. In addition, MMP-9 activation was able to function as a material to increase the synthesis of progesterone by acting on granulosa-lutein. The function of MMPs is known to be formed on the basis of structural changes, but from the results of the study, the expression in PAPP-A on the timing of pregnant corpus luteum has many similarities with expression of MMPs. Although the expression of PAPP-A and together with expression of MMP-9 sharply increased from 30 days to 60 days in the early pregnancy, it showed constant expression after 90 days and 250 days. However, MMP-2 was increased at 250 days in lutein cell, it can be seen as acting as a functional remodeling on the granulosa lutein cell on the late pregnancy (Brenner et al., 1985).

    Expression of MMP-2 increased in the corpus luteum at the 250th day of pregnancy, in addition expression of TIMP2 as a suppressor decreased and expression of PAPP-A was maintained in granulosa-lutein could be seen. This phenomenon seems to work in a different way from the research report that the corpus luteum at the end of pregnancy is rapidly reconstituted and regresses (Huppertz et al., 1998). In other words, the structural remodeling of the pregnant corpus luteum associated with what is not a degradation of corpus luteum by MMPs but is reconstituted to maintain PAPP-A by the interaction between granulosa-lutein and theca-lutein. As a result, the expression of MMP-9 was increased in granulosa-lutein cell, and this result showed in like the reports in Michaluk and Kaczmarek (2007).

    As the result that PAPP-A expression is maintained on 250 days of pregnancy and MMP-9 mainly acts, as the result that the action of cell proliferation plays a role in cell differentiation and activity rather than functional decline of cells. Our think that the corpus luteum reconstruction due to structural change can be remodeling rather than loss of corpus luteum. Therefore, the results of this study suggest that the corpus luteum formed in the early pregnancy is maintained at the functional completion stage, and the luteal body function exists in the retention state in the latter half. But activation of MMPs during pregnancy which seems to be a full-scale change of corpus luteum structure remodeling from 250 days on pregnancy (Mahmoodzadeh et al., 2010).

    The results of this study show that the luteal regression of cattle at the late pregnancy is not regressed radically immediately after birth but gradually changes from the 250th day of pregnancy. Based on the results of this study it can be seen that the expression of MMPs can be used as an indicator for functional observation of normal pregnancy.

    CONCLUSION

    The results of this study show that the luteal regression of cattle at the late pregnancy is not regressed radically immediately after birth but gradually changes from the 250th day of pregnancy. Based on the results of this study it can be seen that the expression of MMPs can be used as an indicator for functional observation of normal pregnancy.

    Figure

    JET-33-61_F1.gif

    In-situ Zymography analysis of MMPs during 30, 90, 250 pregnant days in bovine corpus luteum. A : H&E analysis, B : In-situ zymography, Black arrow is MMPs activation zone.

    JET-33-61_F2.gif

    Expression of MMPs, TIMPs and PAPP-A protein from total protein of 30, 60, 90, 250 days of pregnant in bovine corpus luteum. A: Zymography, B: western blot, C: MMP-2, 9 activation levels (Level analysis from the ImageJ(imagej.nih.gov)), PCL : pregnant corpus luteum.

    JET-33-61_F3.gif

    Expression and localization analysis of MMPs, TIMPs protein on 30, 60, 90, 250 days of pregnant in bovine corpus luteum tissues. A: MMP-2, B: TIMP-2, C: MMP-9, D: TIMP-3, brown pattern is detection of protein. Black arrow is protein detection zone.

    JET-33-61_F4.gif

    Expression and localization analysis of PAPP-A protein on 30, 60, 90, 250 days of pregnant in bovine corpus luteum tissues. Purple pattern is count staining, and brown pattern is detection of protein. Black arrow is protein detection zone.

    Table

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