Paracrine actions of folliculogenesis on ovarian surface epithelial cells ...
Paracrine actions of folliculogenesis on ovarian surface epithelial cells: A murine pilot study.
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Dorcus Ye 1,2,3 , Elizabeth Smith 1,2 , Wensi Tao1,2, Robert Moore 1,2, Ali Sheikhi 4, Xiang-Xi Mike Xu1,2 . 1Department
Of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida; 2Sylvester Comprehensive Cancer Centre ,University of Miami, Miami, Florida 3Department of Obstetrics and Gynaecology, Royal College of Surgeons in Ireland, Ireland. 4Center for Support and Training in Analysis and Research (CSTAR), University College Dublin, Ireland. Correspondence: Professor Xiang-Xi M. Xu, Department of Cell Biology, University of Miami Miller School of Medicine, FL 33136, USA. [email protected].
Introduction:
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The origin of ovarian cancer cells remain elusive. Some suggest high grade serous ovarian cancer cells originate from distal fimbriae of fallopian tubes while others suggest its origination from inclusion cysts derived from ovarian surface epithelium (OSE)1. Our studies on the Wv germ cell deficient mouse model showed tubular adenomas resembling invaginations and papillomatosis2. We postulate that inhibin A positive granulosa cells exert a paracrine inhibitory mechanism on mouse ovarian surface epithelial cells (MOSE). We demonstrated the in vitro actions of early and pre ovulatory granulosa cells on MOSE cell proliferation. (a)
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Illustration a) Anatomical dissection of wild type mouse b) Location of left ovary c) Cell culture primary murine ovarian surface epithelial cells zeiss microscope 20x magnificaition d) Isolation of murine pre antral follicle 40x magnification
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Materials & Methods: Animal ethical approval was obtained from Instituitional Animal Care and Use Committee (IACUC) of University of Miami, Florida. MOSE were isolated from C57BL/6J mice. Mice were primed with diethylstilboestrol to isolate granulosa cells from pre antral follicles. Super ovulation protocols were used to isolate granulosa cells from pre-ovulatory follicles. Conditioned medium of pre antral granulosa cells (CMPAGC) and pre ovulatory granulosa cells (CMPOGC) were collected. Unprimed ovaries were cut into ovarian explants to culture stroma cells. Conditioned media of ovarian explants (CMOE) and stroma cells (CMS) were collected. MOSE was treated with 4 different media in varying concentrations [20%- 80%] over 96 hours. Cell proliferation was performed using WST 1 with absorbance values measured with spectrophotometer. Statistical analysis was performed with SPSS version 20. The significance of the difference between means was assessed using Kruskal Wallis test. The strength of the co relation between the variables was assessed using Pearson correlation co efficient. (f) (e)
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Figure (a,b) Graphs representing cell proliferation effects on MOSE treated with CMPAGC (b,c) CMPOGC (e,f) CMOE
Illustration e) Isolation of murine granulosa cells f) A transwell co culture system3 Results: CMPAGC50 exert suppression on MOSE of 78% and greater suppression with CMPAGC80 of 89% with significant difference between its control and CMPAGC group (p-value=0.019). CMPOGC50 exert a greater suppression on MOSE of 87%. There was no significant change CMPAGC and CMPOGC groups on MOSE suppression. CMOE stimulated MOSE proliferation effect in concentration-dependent manner.
Conclusion: Conditioned media from pre antral and pre ovulatory granulosa cells suppress MOSE cell proliferation. We found no difference between granulosa cells isolated from different follicular stages. The study emphasizes the role of granulosa cells in maintaining the balance of intra ovarian communication with MOSE cells. The loss of granulosa cells lead to OSE hyper proliferation which implicates the increase ovarian cancer risk in postmenopausal women. It is worthwhile identifying the key paracrine mediator responsible for its inhibitory action on MOSE, through a differential secretome analysis and western blotting. This will provide insight into early events of ovarian tumorigenesis & explore potential biomarkers for early cancer detection References: 1) Dubeau L1 The cell of origin of ovarian epithelial tumours Lancet Oncol. 2008 Dec;9(12):1191-7. 2) Cai KQ1Xu XX4 et el. Global deletion of Trp53 reverts ovarian tumor phenotype of the germ cell-deficient white spotting variant (Wv)mice. Neoplasia. 2015 Jan;17(1):89-100. 3)Corning Transwell Culture support.http://www.fischersci.com
A
Dorcus Ye 1,2,3 , Elizabeth Smith 1,2 , Wensi Tao1,2, Robert Moore 1,2, Ali Sheikhi 4, Xiang-Xi Mike Xu1,2 . 1Department
Of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida; 2Sylvester Comprehensive Cancer Centre ,University of Miami, Miami, Florida 3Department of Obstetrics and Gynaecology, Royal College of Surgeons in Ireland, Ireland. 4Center for Support and Training in Analysis and Research (CSTAR), University College Dublin, Ireland. Correspondence: Professor Xiang-Xi M. Xu, Department of Cell Biology, University of Miami Miller School of Medicine, FL 33136, USA. [email protected].
Introduction:
Figure (a)
(b)
(c)
(d)
O
S E - C M
P A G
C
S t u d y
0 .4
M O S E a b s o rb a n c e 4 5 0 n m
The origin of ovarian cancer cells remain elusive. Some suggest high grade serous ovarian cancer cells originate from distal fimbriae of fallopian tubes while others suggest its origination from inclusion cysts derived from ovarian surface epithelium (OSE)1. Our studies on the Wv germ cell deficient mouse model showed tubular adenomas resembling invaginations and papillomatosis2. We postulate that inhibin A positive granulosa cells exert a paracrine inhibitory mechanism on mouse ovarian surface epithelial cells (MOSE). We demonstrated the in vitro actions of early and pre ovulatory granulosa cells on MOSE cell proliferation. (a)
M
c o n tr o l C M
P A G C 0
C M
P A G C 2 0
C M
P A G C 5 0
C M
P A G C 8 0
0 .3
0 .2
0 .1
0 .0 0
2 4
4 8
t im
7 2
9 6
e /h r
Figure (c) M
O
S E - C
M
P O
G
C
S t u d y
Illustration a) Anatomical dissection of wild type mouse b) Location of left ovary c) Cell culture primary murine ovarian surface epithelial cells zeiss microscope 20x magnificaition d) Isolation of murine pre antral follicle 40x magnification
M O S E a b s o rb a n c e 4 5 0 n m
0 .4
c o n tr o l
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P O G C 0
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Figure (e) M
O
S E - C
M
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S t u d y
0 .6
M O S E a b s o rb a n c e 4 5 0 n m
Materials & Methods: Animal ethical approval was obtained from Instituitional Animal Care and Use Committee (IACUC) of University of Miami, Florida. MOSE were isolated from C57BL/6J mice. Mice were primed with diethylstilboestrol to isolate granulosa cells from pre antral follicles. Super ovulation protocols were used to isolate granulosa cells from pre-ovulatory follicles. Conditioned medium of pre antral granulosa cells (CMPAGC) and pre ovulatory granulosa cells (CMPOGC) were collected. Unprimed ovaries were cut into ovarian explants to culture stroma cells. Conditioned media of ovarian explants (CMOE) and stroma cells (CMS) were collected. MOSE was treated with 4 different media in varying concentrations [20%- 80%] over 96 hours. Cell proliferation was performed using WST 1 with absorbance values measured with spectrophotometer. Statistical analysis was performed with SPSS version 20. The significance of the difference between means was assessed using Kruskal Wallis test. The strength of the co relation between the variables was assessed using Pearson correlation co efficient. (f) (e)
C M 0 .3
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O
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C M
O
E 2 0
C M
O
E 5 0
C M
O
E 8 0
0 .2
0 .0 0
2 4
4 8
t im
7 2
9 6
e / h r
Figure (a,b) Graphs representing cell proliferation effects on MOSE treated with CMPAGC (b,c) CMPOGC (e,f) CMOE
Illustration e) Isolation of murine granulosa cells f) A transwell co culture system3 Results: CMPAGC50 exert suppression on MOSE of 78% and greater suppression with CMPAGC80 of 89% with significant difference between its control and CMPAGC group (p-value=0.019). CMPOGC50 exert a greater suppression on MOSE of 87%. There was no significant change CMPAGC and CMPOGC groups on MOSE suppression. CMOE stimulated MOSE proliferation effect in concentration-dependent manner.
Conclusion: Conditioned media from pre antral and pre ovulatory granulosa cells suppress MOSE cell proliferation. We found no difference between granulosa cells isolated from different follicular stages. The study emphasizes the role of granulosa cells in maintaining the balance of intra ovarian communication with MOSE cells. The loss of granulosa cells lead to OSE hyper proliferation which implicates the increase ovarian cancer risk in postmenopausal women. It is worthwhile identifying the key paracrine mediator responsible for its inhibitory action on MOSE, through a differential secretome analysis and western blotting. This will provide insight into early events of ovarian tumorigenesis & explore potential biomarkers for early cancer detection References: 1) Dubeau L1 The cell of origin of ovarian epithelial tumours Lancet Oncol. 2008 Dec;9(12):1191-7. 2) Cai KQ1Xu XX4 et el. Global deletion of Trp53 reverts ovarian tumor phenotype of the germ cell-deficient white spotting variant (Wv)mice. Neoplasia. 2015 Jan;17(1):89-100. 3)Corning Transwell Culture support.http://www.fischersci.com
