Abstract
Prostaglandin endoperoxide synthase-2 (PTGS2), tumour necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor-like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. In this study, we compared the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin (FRS) treatment). These genes were markedly stimulated in GCs isolated from cows 21 h after LH-surge. In isolated GCs, FRS induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins (PGs) in the cascade of ovulatory genes, PTGS2 was silenced with siRNA. PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared with scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA levels but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNA levels, further confirming that these genes are prostanoid dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was investigated. In EndoCs, but not in GCs, chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.
Introduction
Ovulation is triggered by LH surge. The LH receptor activates several families of heterotrimeric G-proteins, but the activation of Gs and cAMP/PKA cascade is widely accepted as an important downstream signalling of LH action (Fan et al. 2011, Breen et al. 2013). It brings about series of changes such as resumption of meiosis in the oocyte, reprogramming of the granulosa cells (GCs) and theca cells (TCs) layer of the follicular wall. These events are accompanied by the expression of new mRNAs and proteins and result in the release of a fertilisable oocyte and the luteinisation of follicular cells (Richards 2001, 2005). In preparation for oocyte expulsion, the extracellular matrix (ECM) of cumulus–oocyte complex (COC) undergoes remodeling (Richards 1994, Salustri et al. 1996). A number of genes mediating these processes have been identified, amongst them are prostaglandin endoperoxide synthase 2 (PTGS2), tumour necrosis factor-alpha-induced protein 6 (TNFAIP6), pentraxin 3 (PTX3), epidermal growth factor (EGF)-like factors: amphiregulin (AREG) and epiregulin (EREG) (Mukhopadhyay et al. 2001, Salustri et al. 2004, Conti et al. 2006, Bridges & Fortune 2007).
LH-induced PTGS2 in the GCs layer increases prostaglandins (PGs), especially PG endoperoxide 2 (PGE2) in the follicular fluid (Sirois & Richards 1992, Sirois 1994, Tsai et al. 1996, Liu et al. 1997). The essential role of PTGS2 is demonstrated in gene-knockout experiments and PTGS inhibitors studies which resulted in failed ovulation with normal luteinisation and corpus luteum formation (Dinchuk et al. 1995, Davis et al. 1999, Peters et al. 2004). Later, it was demonstrated that LH/hCG upregulate EGF-like factors in murine GCs; these peptides mediated LH action in COC expansion and oocyte maturation (Park et al. 2004, Conti et al. 2006, Shimada et al. 2006). During COC expansion, LH surge also induces TNFAIP6, which is a hyaluronan (HA)-binding protein (Lee et al. 1992) critical for the stability of glycosaminoglycan HA-rich ECM (Ochsner et al. 2003). A study of Tnfaip6-knockout mice showed defective COC expansion and infertility (Fulop et al. 2003). Likewise, PTX3, belonging to the long pentraxin family of inflammatory proteins, was expressed in COC (Varani et al. 2002, Salustri et al. 2004). It plays a protective role during the formation of HA-rich matrix of the COC by cross-linking HA polymers through interactions with heavy chains of inter-α trypsin inhibitor (IαI) and/or TNFAIP6 (Ievoli et al. 2011). An abnormal COC expansion characterised by unstable extracellular matrix was reported in Ptx3-deficient mice (Salustri et al. 2004). Thus, PTGS2, EGF-like factors (AREG and EREG) and HA-binding proteins (TNFAIP6 and PTX3) are all the obligatory factors for ECM stability, COC expansion and follicular rupture.
Most of our knowledge on ovulatory genes stems from rodents, especially mice; however reproductive strategies differ in rodents and large mono-ovulatory animals. Therefore, data from rodents cannot always be extrapolated in mono-ovulatory mammals such as bovine (Bahr & Wolf 2012). In addition, these animals are not amendable for gene deletion studies. In such species, siRNA can be used as a means for gene knockdown (Fang et al. 2013). Therefore, we aimed herein to study the induction of ovulatory genes in bovine GCs under in vivo and in vitro conditions. siRNA silencing of PTGS2 was used to critically examine the role of PGE2 in the cascade of events in GCs that leads to ovulation. As a proof of concept we also employed endometrial cells (EndoCs), these cells, similar to GCs, express PTGS2 and their synthesised PGs play significant physiological roles.
Materials and methods
Animals and sample collection
Follicles were collected from Holstein–Friesian cows as described (Vanselow et al. 2010, Christenson et al. 2013). Large dominant follicles before LH surge were collected after carefully, monitoring a growing cohort of follicles in normally cycling cows by transrectal ultrasonography (Aloka SSD-500, Aloka GmbH, Meerbusch, Germany). The animals were slaughtered at days 7 (n=1) and 8 (n=2) of the estrous cycle during the first follicular wave. Only the largest growing, but not stagnating or regressing, follicle of each animal was collected. To collect dominant follicles after the LH surge, normally cycling cows were treated with 500 μg PGF2a (PGF Veyx forte, Veyx Pharma GmbH, Schwarzenborn, Germany) at day 8 of the cycle, to induce luteolysis. Forty-eight hours later, the animals were injected with 100 μg of a gonadotrophin-releasing hormone (GNRH) analogue (GonavetVeyx, Depherelin, Veyx Pharma GmbH) to induce the LH surge. The animals were slaughtered 23 h later and the largest growing follicle of each animal was isolated (n=3). The GCs were collected by aspiration of the follicular fluid with an 18G needle. The fluid was centrifuged (2 min, 400 g) and the sediment cells were frozen in liquid nitrogen and stored at −80 °C for RNA preparation. RNA isolation, RT and qPCR were carried out as described previously (Christenson et al. 2013).
All procedures involving living animals (injections and ultrasonography examinations in cattle) were performed according to the German law for animal protection (8a TierSchG i.V.m. 29 Tierschutz-Versuchstierverordnung). The named procedures are not subjected to specific permit by the governmental authority Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei (LALLF) Rostock.
Isolation and culture of GCs
The ovaries were collected at a local abattoir as described previously (Meidan et al. 1990). The GCs were enzymatically dispersed by using a combination of collagenase I (125 units/ml), hyaluronidase III (36 units/ml) and deoxyribonuclease I (11 units/ml) in DMEM/F-12 containing 2 mM l-glutamine and 100 μg/ml of penicillin/streptomycin. Only large follicles (>10 mm in diameter) containing ≥4 million viable cells were used. The GCs were cultured overnight in a medium containing 3% FCS. The cells were then incubated with DMEM/F-12 containing 1% FCS (control) and with PGE2 (1 μM; Cayman Chemical Co., Ann Arbor, MI, USA) or with forskolin (FRS; 10 μM) for various time points as indicated. Unless otherwise stated all biochemicals were from Sigma–Aldrich and cell culture materials were from Biological Industries, Kibbutz Beit Haemek, Israel.
Isolation and culture of EndoCs
Uterine horns from cows at late luteal phase were collected from the abattoir (Arosh et al. 2002). Endometrial strips were dissected and incubated in an enzyme solution (18 units/ml DNAse and 315 units/ml collagenase I) at 38 °C for 25 min in a shaking water bath. The cell suspension was filtered with a cell strainer, centrifuged and cultured in DMEM/F-12 containing 10% FCS. Confluent plates were trypsinised with trypsin EDTA solution (0.25% trypsin and 0.02% EDTA). The cells of passages 2–3 were utilised in this study. Based on morphology and growth in culture, the cells were identified as endometrial stromal cells (Cherny & Findlay 1990).
Transfection of cells
For transfection experiments, GCs were trypsinised with trypsin EDTA solution (0.05% trypsin and 0.02% EDTA) immediately after isolation from follicles. The trypsinised cells were seeded (3×105 GCs; 0.8×105 EndoCs) in six-well plates and cultured for up to 24 h in 3% FCS. Then cells were transfected using Lipofectamine 2000 reagent (Invitrogen) in 1% FCS, according to the manufacturer's protocol. The cells were transfected with siRNA sequence, targeting (50 nmol/l) naked PTGS2 siRNA or cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA or scrambled siRNA. The sequence of naked PTGS2 siRNA was sense (S), GUGAAAGGCUGUCCCUUUA[dT][dT], antisense (AS), UAAAGGGACAGCCUUUCAC[dT][dT] corresponding to bases 1781–1799 of the bovine PTGS2 mRNA sequence (NM_174445). The sequence for chol-PTGS2 siRNA was same as for naked PTGS2 siRNA with cholesterol conjugation in 3′ end of sense strand: S, GUGAAAGGCUGUCCCUUUA[dA][dT][CholTEG], AS, UAAAGGGACAGCCUUUCAC[dA][dT]. Scrambled siRNA sequence-negative control was S, UUCUCCGAACGUGUCACGU[dT][dT], and AS, ACGUGACACGUUCGGAGAA[dT][dT]. A day after transfection, PTGS2 was induced in GCs by FRS (10 μM) and in EndoCs by phorbol 12-myristate 13-acetate (PMA; 100 ng/ml), then the cells were harvested and total RNA was extracted from cells 48 h after transfection.
For treatment of GCs and EndoCs in the absence of transfection reagent, the cells were incubated with chol-PTGS2 siRNA for 24 h and total RNA was extracted from cells after 48 h.
RNA extraction and real-time PCR
Total RNA was isolated from the cells using TriFast reagent (Peqlab Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer's instructions: 1 μg of total RNA was reverse transcribed using M-MuLV Reverse Transcriptase (200 units/μl), M-MuLV RT Buffer (New England Biolabs, Ipswich, MA, USA), random primer (100 nM), oligo-dT (100 μM) and dNTPs mix (100 mM) (Bioline Reagents Limited, London, UK). Real-time PCRs were carried out using the Mx3000P system (Stratagene, Garden Grove, CA, USA), using Platinum SYBR Green (SuperMix, Invitrogen), as previously described (Klipper et al. 2009). Gene expression (PTGS2, TNFAIP6, PTX3, AREG and EREG) was analysed by quantitative real-time PCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the housekeeping gene. The threshold cycle number (Ct) was used to quantify the relative abundance of the gene; arbitrary units were calculated as 2−ΔCt=2−(Ct target gene−Ct housekeeping gene). The primer sequences used were as follows: GAPDH (NM_001034034) forward: 5′-GTCTTCACTACCATGGAGAAGG-3′, reverse: 5′-TCATGGATGACCTTGGCCAG-3′; PTGS2 (NM_174445) forward: 5′-CAGCGGTGCAGCAAATCCTTG-3′, reverse: 5′-CTGTGTTGGGAGTGGGTTTCA-3′; AREG (NM_001099092) forward: 5′-CTATAGCTGCTTTCGTCTCTGC-3′, reverse: 5′-CGTTCTTCAGCGACACCTTCA-3′; EREG (XM_002688367) forward: 5′-CTGCTGCTCGTCCTGGTTTTC-3′, reverse: 5′-GCTGTGCAGTTATCTCCCGAC-3′; TNFAIP6 (NM_001007813) forward: 5′-ATGGCTTGAACAAGCAGCAGG-3′, reverse: 5′-GCCATCCACCCAGCAGCACA-3′; PTX3 (NM_001076259) forward: 5′-AGCCTCTTGCCTCGTCCCCTC-3′, reverse: 5′-TCTGAGTTCTCCGCCGACACT-3′; VEGF (NM_174216) forward: 5′-CCATGAACTTTCTGCTCTCTTGG-3′, reverse: 5′-TCCATGAACTCCACCACTTCG-3′; SLC2A1 (NM_174602) forward: 5′-CGCTTCCTGCTCATTAACCG-3′, reverse: 5′-CCTTCTTCTCCCGCATCAT-3′; TNFR1 (NM_174674) forward: 5′-GGCGAGACACGGACTGCA-3′, reverse: 5′-TCCCGGTCCACTACACAAGG-3′.
PGE2 analysis
Media from the cell cultures were collected on the day of RNA isolation. The levels of PGE2 were measured by PGE2 enzyme Immunoassay Kit – monoclonal (Cayman Chemical Co.) according to manufacturer's instruction. The standard curve ranged from 6.4 to 1000 pg/ml. Cross reactivity of the assay with PGE2 was 100 and 0.01% for PGF2a.
Statistical analyses
Data are presented as means±s.e.m.; experiments were repeated at least three times. The expression of specific mRNA transcripts was normalised relative to the abundance of GAPDH mRNA. Data were analysed by Student's t-test. Differences were considered significant at P<0.05. Asterisks represent significant differences from their respective controls. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Results
In vitro and in vivo induction of ovulatory genes
hCG (Fig. 1A inset) and LH (data not shown) induced PTGS2; however, LH/hCG response was variable therefore FRS was used instead as an activator of adenylyl cyclase. The primary bovine GCs were cultured in the absence or presence of FRS (10 μM) for 6–24 h. There was a distinct temporal profile of induction for each gene (Fig. 1). A significant induction of PTGS2 (Fig. 1A) was observed at 12 h (threefold), which was further elevated (13-fold compared with its control) at 24 h. Induction of TNFAIP6 followed a similar pattern to that of PTGS2, but a maximal fold stimulation was much higher (30-fold; Fig. 1B). Unlike to these two genes, AREG and EREG were maximally induced already at 6 h and then FRS effect decreased gradually until 24 h (Fig. 1C and D). Amongst the genes studied, EREG was highly expressed. In contrast to other genes, PTX3 was decreased in the presence of FRS at all time point examined (Fig. 1E).
In vitro expression of (A) PTGS2, (B)TNFAIP6, (C) AREG, (D) EREG and (E) PTX3 in bovine GCs. GCs were incubated with or without (control: C) forskolin (FRS; 10 μM) for 6, 12 and 24 h, when cells were collected for RNA extraction. mRNA expression was measured by quantitative real-time PCR. Control levels at 6 h were designated 100%. The results are mean±s.e.m. from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 indicate significant differences from their respective controls. Inset: GCs were incubated with hCG (10 IU) for 24 h.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
In vivo as well, comparing the mRNA expression in GCs collected before and 21 h after LH administration to cows reveals a marked stimulation of these genes by LH. PTGS2, TNFAIP6 and PTX3 were strongly upregulated (Fig. 2A, B and E). AREG and EREG on the other hand were only moderately induced at this time point (Fig. 2C and D).
In vivo expression of (A) PTGS2, (B) TNFAIP6, (C) AREG, (D) EREG and (E) PTX3 in bovine GCs. GCs were aspirated from cows before (n=3) and 21 h after GNRH-induced LH surge (n=3). mRNA expression was measured by quantitative real-time PCR. The results are mean±s.e.m. *P<0.05, **P<0.01 and ****P<0.0001 indicate significant differences from the follicles collected before LH surge.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
Effect of PGE2 on ovulatory genes
To examine the role of endogenous PGs we employed siRNA targeting PTGS2. The mRNA levels of PTGS2 were significantly inhibited. PTGS2 levels were only 30% of those present in GCs treated with scrambled siRNA (Fig. 3A). In accordance, the concentration of PGE2 in the cell culture media was also strongly reduced to <1/3 as compared with scrambled siRNA (Fig. 3B). Silencing was specific to PTGS2 because non-related genes such as vascular endothelial growth factor (VEGF) and solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) were not affected (Fig. 3C). PTGS2 knockdown affected some of the ovulatory genes studied. Along with 70% inhibition of PTGS2, the mRNA levels of TNFAIP6, AREG and EREG were significantly inhibited by 40, 30 and 20% respectively (as compared with 0% inhibition by scrambled siRNA; Fig. 3C). Notably, however, PTX3 was not affected by PTGS2 silencing (Fig. 3C).
Effects of PTGS2 gene silencing on PGE2 concentration and ovulatory gene expression in bovine GCs. Bovine GCs were transfected with 50 nmol/l of scrambled siRNA; designated 100% or siRNA targeting PTGS2 (naked PTGS2 siRNA). 24 h post-transfection forskolin (10 μM) was added. RNA was extracted 48 h post transfection and mRNA levels were measured using quantitative real-time PCR. Scrambled siRNA was designated as 100%. (A) PTGS2 expression (B) PGE2 concentrations in culture media of cells transfected with scrambled siRNA or naked PTGS2 siRNA. (C) Percent inhibition of PTGS2, TNFAIP6, AREG, EREG, PTX3, VEGF and SLC2A1 compared with scrambled siRNA (0%). The data were obtained from five independent experiments. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 indicate significant differences from scrambled siRNA.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
To further strengthen these findings, we examined next the effect of exogenous PGE2 on AREG, EREG, TNFAIP6 and PTX3. Bovine GCs were cultured in presence and absence of PGE2 (1 μM) for 3–24 h. The effect of PGE2 subsided with time. At 6 h, as given in Table 1, maximal effect of PGE2 was observed and expression of AREG, EREG and TNFAIP6 was significantly upregulated. As with PTGS2 silencing, the expression of PTX3 was not affected by PGE2. PGE2 did not modulate PTGS2 expression (data not shown).
Induction of ovulatory genes in bovine granulosa cells by 6 h treatment with PGE2 (1 μM).
| Gene | Fold over control | P value |
|---|---|---|
| AREG | 3.4±1.08 | 0.0016 |
| EREG | 11.8±3.42 | 0.019 |
| TNFAIP6 | 2.2±0.49 | 0.04 |
| PTX3 | 1.0±0.12 | NS |
Expression of each gene is as compared with its control designated as 1. n=4.
PTGS2 silencing with naked siRNA and cholesterol-conjugated siRNA in GCs and EndoCs
We then compared naked siRNA with cholesterol-conjugated siRNA molecule that does not necessitate transfection reagent. First, in bovine GCs, we examined the efficiency of chol-PTGS2 siRNA using a transfection reagent. Similar to the naked siRNA molecule, the mRNA levels of PTGS2 were declined to 65% as compared with scrambled siRNA (Fig. 4A). As expected PGE2 level were also reduced (data not shown). Likewise, TNFAIP6 expression was inhibited by 43% (Fig. 4B). However, when bovine GCs were treated with chol-PTGS2 siRNA without transfection reagent, no inhibition of PTGS2 or TNFAIP6 was observed.
Cholesterol-conjugated PTGS2 siRNA treatment in bovine GCs with and without transfection reagent. Bovine GCs were treated with 50 nmol/l of scrambled siRNA (negative control; designated 100%) or cholesterol-conjugated PTGS2 siRNA (chol-PTGS2 siRNA) with transfection reagent (A and B) or without transfection reagent (C and D). 24 h post transfection/treatment forskolin (10 μM) was added and RNA was extracted 48 h post transfection/treatment the cells. mRNA levels were measured using quantitative real-time PCR. Scrambled siRNA was designated as 100%. The data were obtained from three independent experiments. *P<0.05 indicates significant difference from scrambled siRNA.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
Since bovine EndoCs also express the PTGS2, we next examined the effects of these siRNA molecules in bovine EndoCs. To induce PTGS2 in these cells, PMA (100 ng/ml) (Parent & Fortier 2005) was utilised (Fig. 5A). When transfected with naked siPTGS2, a significant reduction was observed in the mRNA levels and PGE2 concentration by 65 and 91% respectively (Fig. 5B and C). Tumour necrosis factor receptor (TNFR1), another gene expressed in same levels as that of PTGS2 in bovine EndoCs, was unaffected by the siRNA (Fig. 5D), indicating specificity of the silencing process. In a similar manner to naked siPTGS2, transfection of chol-PTGS2 siRNA in EndoCs showed 68% reduction in PTGS2 mRNA levels (Fig. 6A). But in this cell type, chol-PTGS2 siRNA treatment without transfection reagent significantly reduced PTGS2 mRNA levels and PGE2 concentration (25 and 50% respectively) (Fig. 6B and C).
Expression of PTGS2 and PGE2 concentrations in bovine endometrial cells. (A) PTGS2 expression in bovine EndoCs incubated with or without PMA (100 ng/ml) for 24 h. (B) Silencing of PTGS2 in bovine EndoCs. The cells were transfected with 50 nmol/l of scrambled siRNA; designated 100% or siRNA-targeting PTGS2 (naked PTGS2 siRNA). 24 h post transfection PMA (100 g/ml) was added and RNA was extracted 48 h post transfection. mRNA levels were measured using quantitative real-time PCR. (C) PGE2 concentrations in culture media of cells transfected with scrambled siRNA or naked PTGS2 siRNA. (D) TNFR1 expression in scrambled siRNA and naked PTGS2 siRNA transfected cells. The data were obtained from four independent experiments. *P<0.05 and ***P<0.001 indicate significant differences from scrambled siRNA.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
Cholesterol-conjugated PTGS2 siRNA treatment in bovine endometrial cells (bovine EndoCs) with and without transfection reagent. Endometrial cells were transfected or treated with 50 nmol/l of scrambled siRNA; designated 100% or cholesterol-conjugated PTGS2 siRNA (chol-PTGS2 siRNA). 24 h post transfection/treatment PMA (100 g/ml) was added and RNA was extracted 48 h post transfection/treatment for determination of PTGS2 mRNA using quantitative real-time PCR. (A) PTGS2 expression in transfected cells. PTGS2 expression (B) and PGE2 concentrations in culture media (C) of siRNA treated non-transfected cells. The data were obtained from three independent experiments. *P<0.05, **P<0.01 and ****P<0.0001 indicate significant differences from scrambled siRNA.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
Discussion
The present data demonstrated that ovulatory genes, namely PTGS2, TNFAIP6, AREG, EREG and PTX3 were upregulated in bovine GCs 21 h after an induced LH surge. Except PTX3, the other genes studied were also induced by FRS treatment in isolated GCs culture in vitro. AREG and EREG were induced abruptly (at 6 h) during culture and progressively declined until 24 h. PTGS2 and TNFAIP6 showed a different pattern with gradual induction of FRS, reaching a maximum at 24 h. Using siRNA, we achieved a marked and specific silencing of PTGS2 in bovine GCs and another cell type expressing PTGS2, EndoCs. Reduction in PTGS2 mRNA levels in turn results in declined PGE2 concentrations in the culture media of both cell types. PTGS2 knockdown in GCs also inhibited the expression of ovulatory genes TNFAIP6, AREG and EREG, but not PTX3. In agreement, PGE2 treatment of GCs elevated the expression of these genes, except PTX3. Together these studies suggest that PGs, most likely PGE2, which are induced in GCs by LH/cAMP promote the expression of TNFAIP6, AREG and EREG (Fig. 7).
Schematic summary demonstrating induction of ovulatory genes by LH/cAMP and PGE2 in bovine GCs. (A) Role of LH/cAMP on the induction of ovulatory genes. GCs collected from cows 21 h after GNRH-induced LH surge upregulated the expression of following ovulatory genes: PTGS2, TNFAIP6, AREG, EREG and PTX3. Except PTX3, the other genes were also induced in vitro by forskolin, directly activating adenynyl cyclase in isolated GCs culture. (B) Role of PGE2 on the expression of ovulatory gene. The mRNA levels of PTGS2 were significantly inhibited by 70% compared with cells treated with scrambled siRNA. Concentrations of PGE2 in the cell culture media were also markedly reduced to <1/3 as compared with control. Along with the silencing of PTGS2, the expression of TNFAIP6, AREG and EREG was significantly downregulated. PTX3 was not affected by PTGS2 silencing. In agreement with PTGS2 silencing, incubation with PGE2 (1 μM) for 6 h elevated the mRNA levels of TNFAIP6, AREG and EREG, but not PTX3.
Citation: REPRODUCTION 149, 1; 10.1530/REP-14-0337
Interesting observations emerged from the comparison of in vivo and in vitro data. Although AREG and EREG were induced by LH surge, their fold inductions were low, in comparison with PTGS2 and TNFAIP6. The in vitro temporal pattern suggests that this may be due to the fact that AREG and EREG expression had already resided at 24 h after being highly induced at earlier time points. Another important observation is related with PTX3, which is considered as a bona fide ovulatory protein necessary for ECM stability during cumulus expansion (Varani et al. 2002, Salustri et al. 2004). PTX3, as shown before and also in this study, is induced in GCs by the LH surge in mice and bovine (Varani et al. 2002, Christenson et al. 2013). However, our study demonstrates that although GCs do express PTX3, it was not induced in isolated cell culture but rather inhibited by elevated cAMP levels. In fact, the mode of PTX3 induction is not well established yet and warrants further investigation. Yet, lack of induction in isolated GCs culture strongly suggests that other follicular compartments may mediate its induction by LH. Indeed in vitro induction of PTX3 (by cAMP and PGE2) was demonstrated in the past only in murine COC, where the presence of oocyte appears to be essential (Salustri et al. 2004).
The temporal and functional relationship between PGs and EGF-like factors has been inconclusive. Ptgs2-knockout mice have reduced level of Areg and Ereg (Shimada et al. 2006). In agreement, PGE2 induces AREG and EREG in a dose-dependent fashion in human GCs (Ben-Ami et al. 2006). Moreover, injecting intrafollicular indomethacin (INDO) in cows showed reduced AREG expression in GCs (Li et al. 2009). These reports tend to suggest that PTGS2 induces EGF-like factors. Other studies favour the concept of PTGS2 being induced by EGF-like factors. For instance, explants of preovulatory mice follicles treated with LH and AG14780 (EGFR kinase inhibitor) showed no expression of Ptgs2 (Ashkenazi et al. 2005). Similarly, reduced PTGS2 was noted when bovine GCs that were treated with the FRS, an adenylyl cyclase activator and an inhibitor of EGFR tyrosine kinase activity (Sayasith et al. 2013). In a recent study, EGFR knockdown has resulted in reduced expression of PTGS2 and decreased PGE2 (Fang et al. 2013).
We found that PTGS2 knockdown resulted in decreased expression of AREG and EREG. Also, exogenous PGE2 upregulate the expression of both AREG and EREG. Expression of TNFAIP6 was similarly reduced in PTGS2-silenced GCs and induction of TNFAIP6 by exogenous PGE2. Our data therefore support the notion that AREG and EREG are regulated by PTGS2 as observed in Ptgs2-knockout mice. Our data also point out that TNFAIP6 is yet another downstream target of PTGS2. Considering the major effect induced by LH/cAMP on these genes, shown here and in many other studies, a likely scenario of post-LH events would suggest that PGs provide a secondary, autocrine pathway to regulate the expression of EGF-like peptide in GCs. However, it is also plausible that AREG and EREG can in turn provide an additional signal to further induce PTGS2 and PGs. These multiple-positive autocrine loops would ensure a successful ovulatory process.
Previous studies employed PTGS2 inhibitors to examine the role of PTGS2 and PGE2 on ovulatory genes. The inhibitors used such as INDO are non-specific as they also affect PTGS1, but even PTGS2 inhibitors can have non-specific effects if not calibrated precisely. In this study, we employed siRNA silencing of PTGS2 instead, which provides a specific and effective tool to critically examine the role of PGs in GCs in vitro but may also allow manipulation of ovulatory process. Furthermore, by inhibiting the mRNA rather than the protein, a more profound and longer inhibition can be achieved. Introducing the inhibitory sequence into small hairpin RNA (shRNA) plasmid can be used for stable knockdown. Furthermore, siRNA provides a means for a post-transcriptional gene regulation in vitro, but also in vivo in species where gene knockout is not feasible. Under in vivo conditions transfection reagents may exhibit immunostimulatory effects and toxicity (Dass 2004). Conjugation of cholesterol, a lipophilic molecule, with siRNA can facilitate the entry of siRNA without the need of transfection reagent (Yuan et al. 2008, Medvedeva et al. 2009, Wu et al. 2009). Chol-PTGS2 siRNA can be therefore used in various cell types expressing PTGS2. In the reproductive tract, EndoCs are most relevant where PTGS2-derived PGs plays a central role in the regulation of the estrous cycle, pregnancy recognition, pregnancy maintenance and parturition (Charpigny, Reinaud et al. 1997, Asselin, Lacroix et al. 1997, Fuchs, Rust et al. 1999, Liu, Antaya et al. 2001). In this study, we found chol-PTGS2 siRNA without transfection reagent to be effective in EndoCs, suggesting that this molecule can be administered locally in the uterine horn of farm animals to decisively examine the role of PTGS2 during the estrous cycle or pregnancy. In contrast, chol-PTGS2 siRNA was not effective in GCs without transfection reagent, the possible reasons are not clear, but it might be related with differences in membrane composition and/or the fact that EndoCs are proliferating at higher rates as compared with GCs.
In conclusion, our data confirm that the induction of ovulatory genes in bovine GCs, such as PTGS2, AREG, EREG and TNFAIP6, is dependent on LH/cAMP in vivo and in vitro. We had achieved a successful and specific knockdown of PTGS2 in GCs. PTGS2 silencing also caused significant reduction in mRNA levels of AREG, EREG and TNFAIP6. Exogenous PGE2 increased these genes, further confirming that these genes are prostanoid dependent. In isolated GCs culture, neither cAMP nor PGE2 elevated PTX3. PTGS2 knockdown in GCs and EndoCs can be utilised to critically determine the functions of PTGS2 in vitro. siRNA ablation of PTGS2 in the reproductive tract as well as of other genes through local delivery may provide a novel approach for studying gene functions in large animals in vivo.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Supported by a grant from Chief Scientist of the Israeli Ministry of Agriculture (R Meidan and U Moallem).
Acknowledgements
The authors thank Dr A F Parlow and National Hormone and Peptide program for providing human chorionic gonadotropin.
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