- Email: [email protected]
A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro
Available online at www.sciencedirect.com
Agricultural Sciences in China 2007, 6(1): 115-120
*
*’
ScienceDirect
January 2007
A Novel Three-DimensionalMouse Embryonic Implantation Model In Vifro SONG Yu-xuan and CAO Bin-yun Animal Sci-Tech College, Norihwest A & F University, Yangling 712100, P.R.China
Abstract To regenerate three-dimensional endometrium in vitro as a novel model for studying the mechanism of implantation of embryos, the luminal epithelial cells and stromal cells of the rabbit uterus were separated and cultured in vitro. The type I mouse tail collagen was used as scaffolding material. The stromal cells were inoculated in the type I mouse tail collagen, and the luminal epithelial cells were inoculated on the type I mouse tail collagen to regenerate the endometrium in vitro. The regenerated endometrium was cultured in DMEM-F/12 media containing 100 nmol L-1 progesterone, 10 nM P-estradiol, and 10% fetal bovine serum (FBS) for 3 d. The media were then replaced with CZB containing 100 nM progesterone, 10 nmol L-’ P-estradiol, and 10%FBS, and the mouse blastulas were co-cultured with it. The results of scanning electronic micrography showed that the epithelial cells on the surface of the reconstructed endometrium were covered with numerous slender microvilli and some epithelial cells protruded pinopodes. After culturing for 12 h with the mouse blastula, the shedding, attachment, and implantation of the blastula were observed. The blastula can escape from zona pellucida and attach to the three-dimensional endometrium and is then implanted into it. This study showed that the reconstructed three-dimensional endometrium can serve as a robust embryo implantation model in vitro.
Key words: three-dimensional endometrium, embryo, implantation, model, in vitro
INTRODUCTION The zygote enters the uterus after developing in the oviduct for a period of time. It is mobile in the uterus liquid .at the beginning, and then the blastula is shed from the zona pellucida as it expands. The blastula that escaped from the zona pellucida can attach to the endometrium at a suitable position. The trophoblast of the blastula can have a “cross talk” with the endometrium and make a physiological contact with the endometrium. This process is called embryonic implantation. The study of implantationmay completely solve the problem of women’s contraception. So it was considered as one of the most important scientific topics in the research field of reproductive biology. The
implantation is also the key step that controls the fertility and infertility of mammals. Implantation processes involve multiple interactions among secretions of the uterus, corpus luteum, and the embryo, and extensive tissue remodeling, angiogenesis, and apoptosis occurs at the implantation site (Liu 2002). The mechanism of the implantation has not been revealed completely hitherto, and just some hypotheses were put forth for elucidation. This is partly ascribed to the complicacy of the implantation itself, but mostly ascribed to the difficulty to study the implantation because of the limited in vivo study. The model of the regeneration of the endometrium was made in recent years to study diseases related with the endometrium (Gaetje et al. 1995; Fasciani et al. 2003; Park et al. 2003). In this experiment, we used the type I mouse
Received 10 April, 2006 Accepted 28 August, 2006 SONG Yu-xuan,Ph D, E-mail: syx98728@ 163.com; Correspondence CAO Bin-yun, Professor, E-mail: [email protected]
82007,CAAS. All tights reserved. Published by ElsevierLtd.
SONG Yu-xuan et al.
116
tail collagen as scaffolding material to regenerate the three-dimensional endometrium in v i m , and then the murine blastula were co-cultured with it for a period of time to observe the implantation of blastula in the regenerated endometrium. This study aims to establish a robust model to facilitate the elucidation of the mechanism of the implantation.
MATERIALS AND METHODS Cell preparation and culture The uterus tissue was obtained from healthy New Zealand white rabbits aged 4 months undergoing superovulation. Endometrial tissues were isolated by curettage of hysterectomy from the uteri of rabbits after 4 d from the injection of PMSG. The pooled endometrial tissues were agitated in 5 mL of 0.25% uypsin solution (Sigma, USA) at 37°C for 10 min. The enzymatic reaction was stopped by adding 6 mL of Dulbecco's modified Eagle's medium (DMEM)/Fl2 (Gibco-BRL, USA) with 10% fetal bovine serum (FBS) (Shengyuanheng Bio-Tech, China). After centrifugation for 10 min at 600 r/min, the pellet was resuspended in 10 mL of DMEM/F12 plus 10% FBS. The suspension was centrifuged at 300 r/min for 5 min, and the stromal cell-rich fraction (suspension) was collected and inoculated into a 75-cm' culture flask for 24 h. For isolation of epithelial cells, the pellet was resuspended and inoculated into another 75-cm' culture flask for 24 h. The flasks was then rinsed several times with PBS to remove red blood cells, and the old medium was replaced with fresh DMEM/F12 medium containing 100 nmol L ' P-estradiol (Sigma, USA), 10 nmol L' progesterone (Sigma, USA), and 10%FBS. The stromal cells and epithelial cells were allowed to grow to confluence at 37°C in a humidified chamber supplied with 5 % CO,.
Regeneration of endometrium in vitro The cells in passages 3-5 that were cultured in flasks were digested with 0.25% trypsin solution and centrifuged for 5 min at 1000 r/min. First, the liquid type I collagen of mouse tail (1.80 mg I&') and Matrigel were mixed according to the ratio 4: 1, and then this mixture
was mixed with 2 x DMEM/F12 containing 20% FBS according to the ratio 1:1, adjusting the pH value of the final mixed liquid to 7.00-7.20 using 0.1 mol L-' of NaOH solution. The stromal cells was resuspended with mixed collagen liquid, the cell density was adjusted to 106 mL-', and then 0.75 mL of the mixed liquid along with stroma1 cells was transferred to one of the wells of a 24well plate. After 15 min, the epithelial cells were inoculated on the surface of the stromal cell embedded in collagen. The regenerated endometrium was cultured in the DMEM/F12 medium with 100 nmol L-' pestradiol, 10 nmol L-' progesterone, and 10% FBS for 3 d, and was then cultured in the DMEMF12 medium with 100 nmol L-I progesterone and 10 nmol L-I pestradiol and 10% FBS for 3 d.
Electron micrographs After culturing in the DMEM/F12 with sex steroids, the regenerated endometrium was prepared for examination using scanning electronic microscopy. The fixed and dehydrated specimens were further dried using a critical point dryer. They were sputter-coated with gold and observkd under an electron microscope at 25 kv (Philips, S-2380N, the Netherlands).
Collection of mouse embryo Female Kunming 6-8-week white mice underwent ovulation induction by the injection of 10 IU pregnant mare serum gonadotropin (PMSG, Sigma, USA), followed 48 h later by the injection of 10 IU human chorionic gonadotropin (hCG, Sigma, USA). Females were mated with males of the same strain. Mice with vaginal plugs were considered pregnant and sacrificed by cervical dislocation 5 d post-hCG for murine blastula. Embryos were flushed from the uterus with CZB and supplemented with 5 mg mL-' of bovine serum albumin (BSA, Sigma, USA). Morphologically,normal blastocysts were washed and pooled in fresh CZB medium before use.
Mouse blastula co-cultured with regenerated endometrium The regenerated endometrium was cultured in the
82007. CMS. All rightsreserved. Publishedby Elsevier Ltd.
A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro
DMEh4F12 medium with 100 nmol L-I of P-estradiol, 10 nmol L-’ of progesterone, and 10% FBS for 3 d, and then cultured in the DMEM/F12 medium with 100 nmol L-I of progesterone, 10 nmol L-I of P-estradiol, and 10% FBS for 3 d. After this treatment, the medium was replaced by CZB, and the murine blastula was placed into the three-dimensional culture system to observe the implantation of embryos.
117
RESULTS
digestion, and this has permitted a study of these two cell populations under specific experimental culture conditions. The epithelial cells showed epithelial-like characteristics (Fig. l), and stromal cells showed fibroblast-like features (Fig.2). The culture of the stroma1 cell populations was carried out easily, and these cells displayed a limited in vitro life span. In contrast, epithelium only survived in short-term primary culture. But if the epithelial cells were co-cultured with a few stromal cells, they could be subcultured by 4-5 passages.
Culture and morphology of the endometrial cells
Scanning electronic micrographs
Separation of rabbit endometrium into its epithelial and stromal components has been achieved through trypsin
As illustrated by Fig.3, the results of SEM showed that the epithelial cells on the surface of the reconstructed
Fig. 1 The morphology of the endometrial epithelial cells (x 50 times). A, primary epithelial cells; B, epithelial cells in passage 1 .
Fig. 2 The morphology of the endometrial stromal cells (x50 times). A, primary stromal cells; B, stromal cells in passage 1.
Q 2007,CAAS. All nghts reserved.Publishedby Elsevier Ltd.
1 I8
endometrium were covered with numerous slender microvilli and some cells were ciliated. Some epithelial cells protruded pinopodes. The presence of morphologically supports a property of epithelium (Bentin-Ley et al. 1994; Fawcett 1994), and it is known that ciliated columnar epithelium is formed lining the endometrium (Cunha et al. 1983; Ohtake et d.1999) (Fig.3).
SONG Yu-xuan et al.
Implantation of murine embryos in the reconstructed endometrium When the murine blastocysts were co-cultured with the regenerated endometrium for 1 d, it could be observed that the blastula sheds from the zona pellucida. The hatched blastulas begin to implant into the en-
Fig. 3 Scanning electronic micrographs of regenerated endometrium. Scanning electron micrographs of the cultured epithelial cells. Pinopodes (black arrows), microvilli (white arrows), and cilia (C) are seen. The bar scale given at the bottom of the Fig. is 10 pm.
Fig. 4 Mouse embryos implanted into the reconstructed endometrium. A, expanded murine blastocysts flushed from the uterus; B, blastocysts begin to implant into the reconstructed endometrium; C, blastocysts mostly implanted into the reconstructed endometrium; D, blastocysts implanted into the reconstructed endometrium completely.
O m 7 , CAAS.All rights reserved. Published by ElsevierLM.
A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro
dometrium after being co-cultured for 1.5 d (Fig.4).
DISCUSSION It is well known that endometrium is the functional layer of the uterus. In vitro model is convenient for elucidating the mechanism of the physiology of uterus and implantation. The high-purity epithelial cells and stromal cells of rabbit endometrium were obtained using improved centrifugation method, and these cells can be subcultured for 4-5 passages. Isolation and culture of endometrial cells makes possible the study of the physiology of uterus in vitro. The stromal cells of endometrium can be used as an ideal in vitro model for stromal invasion during implantation of the human blastocyst and for the study of the mechanism of implantation of human embryos (Janet et al. 2003). Although monolayer cells cultured in the plate can be used to study some physiological phenomenon in vitro, they cannot better mimic the natural environment of the uterus. Monolayer cells cultured in the plate are different in morphology and function compared with the cells in vivo. The epithelial cells in vivo often have polarized columnar shape. Further, the different cells in a tissue can interact by paracrine mechanism. So three-dimensional model can better mimic the natural uterus in vitro. Three-dimensionalmodel used biocompatible materials for scaffolding, and then the seeding cells were inoculated on it to regenerate the tissue in vitro. Using the type I mouse tail collagen as scaffolding material, in this study, the rabbit endometrium was regenerated in vitro based on the structure of snatural rabbit endometrium. Uterus is one of the most dynamic tissues in vivo. Endometrium periodically changes in morphology and function during menstrual cycle. Uterus is in the “windows periods” for the receptivity of embryos during implantation. Only in “windows periods”, embryos can have the cross-talk with endometrium. Pinopodes (pp) is an important morphological marker in the windows period of uterus. Nikas and Aghajanova (2002) found that the microvilli on the surface of the epithelial cells inosculated to be protuberant pinopodes at the period of implantation instantly and considered that pinopodes is a morphological marker of implantation (Usadi et al. 2003). The expression of pinopodes is strictly regulated by the progesterone. Pinopode be-
119
gin to appear with the increase in the level of progesterone and disappear with decrease in the level of progesterone in vivo (Stavreus-Evers et al. 2001). The SEM results of this study showed that the ultrastructure of pinopode on the surface of epithelial cells appeared after the 100 nmol L-’ progesterone and 10 nmol L-’ pestradiol treatments. This demonstrated that the regenerated endometrium showed similar structure and function compared with the natural endometrium. Implantation of embryo in all mammals involves shedding of the zona pellucida, followed by orientation, apposition, attachment, and adhesion of the blastocyst to the endometrium. In this experiment, it was observed that the mouse blastula co-cultured with regenerated endometrium shed from the zona pellucida and attached to the surface of regenerated endometrium, and then penetrated the epithelial layer and implanted into the stromal layer. Model systems for the study of complex processes, such as implantation, can involve different degrees of complexity. It is likely that each stage of the implantation process involves multiple molecular mechanisms; consequently, the more complex the model, the more of these putative mechanisms may operate within the model. The model presented here will help reveal the functional activity of the molecules involved in implantation and make possible the easy elucidation of the mechanism of the implantation.
Acknowledgements The authors thank National 863 Program of China (2002AA124051) for providing financial support.
References Bentin-Ley U, Pedersen B, Lindenberg S, Larsen J F, Hamberger L, Horn T. 1994. Isolation and culture of human endometrial cells in a three-dimensional culture system. Journal of Reprodution and Fertility, 101,327-332. Cunha G R, Shannon J M, Taguchi 0, Fuji H, Meloy B A. 1983. Epithelial-mesenchymal interactions in hormone-induced development. In: Sawer R H, Fallon J F, eds, Epithelialmesenchymal Interactions in Development. Praeger Publishers, New York. pp. 51-74. Fasciani A, Bocci G, Xu J, Bielecki R, Greenblatt E, Leyland N, Casper R F. 2003. Three dimensional in vitro culture of endomentrial explants minics the early stages of endometriosis.Fertility and Steriliry, 80, 1 1 37-1 143. Fawcett D W. 1994. Epithelium. In: Jensh R, ed, Concise
QW7, CAAS. All rightsreserved. Publishedby ElsevierLM.
120
Histology. Chapman&Hill, New York. pp. 19-30. Gaetje R, Kotzian S, Henmann G, Baumann R, Staninski-Powitz A. 1995. hvasiveness of endometriotic cells in vitro. Lancet, 346,1463. Janet C, Karen M, Isabella S, David B, Ian S, Helen M. 2003. An in-vitro model for stromal invasion during implantation of the human blastocyst. Human Reproduction, 18, 283-290. Liu Y X. 2002. Molecular basis of implantation. Bulletin ofthe Chinese Academy ofsciences, 5,331-333. (in Chinese) Nikas G, Aghajanova L. 2002. Endometrial pinopodes: some more understanding on human implantation. Reproductive Biomedicine Online, 4, 18-23. Ohtake H, Katabuchi H, Matsuura K, Okamura H. 1999. A novel in vitro experimental model for ovarian endometriosis: the three-dimensional culture of human ovarian surface
SONG Yu-xuan et al.
epithelial cells in collagen gels. Fertility and Sterility, 71,5055. Park D W, Choi D S, Ryu H S, Kwon H C, Joo H, Min C K. 2003. A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invision. Cancer Letters, 195, 185-192. Stavreus-Evers A, Nikas G, Sahlin L, Eriksson H, Landgren B M. 2001. Formation of pinopodes in human endometrium is associated with the concentrations of progesterone and progesterone receptor. Fertility and Sterility, 76,782-791. Usadi R S, Murray M J, Bagnell R C. 2003. Temporal and morphologic characteristics of pinopode expression across the secretory phase of the endometrial cycle in normally cycling women with proven fertility. Fertility and Sterility, 79,910-914. (Edited by WANG Lu-han)
02007,CMS. All rightsreserved.Publishedby ElsevierL.!t
Agricultural Sciences in China 2007, 6(1): 115-120
*
*’
ScienceDirect
January 2007
A Novel Three-DimensionalMouse Embryonic Implantation Model In Vifro SONG Yu-xuan and CAO Bin-yun Animal Sci-Tech College, Norihwest A & F University, Yangling 712100, P.R.China
Abstract To regenerate three-dimensional endometrium in vitro as a novel model for studying the mechanism of implantation of embryos, the luminal epithelial cells and stromal cells of the rabbit uterus were separated and cultured in vitro. The type I mouse tail collagen was used as scaffolding material. The stromal cells were inoculated in the type I mouse tail collagen, and the luminal epithelial cells were inoculated on the type I mouse tail collagen to regenerate the endometrium in vitro. The regenerated endometrium was cultured in DMEM-F/12 media containing 100 nmol L-1 progesterone, 10 nM P-estradiol, and 10% fetal bovine serum (FBS) for 3 d. The media were then replaced with CZB containing 100 nM progesterone, 10 nmol L-’ P-estradiol, and 10%FBS, and the mouse blastulas were co-cultured with it. The results of scanning electronic micrography showed that the epithelial cells on the surface of the reconstructed endometrium were covered with numerous slender microvilli and some epithelial cells protruded pinopodes. After culturing for 12 h with the mouse blastula, the shedding, attachment, and implantation of the blastula were observed. The blastula can escape from zona pellucida and attach to the three-dimensional endometrium and is then implanted into it. This study showed that the reconstructed three-dimensional endometrium can serve as a robust embryo implantation model in vitro.
Key words: three-dimensional endometrium, embryo, implantation, model, in vitro
INTRODUCTION The zygote enters the uterus after developing in the oviduct for a period of time. It is mobile in the uterus liquid .at the beginning, and then the blastula is shed from the zona pellucida as it expands. The blastula that escaped from the zona pellucida can attach to the endometrium at a suitable position. The trophoblast of the blastula can have a “cross talk” with the endometrium and make a physiological contact with the endometrium. This process is called embryonic implantation. The study of implantationmay completely solve the problem of women’s contraception. So it was considered as one of the most important scientific topics in the research field of reproductive biology. The
implantation is also the key step that controls the fertility and infertility of mammals. Implantation processes involve multiple interactions among secretions of the uterus, corpus luteum, and the embryo, and extensive tissue remodeling, angiogenesis, and apoptosis occurs at the implantation site (Liu 2002). The mechanism of the implantation has not been revealed completely hitherto, and just some hypotheses were put forth for elucidation. This is partly ascribed to the complicacy of the implantation itself, but mostly ascribed to the difficulty to study the implantation because of the limited in vivo study. The model of the regeneration of the endometrium was made in recent years to study diseases related with the endometrium (Gaetje et al. 1995; Fasciani et al. 2003; Park et al. 2003). In this experiment, we used the type I mouse
Received 10 April, 2006 Accepted 28 August, 2006 SONG Yu-xuan,Ph D, E-mail: syx98728@ 163.com; Correspondence CAO Bin-yun, Professor, E-mail: [email protected]
82007,CAAS. All tights reserved. Published by ElsevierLtd.
SONG Yu-xuan et al.
116
tail collagen as scaffolding material to regenerate the three-dimensional endometrium in v i m , and then the murine blastula were co-cultured with it for a period of time to observe the implantation of blastula in the regenerated endometrium. This study aims to establish a robust model to facilitate the elucidation of the mechanism of the implantation.
MATERIALS AND METHODS Cell preparation and culture The uterus tissue was obtained from healthy New Zealand white rabbits aged 4 months undergoing superovulation. Endometrial tissues were isolated by curettage of hysterectomy from the uteri of rabbits after 4 d from the injection of PMSG. The pooled endometrial tissues were agitated in 5 mL of 0.25% uypsin solution (Sigma, USA) at 37°C for 10 min. The enzymatic reaction was stopped by adding 6 mL of Dulbecco's modified Eagle's medium (DMEM)/Fl2 (Gibco-BRL, USA) with 10% fetal bovine serum (FBS) (Shengyuanheng Bio-Tech, China). After centrifugation for 10 min at 600 r/min, the pellet was resuspended in 10 mL of DMEM/F12 plus 10% FBS. The suspension was centrifuged at 300 r/min for 5 min, and the stromal cell-rich fraction (suspension) was collected and inoculated into a 75-cm' culture flask for 24 h. For isolation of epithelial cells, the pellet was resuspended and inoculated into another 75-cm' culture flask for 24 h. The flasks was then rinsed several times with PBS to remove red blood cells, and the old medium was replaced with fresh DMEM/F12 medium containing 100 nmol L ' P-estradiol (Sigma, USA), 10 nmol L' progesterone (Sigma, USA), and 10%FBS. The stromal cells and epithelial cells were allowed to grow to confluence at 37°C in a humidified chamber supplied with 5 % CO,.
Regeneration of endometrium in vitro The cells in passages 3-5 that were cultured in flasks were digested with 0.25% trypsin solution and centrifuged for 5 min at 1000 r/min. First, the liquid type I collagen of mouse tail (1.80 mg I&') and Matrigel were mixed according to the ratio 4: 1, and then this mixture
was mixed with 2 x DMEM/F12 containing 20% FBS according to the ratio 1:1, adjusting the pH value of the final mixed liquid to 7.00-7.20 using 0.1 mol L-' of NaOH solution. The stromal cells was resuspended with mixed collagen liquid, the cell density was adjusted to 106 mL-', and then 0.75 mL of the mixed liquid along with stroma1 cells was transferred to one of the wells of a 24well plate. After 15 min, the epithelial cells were inoculated on the surface of the stromal cell embedded in collagen. The regenerated endometrium was cultured in the DMEM/F12 medium with 100 nmol L-' pestradiol, 10 nmol L-' progesterone, and 10% FBS for 3 d, and was then cultured in the DMEMF12 medium with 100 nmol L-I progesterone and 10 nmol L-I pestradiol and 10% FBS for 3 d.
Electron micrographs After culturing in the DMEM/F12 with sex steroids, the regenerated endometrium was prepared for examination using scanning electronic microscopy. The fixed and dehydrated specimens were further dried using a critical point dryer. They were sputter-coated with gold and observkd under an electron microscope at 25 kv (Philips, S-2380N, the Netherlands).
Collection of mouse embryo Female Kunming 6-8-week white mice underwent ovulation induction by the injection of 10 IU pregnant mare serum gonadotropin (PMSG, Sigma, USA), followed 48 h later by the injection of 10 IU human chorionic gonadotropin (hCG, Sigma, USA). Females were mated with males of the same strain. Mice with vaginal plugs were considered pregnant and sacrificed by cervical dislocation 5 d post-hCG for murine blastula. Embryos were flushed from the uterus with CZB and supplemented with 5 mg mL-' of bovine serum albumin (BSA, Sigma, USA). Morphologically,normal blastocysts were washed and pooled in fresh CZB medium before use.
Mouse blastula co-cultured with regenerated endometrium The regenerated endometrium was cultured in the
82007. CMS. All rightsreserved. Publishedby Elsevier Ltd.
A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro
DMEh4F12 medium with 100 nmol L-I of P-estradiol, 10 nmol L-’ of progesterone, and 10% FBS for 3 d, and then cultured in the DMEM/F12 medium with 100 nmol L-I of progesterone, 10 nmol L-I of P-estradiol, and 10% FBS for 3 d. After this treatment, the medium was replaced by CZB, and the murine blastula was placed into the three-dimensional culture system to observe the implantation of embryos.
117
RESULTS
digestion, and this has permitted a study of these two cell populations under specific experimental culture conditions. The epithelial cells showed epithelial-like characteristics (Fig. l), and stromal cells showed fibroblast-like features (Fig.2). The culture of the stroma1 cell populations was carried out easily, and these cells displayed a limited in vitro life span. In contrast, epithelium only survived in short-term primary culture. But if the epithelial cells were co-cultured with a few stromal cells, they could be subcultured by 4-5 passages.
Culture and morphology of the endometrial cells
Scanning electronic micrographs
Separation of rabbit endometrium into its epithelial and stromal components has been achieved through trypsin
As illustrated by Fig.3, the results of SEM showed that the epithelial cells on the surface of the reconstructed
Fig. 1 The morphology of the endometrial epithelial cells (x 50 times). A, primary epithelial cells; B, epithelial cells in passage 1 .
Fig. 2 The morphology of the endometrial stromal cells (x50 times). A, primary stromal cells; B, stromal cells in passage 1.
Q 2007,CAAS. All nghts reserved.Publishedby Elsevier Ltd.
1 I8
endometrium were covered with numerous slender microvilli and some cells were ciliated. Some epithelial cells protruded pinopodes. The presence of morphologically supports a property of epithelium (Bentin-Ley et al. 1994; Fawcett 1994), and it is known that ciliated columnar epithelium is formed lining the endometrium (Cunha et al. 1983; Ohtake et d.1999) (Fig.3).
SONG Yu-xuan et al.
Implantation of murine embryos in the reconstructed endometrium When the murine blastocysts were co-cultured with the regenerated endometrium for 1 d, it could be observed that the blastula sheds from the zona pellucida. The hatched blastulas begin to implant into the en-
Fig. 3 Scanning electronic micrographs of regenerated endometrium. Scanning electron micrographs of the cultured epithelial cells. Pinopodes (black arrows), microvilli (white arrows), and cilia (C) are seen. The bar scale given at the bottom of the Fig. is 10 pm.
Fig. 4 Mouse embryos implanted into the reconstructed endometrium. A, expanded murine blastocysts flushed from the uterus; B, blastocysts begin to implant into the reconstructed endometrium; C, blastocysts mostly implanted into the reconstructed endometrium; D, blastocysts implanted into the reconstructed endometrium completely.
O m 7 , CAAS.All rights reserved. Published by ElsevierLM.
A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro
dometrium after being co-cultured for 1.5 d (Fig.4).
DISCUSSION It is well known that endometrium is the functional layer of the uterus. In vitro model is convenient for elucidating the mechanism of the physiology of uterus and implantation. The high-purity epithelial cells and stromal cells of rabbit endometrium were obtained using improved centrifugation method, and these cells can be subcultured for 4-5 passages. Isolation and culture of endometrial cells makes possible the study of the physiology of uterus in vitro. The stromal cells of endometrium can be used as an ideal in vitro model for stromal invasion during implantation of the human blastocyst and for the study of the mechanism of implantation of human embryos (Janet et al. 2003). Although monolayer cells cultured in the plate can be used to study some physiological phenomenon in vitro, they cannot better mimic the natural environment of the uterus. Monolayer cells cultured in the plate are different in morphology and function compared with the cells in vivo. The epithelial cells in vivo often have polarized columnar shape. Further, the different cells in a tissue can interact by paracrine mechanism. So three-dimensional model can better mimic the natural uterus in vitro. Three-dimensionalmodel used biocompatible materials for scaffolding, and then the seeding cells were inoculated on it to regenerate the tissue in vitro. Using the type I mouse tail collagen as scaffolding material, in this study, the rabbit endometrium was regenerated in vitro based on the structure of snatural rabbit endometrium. Uterus is one of the most dynamic tissues in vivo. Endometrium periodically changes in morphology and function during menstrual cycle. Uterus is in the “windows periods” for the receptivity of embryos during implantation. Only in “windows periods”, embryos can have the cross-talk with endometrium. Pinopodes (pp) is an important morphological marker in the windows period of uterus. Nikas and Aghajanova (2002) found that the microvilli on the surface of the epithelial cells inosculated to be protuberant pinopodes at the period of implantation instantly and considered that pinopodes is a morphological marker of implantation (Usadi et al. 2003). The expression of pinopodes is strictly regulated by the progesterone. Pinopode be-
119
gin to appear with the increase in the level of progesterone and disappear with decrease in the level of progesterone in vivo (Stavreus-Evers et al. 2001). The SEM results of this study showed that the ultrastructure of pinopode on the surface of epithelial cells appeared after the 100 nmol L-’ progesterone and 10 nmol L-’ pestradiol treatments. This demonstrated that the regenerated endometrium showed similar structure and function compared with the natural endometrium. Implantation of embryo in all mammals involves shedding of the zona pellucida, followed by orientation, apposition, attachment, and adhesion of the blastocyst to the endometrium. In this experiment, it was observed that the mouse blastula co-cultured with regenerated endometrium shed from the zona pellucida and attached to the surface of regenerated endometrium, and then penetrated the epithelial layer and implanted into the stromal layer. Model systems for the study of complex processes, such as implantation, can involve different degrees of complexity. It is likely that each stage of the implantation process involves multiple molecular mechanisms; consequently, the more complex the model, the more of these putative mechanisms may operate within the model. The model presented here will help reveal the functional activity of the molecules involved in implantation and make possible the easy elucidation of the mechanism of the implantation.
Acknowledgements The authors thank National 863 Program of China (2002AA124051) for providing financial support.
References Bentin-Ley U, Pedersen B, Lindenberg S, Larsen J F, Hamberger L, Horn T. 1994. Isolation and culture of human endometrial cells in a three-dimensional culture system. Journal of Reprodution and Fertility, 101,327-332. Cunha G R, Shannon J M, Taguchi 0, Fuji H, Meloy B A. 1983. Epithelial-mesenchymal interactions in hormone-induced development. In: Sawer R H, Fallon J F, eds, Epithelialmesenchymal Interactions in Development. Praeger Publishers, New York. pp. 51-74. Fasciani A, Bocci G, Xu J, Bielecki R, Greenblatt E, Leyland N, Casper R F. 2003. Three dimensional in vitro culture of endomentrial explants minics the early stages of endometriosis.Fertility and Steriliry, 80, 1 1 37-1 143. Fawcett D W. 1994. Epithelium. In: Jensh R, ed, Concise
QW7, CAAS. All rightsreserved. Publishedby ElsevierLM.
120
Histology. Chapman&Hill, New York. pp. 19-30. Gaetje R, Kotzian S, Henmann G, Baumann R, Staninski-Powitz A. 1995. hvasiveness of endometriotic cells in vitro. Lancet, 346,1463. Janet C, Karen M, Isabella S, David B, Ian S, Helen M. 2003. An in-vitro model for stromal invasion during implantation of the human blastocyst. Human Reproduction, 18, 283-290. Liu Y X. 2002. Molecular basis of implantation. Bulletin ofthe Chinese Academy ofsciences, 5,331-333. (in Chinese) Nikas G, Aghajanova L. 2002. Endometrial pinopodes: some more understanding on human implantation. Reproductive Biomedicine Online, 4, 18-23. Ohtake H, Katabuchi H, Matsuura K, Okamura H. 1999. A novel in vitro experimental model for ovarian endometriosis: the three-dimensional culture of human ovarian surface
SONG Yu-xuan et al.
epithelial cells in collagen gels. Fertility and Sterility, 71,5055. Park D W, Choi D S, Ryu H S, Kwon H C, Joo H, Min C K. 2003. A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invision. Cancer Letters, 195, 185-192. Stavreus-Evers A, Nikas G, Sahlin L, Eriksson H, Landgren B M. 2001. Formation of pinopodes in human endometrium is associated with the concentrations of progesterone and progesterone receptor. Fertility and Sterility, 76,782-791. Usadi R S, Murray M J, Bagnell R C. 2003. Temporal and morphologic characteristics of pinopode expression across the secretory phase of the endometrial cycle in normally cycling women with proven fertility. Fertility and Sterility, 79,910-914. (Edited by WANG Lu-han)
02007,CMS. All rightsreserved.Publishedby ElsevierL.!t