Page 1 of 32Reproduction Advance Publication first posted on 10 April 2015 as Manuscript REP-14-0632
1
Title: hCG increases serum progesterone, number of corpora lutea, and
2
angiogenic factors in pregnant sheep.
3 4
Short Title: Effects of hCG during early pregnancy in sheep
5 6
Authors:
7
Megan P.T. Coleson1,2, Nicole S. Sanchez1,2, Amanda K. Ashley1, Timothy T. Ross1,
8
Ryan L. Ashley1, 3
9
Address: 1Department of Animal and Range Sciences, New Mexico State University,
10
Las Cruces, NM 88003, 2Authors contributed equally to this work, 3Corresponding
11
author. New Mexico State University. Department of Animal and Range Sciences. PO
12
Box 30003, MSC 3I. Las Cruces, NM 88003. Email: [email protected]
13 14
Abstract:
15
Early gestation is a critical period when implantation and placental vascularization are
16
established, processes influenced by progesterone (P4). Although human chorionic
17
gonadotropin (hCG) is not endogenously synthesized by livestock, it binds luteinizing
18
hormone receptor, stimulating P4 synthesis. We hypothesized treating pregnant ewes
19
with hCG would increase serum P4, number of corpora lutea (CLs) and concepti,
20
augment steroidogenic enzymes, and increase membrane P4 receptors (PAQRs) and
21
angiogenic factors in reproductive tissues. The objective was to determine molecular
22
alterations induced by hCG in pregnant sheep that may promote pregnancy. Ewes
23
received either 600 IU of hCG or saline i.m. on day 4 post mating. Blood samples were
1 Copyright © 2015 by the Society for Reproduction and Fertility.
Page 2 of 32
24
collected daily from day 0 until tissue collection for serum P4 analysis. Reproductive
25
tissues were collected on either day 13 or 25 of gestation and analyzed for PAQRs,
26
CXCR4, proangiogenic factors and steroidogenic enzymes. Ewes receiving hCG had
27
more CL and greater serum P4, which remained elevated. On day 25, StAR protein
28
production decreased in CL from hCG-treated ewes while HSD3B1 was unchanged;
29
further, expression of CXCR4 significantly increased and KDR tended to increase.
30
PAQR7 and CXCR4 protein was increased in caruncle tissue from hCG-treated ewes.
31
Maternal hCG exposure influenced fetal extraembryonic tissues, as VEGFA, VEGFB,
32
FLT1, and ANGPT1 expression increased. Our results indicate hCG increases serum
33
P4 due to augmented CL number per ewe. hCG treatment resulted in greater PAQR7
34
and CXCR4 in maternal endometrium and promoted expression of proangiogenic
35
factors in fetal extraembryonic membranes. Supplementing livestock with hCG may
36
boost P4 levels and improve reproductive efficiency.
37 38
Introduction
39
Most pregnancy losses in mammalian species occur during early pregnancy (Edey
40
1969, Reynolds & Redmer 2001, Reynolds et al. 2010, Reynolds et al. 2013). Several
41
factors contribute to embryonic death including insufficient production of progesterone
42
(P4) by the corpus luteum (CL), as suboptimal P4 synthesis is indicative of maternal
43
inability to maintain pregnancy (Kittok et al. 1983). P4 is required for establishment and
44
maintenance of pregnancy in all mammals. Growth and development of the conceptus
45
(embryo/fetus and associated extraembryonic membranes) requires P4 signaling to
46
regulate endometrial functions critical for implantation and placentation. Human
2
Page 3 of 32
47
chorionic gonadotropin (hCG) is a potent luteotropic hormone (Norman & Litwack 1987)
48
responsible for increased P4 synthesis by the CL upon binding the luteinizing hormone
49
receptor (LHCGR). Although livestock do not produce hCG, it binds to the LHCGR,
50
which they do express. Due to LHCGR activation by hCG and the substantially longer
51
half-life than luteinizing hormone (LH) in mammals (Cole 2012), it may be useful for
52
increasing P4 production in livestock species. In beef heifers, serum P4 concentrations
53
and conception rates following artificial insemination increased following hCG
54
administration (Funston et al. 2005). In ewes, treatment with hCG increased P4 and
55
total CL weight 12 and 36 h after administration (Nephew et al. 1994). Further, hCG
56
increased the number and size of corpora lutea (CLs) in ewes and these accessory CLs
57
could be responsible for the dramatic increase in serum P4 levels observed (Farin et al.
58
1988, Shabankareh et al. 2012). However, it is unclear if the increase in serum P4 is
59
exclusively due to increased CL numbers or if hCG is stimulating synthesis of
60
steroidogenic enzymes. Addition of this exogenous hormone may boost serum P4
61
levels and decrease pregnancy loss in livestock, thereby increasing animal reproductive
62
efficiency.
63 64
P4 signaling in the uterus primes the endometrium to ensure a receptive environment
65
for the developing conceptus. P4 elicits its effects through nuclear P4 receptors (PRs)
66
as well as membrane P4 receptors (PAQR family). Paradoxically, the elevated P4
67
during early pregnancy leads to a downregulation of PR and in sheep the PR protein is
68
undetectable in endometrial luminal epithelium and glandular epithelium after days 11
69
and 13 of pregnancy, respectively (Spencer & Bazer 1995, Spencer et al. 1995). While
3
Page 4 of 32
70
PRs have been researched extensively, not much is known about PAQRs. Interestingly,
71
expression of PAQRs is upregulated by P4 (Karteris et al. 2006) and may play
72
functional roles during early pregnancy. Because hCG has noted effects on increasing
73
P4 synthesis, one objective of this study was to investigate the effects of exogenous
74
hCG on expression and synthesis of the PAQR family of receptors.
75 76
In addition to stimulating P4 production, hCG also serves as a proangiogenic factor at
77
the fetal/maternal interface (for a review see Cole, 2012 (Cole 2012). hCG influences
78
vascularization by regulating expression of proangiogenic factors such as vascular
79
endothelial growth factor (VEGFA) and angiopoietin one (ANGPT1) (Laitinen et al.
80
1997, Sugino et al. 2000, Wulff et al. 2000). Further, hCG stimulates proliferation of
81
placental microvascular endothelial cells, which are important for capillary sprout
82
formation during angiogenesis (Herr et al. 2007). Haploinsufficiency of VEGFA or its
83
receptors, FMS-like Tyrosine Kinase Receptor 1 (FLT1) and Kinase Insert Domain-
84
Containing Receptor (KDR), results in defective fetal and placental angiogenesis,
85
culminating in embryonic death by mid-gestation (Fong et al. 1995, Shalaby et al. 1995,
86
Carmeliet et al. 1996, Ferrara et al. 1996). ANGPT1 also regulates angiogenesis, as
87
several studies have shown vascular defects when these factors are deficient in mice
88
(Carmeliet et al. 1996, Ferrara et al. 1996, Maisonpierre et al. 1997). Similar to hCG
89
induced functions, activation of C-X-C chemokine receptor type 4 (CXCR4) by its ligand,
90
C-X-C chemokine 12 (CXCL12) amplifies angiogenesis by inducing VEGFA release. In
91
turn, VEGFA increases expression of CXCR4, but not other chemokine receptors, thus
92
establishing a positive feedback loop in which VEGFA induces CXCR4 and CXCL12
4
Page 5 of 32
93
expression, and conversely CXCL12/CXCR4 signaling enhances VEGFA expression
94
(Salcedo et al. 1999, Mirshahi et al. 2000, Salcedo & Oppenheim 2003). We previously
95
reported that placental expression of the CXCL12/CXCR4 signaling axis during early
96
pregnancy corresponds with placental vascularization with marked induction of VEGFA,
97
FLT1, KDR and ANGPT1 (Ashley et al. 2011, Quinn et al. 2014), yet whether hCG
98
regulates the CXCL12/CXCR4 signaling axis is not known.
99 100
Based on the variety of functions induced by hCG in humans, specifically stimulating P4
101
synthesis and influencing placental vascularization, administration of exogenous hCG
102
may improve pregnancy success rates and enhance reproductive efficiency and overall
103
production in livestock. We hypothesized that ewes treated with hCG would have higher
104
serum P4 concentrations, greater number of CL and concepti, augmented steroidogenic
105
enzymes, and increased expression of PAQRs and angiogenic factors in reproductive
106
tissues. This hypothesis was formulated to cover myriad physiological outcomes
107
induced by hCG to provide a more comprehensive perspective on its role in livestock
108
reproduction. The objectives of the study were to determine molecular alterations
109
induced by hCG administration in pregnant sheep that may serve to promote early
110
pregnancy and elucidate additional enzymatic alterations induced by hCG treatment.
111
Specifically, hCG-induced changes in the regulation and synthesis of angiogenic factors
112
and PAQRs were investigated.
113 114
Materials and Methods
115
5
Page 6 of 32
116
Animal Procedures and Tissue Collection
117
All procedures involving animals were approved by the New Mexico State University
118
Animal Care and Use Committee (IACUC #2012-018). Unless indicated, all reagents
119
were purchased from Sigma Aldrich (Saint Louis, MO). Nineteen mixed-aged western
120
whiteface ewes (70.5 ± 1.5 kg body weight) received an intravaginal pessary
121
impregnated with 20 mg flurogestone acetate (Searle, Skokie, IL, 60076) for 14 days to
122
synchronize estrus. Three vasectomized rams, fitted with marking harnesses, were
123
placed with ewes, for detection of estrus. Ewes were mated with fertile rams upon
124
detection of their second estrus after pessary removal and onset of estrus was
125
determined as the day serum P4 was at or below 1 ng/ml (day 0). Ewes were randomly
126
assigned to one of two treatments. Ewes received 600 IU (4.8 ml) of hCG (ProSpec-
127
Tany Techno Gene Ltd., Ness Ziona, Israel, Cat #: hor-250) i.m. (n = 9) or saline (4.8
128
ml, n = 10) on day 4 post mating. Treatment day and dose was determined by previous
129
studies utilizing pregnant sheep (Nephew et al. 1994, Akif Cam & Kuran 2004) and
130
studies conducted in the laboratory of Dr. Tim Ross, where varying doses of hCG as
131
well as single versus multiple injections of hCG have been evaluated in pregnant ewes
132
(personal communication). This treatment regime was highly effective at increasing
133
serum progesterone concentrations and additional injections were not warranted. Within
134
each treatment, ewes were randomly assigned to one of two groups where half the
135
ewes were euthanized 13 days post mating (control n = 4; hCG-treated n = 5) and the
136
remaining ewes euthanized 25 days post mating (control n = 6; hCG-treated n = 5).
137
Ewes were anesthetized with 20 mg/kg body weight of sodium pentobarbital (Vortech
138
Pharmacy, Dearborn, MI, 48126) via i.v. administration. The surgical area was shorn
6
Page 7 of 32
139
and reproductive tract removed using a mid-ventral laparotomy prior to euthanasia by
140
exsanguination. One group of ewes was euthanized on day 13, and CLs were counted.
141
In addition, uteri were flushed with approximately 20 ml of phosphate buffered saline
142
(PBS) supplemented with 0.25% bovine serum albumin (BSA; pH 7.1) and numbers of
143
concepti were recorded. The second group of ewes was euthanized on day 25 post
144
mating, and CL and concepti were counted and weighed. Caruncles, CLs, and fetal
145
extraembryonic membranes were collected, diced into smaller pieces, and immediately
146
immersed in liquid nitrogen, then stored at -80°C.
147 148
Blood Collection and Serum P4 Assessment
149
Daily blood samples were collected via jugular venipuncture into serum separator tubes
150
(Corvac, Kendall Health Care, St. Louis, MO) from all ewes starting on the date of
151
marking by the ram until euthanasia. Tubes were incubated at room temperature for at
152
least 30 min prior to being centrifuged at 4°C for 15 min at 1,500 x g. Serum was
153
harvested and subsequently stored at -20°C until assayed. Serum P4 concentrations
154
were determined using RIA procedures described by Schneider and Hallford (1996;
155
Coat-A-Count, Siemens Medical Solutions Diagnostics, Los Angeles, CA) and
156
performed by the New Mexico State University Endocrinology Laboratory. Inter- and
157
intra-assay CV were 4.7 and 6.7%, respectively.
158 159
RNA Isolation and cDNA Synthesis
160
Total cellular RNA was extracted from tissues using 1 ml of Tri Reagent BD (Molecular
161
Research Center Inc., Cincinnati, OH) per 100 mg of tissue, according to the
7
Page 8 of 32
162
manufacturer’s directions. RNA was eluted in nuclease-free water, and subsequently
163
treated with DNase using the TURBO DNA-free kit (Ambion, Foster City, CA) to ensure
164
samples were not contaminated with genomic DNA. The quantity and purity of RNA was
165
determined using a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham,
166
MA). RNA samples were stored at -80°C until further analysis. Complementary DNA
167
was synthesized from 1 µg RNA using the iScript cDNA Synthesis Kit (BioRad)
168
according to the manufacturer’s recommendations. The products were diluted to a final
169
volume of 100 µl (10 ng/µl).
170 171
Quantitative Real-Time PCR (qPCR)
172
The qPCR analysis was performed using a CFX96 Touch Real-Time PCR Detection
173
System and components of the iQ SYBR green supermix (BioRad Laboratories,
174
Hercules, CA) as previously described (Quinn et al. 2014). Forward and reverse primers
175
were used at a final concentration of 0.525 µM and 2 µl of cDNA for each sample was
176
assayed. The specific primers employed are shown in Table 1. The efficiency of all
177
primers was tested using varying levels of cDNA (50 – 0.05 ng per reaction); all were
178
within acceptable limits (Livak & Schmittgen 2001), and no primer-dimers were formed.
179
The qPCR conditions were 95◦C for 3 min followed by 40 cycles of 95◦C (30 s), 55◦C (30
180
s), 72◦C (15 s) and then a melt curve was performed to assure no primer-dimers were
181
present. Cq values were attained during logarithmic amplification phase of PCR cycling.
182
The GAPDH amplicon did not change across days or pregnancy status and was used to
183
normalize each target via the 2-∆∆Cq method (Livak & Schmittgen 2001).
184
8
Page 9 of 32
185
Protein Isolation and Immunoblotting
186
Protein was isolated from ovine tissues by homogenizing 100 mg of tissue in 1 ml of
187
RIPA buffer (50 mM Tris (pH 7.4), 2 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl
188
sulfate (SDS), 1.0% TritonX-100) supplemented with phosphatase and protease
189
inhibitor cocktails (Roche Applied Science, Germany). Samples were placed on ice for
190
15 min, then centrifuged at 12,000 × g for 10 min at 4◦C. The supernatant was collected
191
and stored at -80◦C. Concentrations of protein were determined using the BCA protein
192
assay (Pierce, Rockford, IL).
193 194
Unless indicated, all reagents used for western blot analysis were purchased from Bio-
195
Rad Laboratories Inc. (Hercules, CA). Equal protein amounts (ranged from 30-50 µg
196
depending on tissue) were combined with 6X dye (187 mM Tris (pH 6.8), 6% SDS, 30%
197
glycerol, 440mM beta-mercaptoethanol, 0.2% bromophenol blue, deionized water) and
198
denatured on a heat block at 100°C for 5 min, then cooled and subjected to SDS-PAGE.
199
After electrophoresis, protein was transferred to polyvinyl difluoride (PVDF) membranes
200
for immunoblotting. After blocking in 5% non-fat dry milk or 5% BSA (Sigma Aldrich)
201
made in Tris-buffered saline plus tween (TBS-T; 68.4 mM Tris base, 10 mM NaCl,
202
0.10% tween 20, pH 7.6) for 1 h at room temperature, membranes were incubated with
203
primary antibody in blocking solution over night at 4°C. Unless indicated, antibodies
204
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific
205
to FLT1 (sc-316), KDR (sc-315), CXCR4 (sc-9046), VEGFA (sc-152), ANGPT1 (Abcam,
206
ab95230), HSD3B1 (sc-30820), StAR (sc-25806), ISG15 (#2743BC; Cell Signaling
207
Technology), PAQR5 (Abcam, ab123979), PAQR7 (Abcam, ab79517), or ACTB (sc-
9
Page 10 of 32
208
47778) were used to probe membranes. Membranes were washed, and an appropriate
209
secondary antibody (goat anti-rabbit IgG-HRP, sc-2004; donkey anti-goat IgG-HRP, sc-
210
2020; donkey anti-rabbit IgG-HRP, sc-2317) was added in blocking solution for 1 h at
211
ambient temperature. The concentration of antibody varied per tissue, but ranged from
212
1:500 to 1:2000 for primary antibodies and 1:5000 to 1:20,000 for secondary antibodies.
213
Proteins were visualized by Clarity Western ECL Substrate peroxide solution and
214
Luminol/enhancer solution for 5 min and immediately detected using the ChemiDoc™
215
XRS and Image Lab Software Version 3 (BioRad Laboratories, Hercules, CA). If
216
antibody removal was required in order to assess loading controls, membranes were
217
stripped in stripping buffer (TBS-T, 143 µM beta-mercaptoethanol, 2% SDS) heated to
218
65°C, then washed with TBS-T and blocked in 5% non-fat dry milk for 1 h at room
219
temperature. Following the stripping procedure membranes were assessed to assure
220
lack of chemiluminescence in the absence of antibody before being probed for the
221
loading control.
222 223
Statistical Analysis
224
The experimental design was completely randomized. Number of corpora lutea and
225
concepti per ewe were analyzed by Chi-Square using the frequency procedure of SAS
226
(SAS Inst. Inc., Cary, NC). P4 concentrations were analyzed using the mixed procedure
227
of SAS with repeated function. Treatment and ewe were in the whole plot and day and
228
day by treatment interaction were in the subplot. For qPCR the Cq value of the gene of
229
interest was normalized to the Cq value of GAPDH for each sample and analysis was
230
run on the 2-∆∆Cq values using GraphPad Prism (Version 6 from GraphPad Software,
10
Page 11 of 32
231
Inc.). The chemiluminescent signals for western blots were quantified using the mean
232
value (intensity) using Image Lab software (Version 4.1). Each band of interest was
233
normalized by dividing by mean value (intensity) for the protein of interest divided by
234
mean value (intensity) of ACTB. Significant changes were determined at P < 0.05 using
235
an unpaired, two-tailed student’s t-test. If the variance significant differed, Welch’s
236
correction was used.
237 238
Results
239
Serum Progesterone Concentrations and Corpora Lutea and Concepti Numbers.
240
A day by treatment interaction (P < 0.05) was observed for serum P4 concentrations for
241
all ewes until day 13 (Fig. 1A). Serum P4 concentrations were similar among treatments
242
through day 3. Ewes receiving hCG on day 4 had greater (P < 0.05) serum P4
243
concentrations than control ewes from days 4 to 13. No treatment by day interaction
244
was detected (P > 0.20) for serum P4 concentration between days 14 and 25; therefore,
245
treatment effects were examined across sampling days. During this period hCG-treated
246
ewes maintained elevated (P = 0.001) serum P4 values (10.9 ± 0.6 ng/mL) compared to
247
controls (6.0 ± 0.6 ng/mL). Treatment with hCG tended (P = 0.108) to increase number
248
of concepti (Fig 1B). On day 25, in the control group, three ewes had singletons, two
249
had twins, and one bore triplets, whereas all hCG treated ewes had twins. No difference
250
was observed between mean weight of concepti for control and hCG-treated ewes
251
(339.7 and 473.6 ± 74.7 mg, respectively, P = 0.20). Ewes receiving hCG had greater
252
number of CL (P = 0.006) compared to control ewes (Fig 1C). Control ewes had ≤ 3 CL,
253
whereas, 87.5% of ewes that received hCG had ≥ 3 CL (Fig 1C). Further, CL weight (g)
11
Page 12 of 32
254
per concepti was significantly greater (P
1
Title: hCG increases serum progesterone, number of corpora lutea, and
2
angiogenic factors in pregnant sheep.
3 4
Short Title: Effects of hCG during early pregnancy in sheep
5 6
Authors:
7
Megan P.T. Coleson1,2, Nicole S. Sanchez1,2, Amanda K. Ashley1, Timothy T. Ross1,
8
Ryan L. Ashley1, 3
9
Address: 1Department of Animal and Range Sciences, New Mexico State University,
10
Las Cruces, NM 88003, 2Authors contributed equally to this work, 3Corresponding
11
author. New Mexico State University. Department of Animal and Range Sciences. PO
12
Box 30003, MSC 3I. Las Cruces, NM 88003. Email: [email protected]
13 14
Abstract:
15
Early gestation is a critical period when implantation and placental vascularization are
16
established, processes influenced by progesterone (P4). Although human chorionic
17
gonadotropin (hCG) is not endogenously synthesized by livestock, it binds luteinizing
18
hormone receptor, stimulating P4 synthesis. We hypothesized treating pregnant ewes
19
with hCG would increase serum P4, number of corpora lutea (CLs) and concepti,
20
augment steroidogenic enzymes, and increase membrane P4 receptors (PAQRs) and
21
angiogenic factors in reproductive tissues. The objective was to determine molecular
22
alterations induced by hCG in pregnant sheep that may promote pregnancy. Ewes
23
received either 600 IU of hCG or saline i.m. on day 4 post mating. Blood samples were
1 Copyright © 2015 by the Society for Reproduction and Fertility.
Page 2 of 32
24
collected daily from day 0 until tissue collection for serum P4 analysis. Reproductive
25
tissues were collected on either day 13 or 25 of gestation and analyzed for PAQRs,
26
CXCR4, proangiogenic factors and steroidogenic enzymes. Ewes receiving hCG had
27
more CL and greater serum P4, which remained elevated. On day 25, StAR protein
28
production decreased in CL from hCG-treated ewes while HSD3B1 was unchanged;
29
further, expression of CXCR4 significantly increased and KDR tended to increase.
30
PAQR7 and CXCR4 protein was increased in caruncle tissue from hCG-treated ewes.
31
Maternal hCG exposure influenced fetal extraembryonic tissues, as VEGFA, VEGFB,
32
FLT1, and ANGPT1 expression increased. Our results indicate hCG increases serum
33
P4 due to augmented CL number per ewe. hCG treatment resulted in greater PAQR7
34
and CXCR4 in maternal endometrium and promoted expression of proangiogenic
35
factors in fetal extraembryonic membranes. Supplementing livestock with hCG may
36
boost P4 levels and improve reproductive efficiency.
37 38
Introduction
39
Most pregnancy losses in mammalian species occur during early pregnancy (Edey
40
1969, Reynolds & Redmer 2001, Reynolds et al. 2010, Reynolds et al. 2013). Several
41
factors contribute to embryonic death including insufficient production of progesterone
42
(P4) by the corpus luteum (CL), as suboptimal P4 synthesis is indicative of maternal
43
inability to maintain pregnancy (Kittok et al. 1983). P4 is required for establishment and
44
maintenance of pregnancy in all mammals. Growth and development of the conceptus
45
(embryo/fetus and associated extraembryonic membranes) requires P4 signaling to
46
regulate endometrial functions critical for implantation and placentation. Human
2
Page 3 of 32
47
chorionic gonadotropin (hCG) is a potent luteotropic hormone (Norman & Litwack 1987)
48
responsible for increased P4 synthesis by the CL upon binding the luteinizing hormone
49
receptor (LHCGR). Although livestock do not produce hCG, it binds to the LHCGR,
50
which they do express. Due to LHCGR activation by hCG and the substantially longer
51
half-life than luteinizing hormone (LH) in mammals (Cole 2012), it may be useful for
52
increasing P4 production in livestock species. In beef heifers, serum P4 concentrations
53
and conception rates following artificial insemination increased following hCG
54
administration (Funston et al. 2005). In ewes, treatment with hCG increased P4 and
55
total CL weight 12 and 36 h after administration (Nephew et al. 1994). Further, hCG
56
increased the number and size of corpora lutea (CLs) in ewes and these accessory CLs
57
could be responsible for the dramatic increase in serum P4 levels observed (Farin et al.
58
1988, Shabankareh et al. 2012). However, it is unclear if the increase in serum P4 is
59
exclusively due to increased CL numbers or if hCG is stimulating synthesis of
60
steroidogenic enzymes. Addition of this exogenous hormone may boost serum P4
61
levels and decrease pregnancy loss in livestock, thereby increasing animal reproductive
62
efficiency.
63 64
P4 signaling in the uterus primes the endometrium to ensure a receptive environment
65
for the developing conceptus. P4 elicits its effects through nuclear P4 receptors (PRs)
66
as well as membrane P4 receptors (PAQR family). Paradoxically, the elevated P4
67
during early pregnancy leads to a downregulation of PR and in sheep the PR protein is
68
undetectable in endometrial luminal epithelium and glandular epithelium after days 11
69
and 13 of pregnancy, respectively (Spencer & Bazer 1995, Spencer et al. 1995). While
3
Page 4 of 32
70
PRs have been researched extensively, not much is known about PAQRs. Interestingly,
71
expression of PAQRs is upregulated by P4 (Karteris et al. 2006) and may play
72
functional roles during early pregnancy. Because hCG has noted effects on increasing
73
P4 synthesis, one objective of this study was to investigate the effects of exogenous
74
hCG on expression and synthesis of the PAQR family of receptors.
75 76
In addition to stimulating P4 production, hCG also serves as a proangiogenic factor at
77
the fetal/maternal interface (for a review see Cole, 2012 (Cole 2012). hCG influences
78
vascularization by regulating expression of proangiogenic factors such as vascular
79
endothelial growth factor (VEGFA) and angiopoietin one (ANGPT1) (Laitinen et al.
80
1997, Sugino et al. 2000, Wulff et al. 2000). Further, hCG stimulates proliferation of
81
placental microvascular endothelial cells, which are important for capillary sprout
82
formation during angiogenesis (Herr et al. 2007). Haploinsufficiency of VEGFA or its
83
receptors, FMS-like Tyrosine Kinase Receptor 1 (FLT1) and Kinase Insert Domain-
84
Containing Receptor (KDR), results in defective fetal and placental angiogenesis,
85
culminating in embryonic death by mid-gestation (Fong et al. 1995, Shalaby et al. 1995,
86
Carmeliet et al. 1996, Ferrara et al. 1996). ANGPT1 also regulates angiogenesis, as
87
several studies have shown vascular defects when these factors are deficient in mice
88
(Carmeliet et al. 1996, Ferrara et al. 1996, Maisonpierre et al. 1997). Similar to hCG
89
induced functions, activation of C-X-C chemokine receptor type 4 (CXCR4) by its ligand,
90
C-X-C chemokine 12 (CXCL12) amplifies angiogenesis by inducing VEGFA release. In
91
turn, VEGFA increases expression of CXCR4, but not other chemokine receptors, thus
92
establishing a positive feedback loop in which VEGFA induces CXCR4 and CXCL12
4
Page 5 of 32
93
expression, and conversely CXCL12/CXCR4 signaling enhances VEGFA expression
94
(Salcedo et al. 1999, Mirshahi et al. 2000, Salcedo & Oppenheim 2003). We previously
95
reported that placental expression of the CXCL12/CXCR4 signaling axis during early
96
pregnancy corresponds with placental vascularization with marked induction of VEGFA,
97
FLT1, KDR and ANGPT1 (Ashley et al. 2011, Quinn et al. 2014), yet whether hCG
98
regulates the CXCL12/CXCR4 signaling axis is not known.
99 100
Based on the variety of functions induced by hCG in humans, specifically stimulating P4
101
synthesis and influencing placental vascularization, administration of exogenous hCG
102
may improve pregnancy success rates and enhance reproductive efficiency and overall
103
production in livestock. We hypothesized that ewes treated with hCG would have higher
104
serum P4 concentrations, greater number of CL and concepti, augmented steroidogenic
105
enzymes, and increased expression of PAQRs and angiogenic factors in reproductive
106
tissues. This hypothesis was formulated to cover myriad physiological outcomes
107
induced by hCG to provide a more comprehensive perspective on its role in livestock
108
reproduction. The objectives of the study were to determine molecular alterations
109
induced by hCG administration in pregnant sheep that may serve to promote early
110
pregnancy and elucidate additional enzymatic alterations induced by hCG treatment.
111
Specifically, hCG-induced changes in the regulation and synthesis of angiogenic factors
112
and PAQRs were investigated.
113 114
Materials and Methods
115
5
Page 6 of 32
116
Animal Procedures and Tissue Collection
117
All procedures involving animals were approved by the New Mexico State University
118
Animal Care and Use Committee (IACUC #2012-018). Unless indicated, all reagents
119
were purchased from Sigma Aldrich (Saint Louis, MO). Nineteen mixed-aged western
120
whiteface ewes (70.5 ± 1.5 kg body weight) received an intravaginal pessary
121
impregnated with 20 mg flurogestone acetate (Searle, Skokie, IL, 60076) for 14 days to
122
synchronize estrus. Three vasectomized rams, fitted with marking harnesses, were
123
placed with ewes, for detection of estrus. Ewes were mated with fertile rams upon
124
detection of their second estrus after pessary removal and onset of estrus was
125
determined as the day serum P4 was at or below 1 ng/ml (day 0). Ewes were randomly
126
assigned to one of two treatments. Ewes received 600 IU (4.8 ml) of hCG (ProSpec-
127
Tany Techno Gene Ltd., Ness Ziona, Israel, Cat #: hor-250) i.m. (n = 9) or saline (4.8
128
ml, n = 10) on day 4 post mating. Treatment day and dose was determined by previous
129
studies utilizing pregnant sheep (Nephew et al. 1994, Akif Cam & Kuran 2004) and
130
studies conducted in the laboratory of Dr. Tim Ross, where varying doses of hCG as
131
well as single versus multiple injections of hCG have been evaluated in pregnant ewes
132
(personal communication). This treatment regime was highly effective at increasing
133
serum progesterone concentrations and additional injections were not warranted. Within
134
each treatment, ewes were randomly assigned to one of two groups where half the
135
ewes were euthanized 13 days post mating (control n = 4; hCG-treated n = 5) and the
136
remaining ewes euthanized 25 days post mating (control n = 6; hCG-treated n = 5).
137
Ewes were anesthetized with 20 mg/kg body weight of sodium pentobarbital (Vortech
138
Pharmacy, Dearborn, MI, 48126) via i.v. administration. The surgical area was shorn
6
Page 7 of 32
139
and reproductive tract removed using a mid-ventral laparotomy prior to euthanasia by
140
exsanguination. One group of ewes was euthanized on day 13, and CLs were counted.
141
In addition, uteri were flushed with approximately 20 ml of phosphate buffered saline
142
(PBS) supplemented with 0.25% bovine serum albumin (BSA; pH 7.1) and numbers of
143
concepti were recorded. The second group of ewes was euthanized on day 25 post
144
mating, and CL and concepti were counted and weighed. Caruncles, CLs, and fetal
145
extraembryonic membranes were collected, diced into smaller pieces, and immediately
146
immersed in liquid nitrogen, then stored at -80°C.
147 148
Blood Collection and Serum P4 Assessment
149
Daily blood samples were collected via jugular venipuncture into serum separator tubes
150
(Corvac, Kendall Health Care, St. Louis, MO) from all ewes starting on the date of
151
marking by the ram until euthanasia. Tubes were incubated at room temperature for at
152
least 30 min prior to being centrifuged at 4°C for 15 min at 1,500 x g. Serum was
153
harvested and subsequently stored at -20°C until assayed. Serum P4 concentrations
154
were determined using RIA procedures described by Schneider and Hallford (1996;
155
Coat-A-Count, Siemens Medical Solutions Diagnostics, Los Angeles, CA) and
156
performed by the New Mexico State University Endocrinology Laboratory. Inter- and
157
intra-assay CV were 4.7 and 6.7%, respectively.
158 159
RNA Isolation and cDNA Synthesis
160
Total cellular RNA was extracted from tissues using 1 ml of Tri Reagent BD (Molecular
161
Research Center Inc., Cincinnati, OH) per 100 mg of tissue, according to the
7
Page 8 of 32
162
manufacturer’s directions. RNA was eluted in nuclease-free water, and subsequently
163
treated with DNase using the TURBO DNA-free kit (Ambion, Foster City, CA) to ensure
164
samples were not contaminated with genomic DNA. The quantity and purity of RNA was
165
determined using a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham,
166
MA). RNA samples were stored at -80°C until further analysis. Complementary DNA
167
was synthesized from 1 µg RNA using the iScript cDNA Synthesis Kit (BioRad)
168
according to the manufacturer’s recommendations. The products were diluted to a final
169
volume of 100 µl (10 ng/µl).
170 171
Quantitative Real-Time PCR (qPCR)
172
The qPCR analysis was performed using a CFX96 Touch Real-Time PCR Detection
173
System and components of the iQ SYBR green supermix (BioRad Laboratories,
174
Hercules, CA) as previously described (Quinn et al. 2014). Forward and reverse primers
175
were used at a final concentration of 0.525 µM and 2 µl of cDNA for each sample was
176
assayed. The specific primers employed are shown in Table 1. The efficiency of all
177
primers was tested using varying levels of cDNA (50 – 0.05 ng per reaction); all were
178
within acceptable limits (Livak & Schmittgen 2001), and no primer-dimers were formed.
179
The qPCR conditions were 95◦C for 3 min followed by 40 cycles of 95◦C (30 s), 55◦C (30
180
s), 72◦C (15 s) and then a melt curve was performed to assure no primer-dimers were
181
present. Cq values were attained during logarithmic amplification phase of PCR cycling.
182
The GAPDH amplicon did not change across days or pregnancy status and was used to
183
normalize each target via the 2-∆∆Cq method (Livak & Schmittgen 2001).
184
8
Page 9 of 32
185
Protein Isolation and Immunoblotting
186
Protein was isolated from ovine tissues by homogenizing 100 mg of tissue in 1 ml of
187
RIPA buffer (50 mM Tris (pH 7.4), 2 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl
188
sulfate (SDS), 1.0% TritonX-100) supplemented with phosphatase and protease
189
inhibitor cocktails (Roche Applied Science, Germany). Samples were placed on ice for
190
15 min, then centrifuged at 12,000 × g for 10 min at 4◦C. The supernatant was collected
191
and stored at -80◦C. Concentrations of protein were determined using the BCA protein
192
assay (Pierce, Rockford, IL).
193 194
Unless indicated, all reagents used for western blot analysis were purchased from Bio-
195
Rad Laboratories Inc. (Hercules, CA). Equal protein amounts (ranged from 30-50 µg
196
depending on tissue) were combined with 6X dye (187 mM Tris (pH 6.8), 6% SDS, 30%
197
glycerol, 440mM beta-mercaptoethanol, 0.2% bromophenol blue, deionized water) and
198
denatured on a heat block at 100°C for 5 min, then cooled and subjected to SDS-PAGE.
199
After electrophoresis, protein was transferred to polyvinyl difluoride (PVDF) membranes
200
for immunoblotting. After blocking in 5% non-fat dry milk or 5% BSA (Sigma Aldrich)
201
made in Tris-buffered saline plus tween (TBS-T; 68.4 mM Tris base, 10 mM NaCl,
202
0.10% tween 20, pH 7.6) for 1 h at room temperature, membranes were incubated with
203
primary antibody in blocking solution over night at 4°C. Unless indicated, antibodies
204
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific
205
to FLT1 (sc-316), KDR (sc-315), CXCR4 (sc-9046), VEGFA (sc-152), ANGPT1 (Abcam,
206
ab95230), HSD3B1 (sc-30820), StAR (sc-25806), ISG15 (#2743BC; Cell Signaling
207
Technology), PAQR5 (Abcam, ab123979), PAQR7 (Abcam, ab79517), or ACTB (sc-
9
Page 10 of 32
208
47778) were used to probe membranes. Membranes were washed, and an appropriate
209
secondary antibody (goat anti-rabbit IgG-HRP, sc-2004; donkey anti-goat IgG-HRP, sc-
210
2020; donkey anti-rabbit IgG-HRP, sc-2317) was added in blocking solution for 1 h at
211
ambient temperature. The concentration of antibody varied per tissue, but ranged from
212
1:500 to 1:2000 for primary antibodies and 1:5000 to 1:20,000 for secondary antibodies.
213
Proteins were visualized by Clarity Western ECL Substrate peroxide solution and
214
Luminol/enhancer solution for 5 min and immediately detected using the ChemiDoc™
215
XRS and Image Lab Software Version 3 (BioRad Laboratories, Hercules, CA). If
216
antibody removal was required in order to assess loading controls, membranes were
217
stripped in stripping buffer (TBS-T, 143 µM beta-mercaptoethanol, 2% SDS) heated to
218
65°C, then washed with TBS-T and blocked in 5% non-fat dry milk for 1 h at room
219
temperature. Following the stripping procedure membranes were assessed to assure
220
lack of chemiluminescence in the absence of antibody before being probed for the
221
loading control.
222 223
Statistical Analysis
224
The experimental design was completely randomized. Number of corpora lutea and
225
concepti per ewe were analyzed by Chi-Square using the frequency procedure of SAS
226
(SAS Inst. Inc., Cary, NC). P4 concentrations were analyzed using the mixed procedure
227
of SAS with repeated function. Treatment and ewe were in the whole plot and day and
228
day by treatment interaction were in the subplot. For qPCR the Cq value of the gene of
229
interest was normalized to the Cq value of GAPDH for each sample and analysis was
230
run on the 2-∆∆Cq values using GraphPad Prism (Version 6 from GraphPad Software,
10
Page 11 of 32
231
Inc.). The chemiluminescent signals for western blots were quantified using the mean
232
value (intensity) using Image Lab software (Version 4.1). Each band of interest was
233
normalized by dividing by mean value (intensity) for the protein of interest divided by
234
mean value (intensity) of ACTB. Significant changes were determined at P < 0.05 using
235
an unpaired, two-tailed student’s t-test. If the variance significant differed, Welch’s
236
correction was used.
237 238
Results
239
Serum Progesterone Concentrations and Corpora Lutea and Concepti Numbers.
240
A day by treatment interaction (P < 0.05) was observed for serum P4 concentrations for
241
all ewes until day 13 (Fig. 1A). Serum P4 concentrations were similar among treatments
242
through day 3. Ewes receiving hCG on day 4 had greater (P < 0.05) serum P4
243
concentrations than control ewes from days 4 to 13. No treatment by day interaction
244
was detected (P > 0.20) for serum P4 concentration between days 14 and 25; therefore,
245
treatment effects were examined across sampling days. During this period hCG-treated
246
ewes maintained elevated (P = 0.001) serum P4 values (10.9 ± 0.6 ng/mL) compared to
247
controls (6.0 ± 0.6 ng/mL). Treatment with hCG tended (P = 0.108) to increase number
248
of concepti (Fig 1B). On day 25, in the control group, three ewes had singletons, two
249
had twins, and one bore triplets, whereas all hCG treated ewes had twins. No difference
250
was observed between mean weight of concepti for control and hCG-treated ewes
251
(339.7 and 473.6 ± 74.7 mg, respectively, P = 0.20). Ewes receiving hCG had greater
252
number of CL (P = 0.006) compared to control ewes (Fig 1C). Control ewes had ≤ 3 CL,
253
whereas, 87.5% of ewes that received hCG had ≥ 3 CL (Fig 1C). Further, CL weight (g)
11
Page 12 of 32
254
per concepti was significantly greater (P
