Page 1 of 31 Reproduction Advance Publication first posted on 16 October 2014 as Manuscript REP-14-0337
REGULATION OF OVULATORY GENES IN BOVINE GRANULOSA CELLS: LESSONS FROM siRNA SILENCING OF PTGS2 Ketan Shrestha1, Karolina Lukasik2, Anja Baufeld3, Jens Vanselow3, Uzi Moallem4 and Rina Meidan1§ 1
Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food and
Environment, The Hebrew University of Jerusalem, Rehovot, Israel. 2
Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction
and Food Research, Polish Academy of Science, Olsztyn, Poland. 3
Department of Reproductive Biology, Leibniz Institute of Farm Animal Biology,
Dummerstorf, Germany. 4
Department of Ruminant Science, Agricultural Research Organization, Bet-Dagan, Israel.
Running title: Silencing of prostaglandin endoperoxide synthase 2. Keywords – ovulation, granulosa cells, follicle, RNA interference, endometrial cells.
Supported by a grant from Chief Scientist of the Israeli Ministry of Agriculture (RM and UM). The authors have nothing to disclose. §
Corresponding author: Prof. Rina Meidan, Department of Animal Sciences, The Robert
H. Smith Faculty of Agriculture, Food and Environment. The Hebrew University of Jerusalem, Rehovot 76100 Israel, email:
[email protected], phone number: +97289489394, fax number: 97289465763.
0
Copyright © 2014 by the Society for Reproduction and Fertility.
Page 2 of 31
1
Abstract
2
Prostaglandin endoperoxide synthase 2 (PTGS2), tumor necrosis factor-alpha-induced protein-6
3
(TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor like factors: amphiregulin (AREG) and
4
epiregulin (EREG) are essential for successful ovulation. We compared here the induction of
5
these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro
6
(forskolin treatment). These genes were markedly stimulated in GCs isolated from cows 21hr
7
after LH- surge. In isolated GCs, forskolin induced a distinct temporal profile for each gene.
8
Generally, there was a good agreement between the in vivo and in vitro inductions of these genes
9
except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular
10
compartments may mediate its induction by LH. Next, to study the role of PTGS2 and
11
prostaglandins in the cascade of ovulatory genes, PTGS2 was silenced with small interfering
12
RNA (siRNA). PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and
13
PGE2 production (70% compared to scrambled siRNA) in bovine GCs. Importantly, PTGS2
14
silencing also reduced AREG, EREG and TNFAIP6 mRNA but not PTX3. Exogenous PGE2
15
increased AREG, EREG and TNFAIP6 mRNAs, further confirming that these genes are
16
prostanoid-dependent. A successful and specific knockdown of PTGS2 was also achieved in
17
endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-
18
PTGS2) siRNA that facilitates cells’ entry was examined. In EndoCs, but not in GCs, Chol-
19
PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent.
20
PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the
21
reproductive tract.
22
1
Page 3 of 31
23
Introduction
24
Ovulation is triggered by LH surge. The LH receptor activates several families of
25
heterotrimeric G-proteins, but the activation of Gs and cAMP/PKA cascade is widely accepted
26
as important downstream signaling of LH action (Fan et al. 2011, Breen et al. 2013). It brings
27
about series of changes such as resumption of meiosis in the oocyte, reprogramming of the
28
granulosa cells (GCs) and theca cells (TCs) layer of the follicular wall. These events are
29
accompanied by the expression of new mRNAs and proteins and result in the release of a
30
fertilizable oocyte and the luteinization of follicular cells (Richards 2001, Richards 2005). In
31
preparation for oocyte expulsion, the extracellular matrix (ECM) of cumulus oocyte complex
32
(COC) undergoes remodeling (Richards 1994, Salustri et al. 1996). A number of genes
33
mediating these processes have been identified; amongst them are Prostaglandin endoperoxide
34
synthase 2 (PTGS2), tumor necrosis factor-alpha-induced protein 6 (TNFAIP6), pentraxin 3
35
(PTX3), epidermal growth factor (EGF) like factors: amphiregulin (AREG) and epiregulin
36
(EREG) (Mukhopadhyay et al. 2001, Salustri et al. 2004, Conti et al. 2006, Bridges & Fortune
37
2007).
38
LH-induced PTGS2 in the GCs layer increases prostaglandins (PGs), especially prostaglandin
39
endoperoxide 2 (PGE2) in the follicular fluid (Sirois & Richards 1992, Sirois 1994, Tsai et al.
40
1996, Liu et al. 1997). The essential role of PTGS2 is demonstrated in gene knockout
41
experiments and PTGS inhibitors studies which resulted in failed ovulation with normal
42
luteinization and corpus luteum formation (Dinchuk et al. 1995, Davis et al. 1999, Peters et al.
43
2004). Later, it was demonstrated that LH/hCG upregulate EGF-like factors in murine GCs;
44
these peptides mediated LH action in COC expansion and oocyte maturation (Park et al. 2004,
45
Conti et al. 2006, Shimada et al. 2006). During COC expansion, LH surge also induces 2
Page 4 of 31
46
TNFAIP6 which is a hyaluronan binding protein (Lee et al. 1992) critical for the stability of
47
glycosaminoglycan hyaluronan (HA) rich ECM (Ochsner et al. 2003). Study from TNFAIP6
48
knockout mice showed defective COC expansion and infertility (Fulop et al. 2003). Likewise,
49
PTX3, belonging to the long pentraxin family of inflammatory proteins, was expressed in COC
50
(Varani et al. 2002, Salustri et al. 2004). It plays protective role during the formation of HA
51
rich matrix of the COC by cross-linking HA polymers through interactions with heavy chains
52
of inter-α trypsin inhibitor (IαI) and/or TNFAIP6 (Ievoli et al. 2011). An abnormal COC
53
expansion characterized by unstable extracellular matrix was reported in PTX3 deficient mice
54
(Salustri et al. 2004). Thus, PTGS2, EGF like factors (AREG and EREG) and HA binding
55
proteins (TNFAIP6 and PTX3) are all the obligatory factors for ECM stability, COC expansion
56
and follicular rupture.
57
Most of our knowledge on ovulatory genes stems from rodents especially mice, however
58
reproductive strategies differ in rodents and large mono-ovulatory animals. Therefore, data
59
from rodents cannot always be extrapolated in mono-ovulatory mammals such as bovine (Bahr
60
& Wolf 2012). In addition, these animals are not amendable for gene deletion studies. In such
61
species, small interfering RNA (siRNA) can be used as means for gene knockdown (Fang et al.
62
2013). Therefore, we aimed here to study induction of ovulatory genes in bovine GCs under in
63
vivo and in vitro conditions. siRNA silencing of PTGS2 was used to critically examine the role
64
of PGE2 in the cascade of events in GCs that leads to ovulation. As a proof of concept we also
65
employed endometrial cells (EndoCs), these cells similarly to GCs, express PTGS2 and their
66
synthesized PGs play significant physiological roles.
67
3
Page 5 of 31
68
Materials and Methods
69
Animals and sample collection
70
Follicles were collected from Holstein-Friesian cows as described (Vanselow et al. 2010,
71
Christenson et al. 2013). Large dominant follicles before LH surge were collected after
72
carefully monitoring a growing cohort of follicles in normally cycling cows by transrectal
73
ultrasonography (Aloka SSD-500, Aloka GmbH, Meerbusch, Germany). Animals were
74
slaughtered at days 7 (n=1) and 8 (n=2) of the estrous cycle during the first follicular wave.
75
Only the largest growing, but not stagnating or regressing follicle of each animal was collected.
76
To collect dominant follicles after the LH surge, normally cycling cows were treated with
77
500µg prostaglandin F2 alpha (PGF2a) (PGF Veyx® forte, Veyx Pharma GmbH, Germany) at
78
day 8 of the cycle, to induce luteolysis. Forty-eight hours later, animals were injected with 100
79
µg of a GnRH analogue (Gonavet Veyx®,Depherelin, Veyx Pharma GmbH) to induce the LH
80
surge. The animals were slaughtered 23 hours later and the largest growing follicle of each
81
animal was isolated (n=3). GCs were collected by aspiration of the follicular fluid with an 18G
82
needle. The fluid was centrifuged (2min, 400rcf) and the sediment cells were frozen in liquid
83
nitrogen and stored at -80°C for RNA preparation. RNA isolation, reverse transcription and
84
qPCR were carried out as described previously (Christenson et al. 2013).
85
All procedures involving living animals (injections and ultrasonography examinations in cattle)
86
were performed according to the German law for animal protection (8a TierSchG i.V.m. 29
87
Tierschutz-Versuchstierverordnung). The named procedures are not subjected to specific
88
permit by the governmental authority Landesamt für Landwirtschaft, Lebensmittelsicherheit
89
und Fischerei (LALLF) Rostock. 4
Page 6 of 31
90
Isolation and culture of granulosa cells
91
Ovaries were collected at a local abattoir as previously described (Meidan et al. 1990). GCs
92
were enzymatically dispersed by using a combination of collagenase I (125 units/ml),
93
hyaluronidase III (36 units/ml) and deoxyribonuclease I (11 units/ml) in Dulbecco's modified
94
Eagle medium (DMEM)/F-12 containing 2 mM L-Glutamine and 100 µg/ml of
95
penicillin/streptomycin. Only large follicles (>10 mm in diameter) containing ≥ 4 million
96
viable cells were used. GCs were cultured overnight in medium containing 3% fetal calf serum
97
(FCS). Cells were then incubated with DMEM/F-12 containing 1% FCS (control) and with
98
PGE2 (1 µM; Cayman Chemical Co) or with forskolin (FRS; 10 µM) for various time points as
99
indicated. Unless otherwise stated all biochemical were from Sigma-Aldrich, St. Louis, MO
100
and cell culture materials were from Biological Industries, Kibbutz Beit Haemek, Israel.
101
Isolation and culture of endometrial cells (EndoCs)
102
Uterine horns from cows at late luteal phase were collected from the abattoir (Arosh et al.
103
2002). Endometrial strips were dissected and incubated in enzyme solution (18 units/ml DNAse
104
and 315 units/ml collagenase I) at 38o C for 25 minutes in shaking water bath. Cell suspension
105
was filtered with cell strainer, centrifuged and cultured in DMEM/F-12 containing 10% FCS.
106
Confluent plates were trypsinized with trypsin EDTA solution (0.25% trypsin and 0.02%
107
EDTA). Cells of passages 2-3 were utilized in this study. Based on morphology and growth in
108
culture the cells were identified as endometrial stromal cells (Cherny & Findlay 1990).
5
Page 7 of 31
109
Transfection of cells
110
For transfection experiments GCs were trypsinized with trypsin EDTA solution (0.05% trypsin
111
and 0.02% EDTA) immediately after isolation from follicles. Trypsinized cells were seeded
112
(3x105 GCs; 0.8X105 EndoCs) in 6-well plates and cultured for up to 24 hr in 3% FCS. Then
113
cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according
114
to the manufacturer’s protocol in 1% FCS. Cells were transfected with siRNA sequence
115
targeting (50 nmol/L) naked PTGS2 siRNA or cholesterol conjugated PTGS2 siRNA or
116
scrambled
117
GUGAAAGGCUGUCCCUUUA[dT][dT],
118
UAAAGGGACAGCCUUUCAC[dT][dT] corresponding to bases 1781-1799 of the bovine
119
PTGS2 mRNA sequence (NM_174445). Sequence for cholesterol conjugated PTGS2 siRNA
120
was same as for naked PTGS2 siRNA with cholesterol conjugation in 3’end of sense strand: S,
121
GUGAAAGGCUGUCCCUUUA[dA][dT][CholTEG],
122
UAAAGGGACAGCCUUUCAC[dA][dT]. Scrambled siRNA sequence - negative control was
123
S,
124
ACGUGACACGUUCGGAGAA[dT][dT]. A day after transfection PTGS2 was induced in
125
GCs by FRS (10 µM) and in EndoCs by phorbol 12-myristate 13-acetate (PMA; 100ng/ml),
126
then cells were harvested and total RNA was extracted from cells 48hr after transfection.
127
For treatment of GCs and EndoCs in absence of transfection reagent, cells were incubated with
128
cholesterol-conjugated PTGS2 siRNA for 24hr and total RNA was extracted from cells after
129
48hr.
siRNA.
Sequence
of
naked
PTGS2
UUCUCCGAACGUGUCACGU[dT][dT],
siRNA
was
sense
antisense
(S), (AS),
AS,
and
AS,
6
Page 8 of 31
130
RNA extraction and real-time PCR
131
Total RNA was isolated from cells using TriFast reagent (Peqlab Biotechnologie GmbH,
132
Erlangen, Germany) according to the manufacturer's instructions. 1 µg of total RNA was
133
reverse transcribed using M-MuLV Reverse Transcriptase (200 units/uL), M-MuLV RT Buffer
134
(New England Biolabs, Ipswich, MA), random primer (100 nM), oligo-dT (100 µM), and
135
dNTPs mix (100 mM) (Bioline Reagents Limited, London). Real-time PCRs were performed
136
using the Mx3000P system (Stratagene, Garden Grove, CA), using Platinum SYBR Green
137
(SuperMix, Invitrogen, Carlsbad, CA), as previously described (Klipper et al. 2009). Gene
138
expression (PTGS2, TNFAIP6, PTX3, AREG and EREG) was analyzed by quantitative real-
139
time PCR. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the
140
housekeeping gene. The threshold cycle number (Ct) was used to quantify the relative
141
abundance of the gene; arbitrary units were calculated as 2-∆Ct=2- (Ct target gene - Ct housekeeping gene).
142
The primer sequences used were as follows: GAPDH (NM_001034034) forward, 5’-
143
GTCTTCACTACCATGGAGAAGG -3’, reverse, 5’- TCATGGATGACCTTGGCCAG -3’;
144
PTGS2 (NM_174445) forward, 5’- CAGCGGTGCAGCAAATCCTTG -3’,
145
CTGTGTTGGGAGTGGGTTTCA
146
CTATAGCTGCTTTCGTCTCTGC -3’, reverse, 5’- CGTTCTTCAGCGACACCTTCA -3’;
147
EREG (XM_002688367) forward, 5’- CTGCTGCTCGTCCTGGTTTTC -3’, reverse, 5’-
148
GCTGTGCAGTTATCTCCCGAC
149
ATGGCTTGAACAAGCAGCAGG -3’, reverse, 5’- GCCATCCACCCAGCAGCACA -3’;
150
PTX3 (NM_001076259) forward, 5’- AGCCTCTTGCCTCGTCCCCTC -3’, reverse, 5’-
151
TCTGAGTTCTCCGCCGACACT -3’.
-3’;
-3’;
AREG
(NM_001099092)
TNFAIP6(NM_001007813)
reverse, 5’-
forward,
forward,
5’-
5’-
152 7
Page 9 of 31
153
Prostaglandin E2 analysis
154
Media from the cell cultures were collected on the day of RNA isolation. Levels of PGE2 were
155
measured by PGE2 enzyme immunoassay kit- monoclonal (Cayman Chemical Co) according to
156
manufacturer’s instruction. The standard curve ranged from 6.4 pg/ml to 1000 pg/ml. Cross
157
reactivity of the assay with PGE2 was 100% and 0.01 % for PGF-2a.
158
Statistical analyses
159
Data are presented as means ± SEM; experiments were repeated at least three times.
160
Expressions of specific mRNA transcripts were normalized relative to the abundance of
161
GAPDH mRNA. Data were analyzed by Student t-test. Differences were considered significant
162
at P < 0.05. Asterisks represent significant differences from their respective controls. *P
