The mRNA expression of prostaglandin E receptors EP2 and

EP4 and the changes in glycosaminoglycans in the sheep

cervix during the estrous cycle

C.M. Kershaw-Young a,b,*, M. Khalid a, M.R. McGowan a,A.A. Pitsillides c, R.J. Scaramuzzi c

a Department of Veterinary Clinical Sciences, The Royal Veterinary College, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdomb Faculty of Veterinary Science, The University of Sydney, Camperdown, Sydney, NSW 2006, Australia

c Department of Veterinary Basic Sciences, The Royal Veterinary College, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom

Received 23 November 2008; received in revised form 24 February 2009; accepted 25 February 2009

Abstract

Transcervical artificial insemination in sheep is limited by the inability to completely penetrate the cervix with an inseminating

pipette. Penetration is partially enhanced at estrus due to a degree of cervical relaxation, which is probably regulated by cervical

prostaglandin synthesis and extracellular matrix remodeling. Prostaglandin E2 acts via prostaglandin E receptors EP1 to EP4, and

EP2 and EP4 stimulate smooth muscle relaxation and glycosaminoglycan synthesis. This study investigated the expression of EP2

and EP4 mRNA and glycosaminoglycans in the sheep cervix during the estrous cycle. Sheep cervices were collected prior to, during,

and after the luteinizing hormone (LH) surge and during the luteal phase. The mRNA expression of EP2 and EP4 was determined by

in situ hybridization, glycosaminoglycan composition was assessed by Alcian blue staining, and hyaluronan concentration was

investigated by ELISA. The expression of EP2 mRNA was greatest prior to the LH surge (P = 0.02), although EP2 and EP4 were

expressed throughout the estrous cycle. Hyaluronan was the predominant glycosaminoglycan, and hyaluronan content increased

prior to the LH surge (P < 0.05). Cervical EP2 mRNA expression changed throughout the estrous cycle and was greatest prior to the

LH surge. We propose that prostaglandin E2 binds to EP2 and EP4 stimulating hyaluronan synthesis, which may cause remodeling of

the cervical extracellular matrix, culminating in cervical relaxation.

# 2009 Elsevier Inc. All rights reserved.

Keywords: Artificial insemination; Cervix; Extracelluar Matrix; Hyaluronan; Prostaglandin E Receptors

www.theriojournal.com

Available online at www.sciencedirect.com

Theriogenology 72 (2009) 251–261

1. Introduction

Sheep-breeding programs use reproductive technol-

ogies such as artificial insemination (AI) to maximize

the use of superior rams and produce improved

offspring by the introduction of superior genotypes

* Corresponding author. Tel.: +61 02 93513463.

E-mail address: claire.young@usyd.edu.au

(C.M. Kershaw-Young).

0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2009.02.018

[1]. The success of these schemes is largely dependent

on the use of AI with frozen-thawed semen, because

fresh semen, once collected, is only viable for 24 h [1].

In the sheep industry, the use of AI with frozen-thawed

semen is limited because fertility and lambing rates

after cervical AI with frozen-thawed semen are not

commercially viable [2]. For fertility rates with frozen-

thawed semen to approach those achieved with fresh

semen, intrauterine deposition of semen is required.

Laparoscopic intrauterine insemination achieves ferti-

lity rates of 70% [3], although differences in fertility

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261252

rates are observed among breeds [1]. However,

laparoscopic AI is costly, time consuming, requires

technical ability, and is not considered welfare friendly

[1,4]. The alternative technique, transcervical AI, would

overcome the constraints associated with laparoscopic

AI and the low fertility rates after cervical AI with

frozen-thawed semen. However, the use of transcervical

AI in sheep is limited because the anatomy of the sheep

cervix restricts the passage of an inseminating pipette

into the uterine lumen. There is a degree of natural

cervical relaxation during the follicular phase of the

estrous cycle that enables deeper penetration of the

cervix with an inseminating pipette [5]. This relaxation

is most likely mediated by changes in periovulatory

hormones, prostaglandin synthesis, and remodeling of

the cervical extracellular matrix. There is an increase in

COX-2 mRNA expression in the sheep cervix at estrus

that is associated with the remodeling of collagen [6],

suggesting that increased COX-2 expression may

increase prostaglandin E2 (PGE2) synthesis, which acts

upon the cervical extracellular matrix to induce cervical

relaxation. The mechanism by which PGE2 may

regulate changes in the cervical extracellular matrix

of the sheep is not fully understood.

Prostaglandin E2 mediates its effects through four

prostaglandin E receptors, EP1 to EP4. Receptors EP1 and

EP3 are involved in the contraction of smooth muscle,

whereas EP2 and EP4 receptors induce the relaxation of

smooth muscle. Therefore, PGE2 may act via EP2 and

EP4 receptors to stimulate cervical relaxation through the

relaxation of smooth muscle. Furthermore, stimulation of

the EP2 and EP4 receptors may induce cervical relaxation

via changes in the cervical extracellular matrix.

Glycosaminoglycans are a major component of the

cervical extracellular matrix and have been implicated in

cervical relaxation at parturition [7]. There are five

glycosaminoglycans—hyaluronan, dermatan sulfate,

chondroitin sulfate, heparan sulfate, and keratan sul-

fate—and all five are present in the nonpregnant human

cervix [8]. Stimulation of the EP4 receptor by PGE2

increases glycosaminoglycan synthesis in human cervi-

cal fibroblasts [9] suggesting that PGE2 may regulate

cervical relaxation in the sheep via changes in

glycosaminoglycan composition.

To gain an understanding of the role of steroid

hormones in the natural mechanism of cervical

relaxation in the nonpregnant ewe, it is necessary to

determine the expression of PGE2 receptors and

glycosaminoglycans in the sheep cervix during the

periovulatory period and during the luteal phase of the

estrous cycle. In addition, focusing on the periovulatory

period will enable the timing of cervical relaxation at

estrus to be determined and may reveal a role for

gonadotropins in cervical relaxation.

The current study aimed to localize and investigate

the expression of EP2 and EP4 receptor mRNAs and

determine the composition of cervical glycosamino-

glycans at four defined stages of the estrous cycle to

examine the hypothesis that cervical relaxation in the

sheep at estrus is mediated by EP2 and EP4 receptor

expression and changes in the composition of tissue

extracellular matrix glycosaminoglycan.

2. Materials and methods

2.1. Animals and tissues

The study was performed on 17 Welsh Mountain

ewes under Home Office authorization, in compliance

with the Animal (Scientific Procedures) Act (UK),

1986. Ewes were synchronized to a common day of

estrus using intravaginal Chronogest sponges for 12 d

and 250 IU PMSG at the time of sponge removal

(Intervet UK Ltd, Cambridge, UK). All ewes were in

estrus within 48 h of sponge withdrawal, which was

designated Day 0 of the estrous cycle. On Day 9

of the synchronized cycle, during the luteal phase

when plasma estradiol concentrations were low

(1.8 � 0.30 pg/mL), the reproductive tracts of five

animals were collected after death by captive bolt and

exsanguination. On Day 11, the remaining animals were

given 125 mg cloprostenol (prostaglandin F2a [PGF2a]

analogue; Estrumate; Schering-Plough Animal Health,

Welwyn Garden City, UK), and the tracts were collected

36 h post-PGF2a prior to the luteinizing hormone (LH)

surge (n = 5) when plasma estradiol concentrations

were high (4.3 � 0.20 pg/mL), 45 h post-PGF2a during

the LH surge (n = 3) when plasma estradiol concentra-

tions were declining (4.1 � 1.77 pg/mL), and 55 h post-

PGF2a following the LH surge (n = 4) when plasma

estradiol concentrations were low (2.1 � 1.63 pg/mL).

Plasma progesterone, LH, and follicle-stimulating

hormone (FSH) concentrations were representative of

the sheep estrous cycle, as described previously [6].

Cervices were assigned to the stage of the estrous cycle

based on plasma LH, estradiol, and progesterone

concentrations. The cervix was separated from the

anterior vagina and uterine body then divided trans-

versely into six equal sections. Alternate cervix sections

comprising the uterine region, midregion, and vaginal

region were fixed in neutral buffered formalin, wax

embedded, sectioned at 9 mm, and mounted onto

Superfrost Plus slides (BDH, Poole, UK). The remain-

ing three sections were stored at �80 8C.

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261 253

2.2. Riboprobe synthesis

Total RNA was extracted from sheep liver using

RNA Stat 60 (AMS Biotechnology Ltd., Oxon, UK)

and RT-PCR was performed. The primers for EP2

amplified a 325-bp product and had the following

sequences; forward: 50-CTGGCTTCGTATGCGCA-

GAA-30, reverse: 50-GCAGGTACGTGGTCTGCTTG-

TG-30. The primers for EP4 amplified a 317-bp DNA

product and were sequenced as follows: forward: 50-ATCTTCGGGGTGGTGGGCAA-30, reverse: 50-CG-

CTTGTCCACGTAGTGGCT-30. The PCR conditions

were 94 8C for 60 s, 55 8C (EP2) or 56 8C (EP4) for 60 s,

and 72 8C for 60 s for 40 cycles. The determined cDNA

sequences for ovine EP2 and EP4 were published in the

EMBL nucleotide sequence database with accession

numbers AJ876410 and AJ617580, respectively. The

EP2 and EP4 PCR products were cloned into the PGEM-

Teasy plasmid (Promega Corporation, Madison, WI,

USA) then riboprobes were synthesized with the SP6

(sense) and T7 (antisense) MEGAscript transcription

kits (Ambion Ltd, Huntingdon, UK) and labeled with

digoxigenin-11-UTP (Roche Diagnostics, Mannheim,

Germany).

2.3. In situ hybridization

In situ hybridization for EP2 and EP4 mRNAs was

performed on four cervix sections (one sense and three

antisense) at the uterine, mid, and vaginal region of the

cervix for each ewe (n = 17). The riboprobes were

hybridized to the cervix sections at 65 8C for 6 h, as

described previously [6].

The expression of EP2 and EP4 mRNA was

quantified as described previously [6]. Briefly, the

cervix was histologically divided into six cell layers

including the luminal epithelium, subepithelial

stroma, irregular smooth muscle layer, longitudinal

smooth muscle layer, transverse smooth muscle layer,

and serosa. Each cervical section was observed at

�100 magnification, then the proportion of cells

staining positive within each cell layer (to the nearest

5%) and the intensity of staining, graded from 0 to 4

as used previously [10], were determined. Each

cell layer within each cervix section at each region of

the cervix for each ewe was assessed and used to

generate an index score as follows: In situ hybridiza-

tion index score = [percentage expression � intensity

score]/100. A mean index score for each cell layer at

each cervical region for each individual ewe was

calculated from the replicate cervix sections (n = 3

per region).

2.4. Alcian blue 8GX staining for

glycosaminoglycans

Critical electrolyte Alcian blue staining was per-

formed at each cervical region of the cervix for each

ewe as described previously [11]. Briefly, paraffin-

embedded serial cervix sections (n = 6 per region per

ewe) were de-waxed, rehydrated, and immersed in

0.025 M sodium acetate buffer pH 5.8, containing

0.05% Alcian blue 8GX (Sigma, Poole, UK) and either

0.05 M (n = 2), 0.4 M (n = 2), or 0.8 M (n = 2) MgCl2for 18 h at room temperature. Sections were washed,

dehydrated, and mounted in DePeX.

Alcian blue stains glycosaminoglycans with increas-

ing selectivity as increasing amounts of MgCl2 are

incorporated into the solution. At low electrolyte

concentrations (0.05 M MgCl2), all glycosaminogly-

cans were stained; at 0.4 M MgCl2, only sulfated

glycosaminoglycans (chondroitin sulfate, dermatan

sulfate, heparan sulfate, and keratan sulfate) were

stained; and at 0.8 M MgCl2, only keratan sulfate and

heparan sulfate were stained.

Alcian blue staining intensity was measured using a

Vickers M85A scanning and integrating microdensit-

ometer [12]. Cervical cell layers were defined as

luminal epithelium, subepithelial stroma, irregular

smooth muscle layer, and longitudinal and transverse

smooth muscle layers, which were not discriminated

from one another. At each MgCl2 concentration, 10

measurements were taken at each cervical cell layer for

each region of the cervix for each ewe. Absolute Alcian

blue staining intensity was expressed as the mean

integrated extinction (MIE � 100) per unit area. The

amount of staining (MIE � 100) for sulfated glycosa-

minoglycans (retained at concentrations of 0.4 M

MgCl2) was subtracted from that disclosed under

conditions that allowed staining of all glycosaminogly-

cans (0.05 M MgCl2) to determine the amount of

staining for ‘‘hyaluronan-like’’ glycosaminoglycan, as

hyaluronan is the only nonsulfated glycosaminoglycan.

2.5. Measurement of hyaluronan concentration by

ELISA

Frozen cervical tissues at the uterine, mid, and vaginal

region were digested in 0.1 M sodium acetate buffer pH

5.8 containing 0.25 mg/mL papain (Roche Applied

Science, Mannheim, Germany) at 60 8C for 18 h, as

described previously [13]. Hyaluronan concentration in

the digested tissue supernatant was assayed in duplicate

by ELISA based on a method by Fosang et al. [14].

Briefly, Nunc-Immuno MaxiSorp 96 well plates were

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261254

Fig. 1. EP2 mRNA expression in the sheep cervix (A–F) prior to the LH surge and (G–L) after the LH surge at the (A, D, G, J: column 1) uterine

region, (B, E, H, K: column 2) midregion, and (C, F, I, L: column 3) vaginal region. At the uterine region, EP2 mRNA expression was greater prior to

the LH surge compared with that after the LH surge (D vs. J: P = 0.015). There were no differences in EP2 mRNA expression between cervices

collected prior to and after the LH surge at the midregion (E vs. K) or vaginal region (F vs. L). (K) Cervical cell layers are labeled: luminal epithelium

(LE), subepithelial stroma (SES), irregular smooth muscle (ISM), longitudinal smooth muscle (LSM), and transverse smooth muscle (TSM). Stroma

is not shown. (A, B, C, G, H, I) As expected, there was no staining for EP2 mRNA in the sense (negative controls). Scale bar = 850 mm.

coated with human umbilical cord hyaluronan (Sigma-

Aldrich, Poole, UK), then 50 mL of standard or sample

was added to the wells, with 50 mL 0.33 mg/mL bio-

tinylated hyaluronic acid binding protein (Seikagaku

America, Falmouth, USA) and incubated overnight at

room temperature. Next, 100 mL streptavidin-biotiny-

lated horseradish peroxidase complex (Amersham Bio-

sciences UK Ltd., Buckinghamshire, UK) was incubated

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261 255

Table 1

EP2 mRNA index score (mean � SEM) at each region of the sheep cervix during the estrous cycle.

Cervical region Stage of estrous cycle

Luteal Pre-LH During-LH Post-LH

Uterine 0.64 � 0.142ax 1.28 � 0.159b

x 0.53 � 0.175ax 0.39 � 0.134a

x

Midregion 1.12 � 0.168ay 1.23 � 0.145a

x 0.87 � 0.213ax 0.97 � 0.176a

y

Vaginal 1.24 � 0.182ay 1.39 � 0.151a

x 0.51 � 0.118ax 1.19 � 0.219a

y

a,bWithin a row, means without a common superscript differed (P < 0.05).x,yWithin a column, means without a common subscript differed (P < 0.05).

Fig. 2. Mean EP2 mRNA expression in each cell layer of the sheep

cervix at the (~) uterine region, (&) midregion, and (&) vaginal

region. a,b,cMeans without a common superscript differed. P values are

indicated in the text.

for 30 min at 37 8C, followed by 100 mL 2,20-azino-bis-

(3-ethylbenzthiazoline-6-sulfonic acid) diammonium

salt (ABTS) substrate at 37 8C for 1 h, and the optical

density was read at OD405. The concentration of cervical

hyaluronan was expressed as mg hyaluronan/mg cervix

(wet weight). The range of the assay was 0.01 to 1.25 mg/

mL. The intra-assay coefficient of variation was 5.6%,

and the interassay coefficient of variation was 17.6%.

2.6. Statistical analysis

In situ hybridization, Alcian blue, and hyaluronan

concentration data were analyzed in SPSS version 13.0

(SPSS Inc., Chicago, IL, USA) by repeated-measures

ANOVA using a linear mixed model, with post hoc tests

using the LSD test where appropriate. Cell layer,

cervical region, and stage of cycle were fixed factors,

and sheep ID were included as a random factor to ensure

that repeated measurements from the same animal were

not taken as independent measurements. Alcian blue

staining data were skewed as determined by skewness

and kurtosis tests, and so statistical analysis was

performed on log-transformed MIE values. Hyaluronan

concentration data were also skewed as determined by

skewness and kurtosis tests; therefore, they were

transformed using the square root to give a normal

distribution prior to statistical analysis.

3. Results

3.1. Prostaglandin E2 EP2 receptor mRNA

expression in the sheep cervix

3.1.1. Expression of EP2 mRNA was greatest prior

to the LH surge

Theexpression of EP2 mRNAwas regulated during the

estrous cycle (P = 0.02), but not at the mid or vaginal

regions of the cervix (Fig. 1). At the uterine region, EP2

mRNA expression was greater prior to the LH surge

compared with that during the LH surge (P = 0.048), after

the LH surge (P = 0.015), and during the luteal phase

(P = 0.04; Table 1). The increase in EP2 mRNA was

observed in all the cervical cell layers, except the luminal

epithelium and serosa, where expression was minimal.

3.1.2. Expression of EP2 mRNA differed between

cervical regions

The expression of EP2 mRNA declined from the

vaginal region to the uterine region of the cervix

(P < 0.001; Fig. 1). This gradient effect was observed

after the LH surge (P = 0.001) and during the luteal

phase (P = 0.008) but not during or prior to the LH surge

(Table 1).

3.1.3. Expression of EP2 mRNA was predominately

in the smooth muscle layers

The expression of EP2 mRNA was greatest in the

irregular (P < 0.01) and longitudinal (P < 0.05)

smooth muscle layers, intermediate in the serosa,

subepithelial stroma, and transverse smooth muscle

layer, and lowest in the luminal epithelium (P < 0.001;

Fig. 2). This pattern of expression was observed in all

regions and at each stage of the cycle.

3.2. Prostaglandin E2 EP4 receptor mRNA

expression in the sheep cervix

3.2.1. Expression of EP4 mRNA was not regulated

during the estrous cycle

The expression of EP4 mRNA (mean � SEM) in the

sheep cervix did not differ between cervices during the

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261256

Fig. 3. EP4 mRNA expression in the sheep cervix during the luteal phase at the (A, D) uterine region, (B, E) midregion, and (C, F) vaginal region.

The expression of EP4 mRNA was greater at the vaginal region compared with that at the uterine region (F vs. D: P = 0.004). The (E) midregion had

intermediate expression and was not different from the (D) uterine or (F) vaginal regions. (F) Cervical cell layers are labeled: luminal epithelium

(LE), subepithelial stroma (SES), irregular smooth muscle (ISM), longitudinal smooth muscle (LSM), and transverse smooth muscle (TSM). Stroma

is not shown. (A, B, C) As expected, there was no staining for EP4 mRNA in the sense (negative controls). Scale bar = 850 mm.

Table 2

EP4 mRNA index score (mean � SEM) at each region of the sheep cervix during the estrous cycle.

Cervical region Stage of estrous cycle

Luteal Pre-LH During-LH Post-LH

Uterine 0.40 � 0.311ax 0.86 � 0.370a

xy 0.14 � 0.221ax 0.38 � 0.325a

x

Midregion 0.81 � 0.453axy 0.52 � 0.264a

x 0.82 � 0.429ay 0.51 � 0.404a

x

Vaginal 1.13 � 0.426ay 0.99 � 0.378a

y 1.03 � 0.471ay 0.64 � 0.327a

x

a,bWithin a row, means without a common superscript differed (P < 0.05).x,yWithin a column, means without a common subscript differed (P < 0.05).

Fig. 4. Mean EP4 mRNA expression in each cell layer of the sheep

cervix at the (&) uterine region, (&) midregion, and (~) vaginal

region. a,b,cMeans without a common superscript differed. P values are

indicated in the text.

luteal phase (0.76 � 0.094), prior to the LH surge

(0.78 � 0.347), during the LH surge (0.64 � 0.439),

and after the LH surge (0.50 � 0.351).

3.2.2. Expression of EP4 mRNA between cervical

regions was dependent on the stage of cycle

The expression of EP4 mRNA differed between

cervical regions (P < 0.001; Fig. 3) and declined from

the vaginal region to the uterine region during the LH

surge (P < 0.001), during the luteal phase (P = 0.004),

and prior to the LH surge (P < 0.001; Table 2). A

similar trend was observed after the LH surge (Table 2).

These differences were observed in all cell layers,

except the luminal epithelium and serosa, where

expression was minimal (P < 0.05).

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261 257

Table 3

Sulfated glycosaminoglycan staining intensity (MIE; mean � SEM) in each cell layer at each region of the sheep cervix.

Cell layer Cervical region

Uterine Midregion Vaginal

Luminal epithelium 0.85 � 0.075abx 0.72 � 0.055a

x 1.00 � 0.119bx

Subepithelial stroma 1.01 � 0.074ax 1.19 � 0.122a

y 2.05 � 0.196by

Irregular smooth muscle 0.90 � 0.081ax 0.62 � 0.045a

x 1.75 � 0.170by

Longitudinal/transverse smooth muscle 0.88 � 0.097ax 0.75 � 0.058a

x 1.65 � 0.133ay

a,bWithin a row, means without a common superscript differed (P < 0.05).x,yWithin a column, means without a common subscript differed (P < 0.05).

Table 4

Hyaluronan-like staining intensity (MIE; mean � SEM) in each cell layer at each region of the sheep cervix.

Cell layer Cervical region

Uterine Midregion Vaginal

Luminal epithelium 13.60 � 0.422ax 19.43 � 0.349b

x 24.34 � 0.284cx

Subepithelial stroma 8.08 � 0.369ay 10.83 � 0.346b

y 7.99 � 0.352ay

Irregular smooth muscle 8.34 � 0.379ay 6.61 � 0.359b

y 9.47 � 0.309ay

Longitudinal/transverse smooth muscle 8.35 � 0.405ay 6.44 � 0.344b

y 9.63 � 0.316ay

a,bWithin a row, means without a common superscript differed (P < 0.05).x,yWithin a column, means without a common subscript differed (P < 0.05).

3.2.3. Expression of EP4 mRNA was predominately

in smooth muscle and subepithelial stroma

The expression of EP4 mRNA was greatest in the

irregular smooth muscle layer (P < 0.01), intermediate

in the subepithelial stroma and longitudinal smooth

muscle layer (P < 0.05), and lowest in the transverse

smooth muscle layer, luminal epithelium, and serosa

(P < 0.05; Fig. 4). This pattern of expression was

observed at all cervical regions (Fig. 4) and at each stage

of the cycle.

Fig. 5. Alcian blue staining in the sheep cervix. (A) Total glycosaminoglyca

(C) Keratan sulfate and heparan sulfate (0.8 M MgCl2). There is a shar

glycosaminoglycans, indicating that the predominant glycosaminoglycan in t

luminal epithelium (LE), subepithelial stroma (SES), irregular smooth musc

muscle (TSM). Stroma is not shown. Scale bar = 850 mm.

3.3. Glycosaminoglycan content in the sheep cervix

using Alcian blue staining

3.3.1. Hyaluronan was the predominant

glycosaminoglycan in the sheep cervix

Hyaluronan-like glycosaminoglycan comprised 80%

to 90% of the total glycosaminoglycans in the cervix,

and sulfated glycosaminoglycans comprised 10% to

20% (Fig. 5). Microdensitometry was not able to detect

differences between staining intensities at 0.4 and 0.8 M

ns (0.05 M MgCl2). (B) Sulfated glycosaminoglycans (0.4 M MgCl2).

p decline in staining intensity between (A) total and (B) sulfated

he sheep cervix is hyaluronan-like. (A) Cervical cell layers are labeled:

le (ISM), longitudinal smooth muscle (LSM), and transverse smooth

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261258

Fig. 6. Mean hyaluronan-like glycosaminoglycan staining intensity

(MIE) at each stage of the estrous cycle at the (~) uterine region,

(&) midregion, and (&) vaginal region of the sheep cervix.

Hyaluronan-like staining intensity is greater prior to the LH surge

compared with other stages of the estrous cycle. P values are

indicated in the text.

MgCL2 despite differences being observed microsco-

pically (Fig. 5). Therefore, statistical analyses were

performed on nonsulfated (0.05 M) and sulfated

(0.4 M) log-transformed data only.

3.3.2. Hyaluronan-like glycosaminoglycan content

was regulated during the estrous cycle

Hyaluronan-like glycosaminoglycan staining inten-

sity was greater in pre-LH cervices compared with

during-LH (P = 0.017), post-LH (P = 0.01), and luteal

(P = 0.001) cervices at the uterine region (Fig. 6) but

was only greater than luteal (P < 0.01) and post-LH

(P = 0.006) cervices at the mid and vaginal regions

(Fig. 6). Sulfated glycosaminoglycan staining intensity

(mean � SEM) was similar during the luteal phase

(1.06 � 0.305), prior to the LH surge (1.17 � 0.236),

during the LH surge (1.14 � 0.362), and after the LH

surge (1.11 � 0.347).

3.3.3. There is a Gradient of glycosaminoglycans

among cervical regions

Sulfated glycosaminoglycan staining was greater at

the vaginal region of the cervix compared with that at

the uterine (P < 0.001) and mid regions (P < 0.001) in

each cell layer (P < 0.001), except the luminal

epithelium (Table 3). Likewise, hyaluronan-like stain-

ing was greater at the vaginal region than at the uterine

region during the LH surge (P = 0.001), and during the

luteal phase (P < 0.001), and the same trend was

observed after the LH surge (Fig. 6). Prior to the LH

surge, there was no difference between cervical regions,

due to the increase in hyaluronan-like glycosaminogly-

can at the uterine region of the cervix (Fig. 6). The

differences between cervical regions were mainly

observed in the luminal epithelium (Table 4).

3.3.4. Hyaluronan-like glycosaminoglycan was

predominately localized to the luminal epithelium

Hyaluronan-like staining was greatest in the luminal

epithelium compared with that of all other cell layers

(P < 0.001; Table 4). Strong staining in the luminal

epithelium was present (Fig. 5). The remaining cell

layers did not differ from each other. Sulfated

glycosaminoglycan staining differed between cell

layers at the midregion and at the vaginal regions of

the cervix only (P < 0.001; Table 3).

3.4. Hyaluronan concentration in the sheep cervix

using ELISA

The mean (�SEM) concentration of hyaluronan (mg/

mg cervix) was not significantly different during the

luteal phase (0.67 � 0.251), prior to the LH surge

(0.46 � 0.175), during the LH surge (0.70 � 0.419), or

after the LH surge (0.76 � 0.305). The concentration of

hyaluronan (mg/mg cervix; mean � SEM) was greatest

at the vaginal region (1.03 � 0.166), intermediate at the

midregion (0.58 � 0.087), and lowest at the uterine

region (0.29 � 0.066). Hyaluronan concentration was

greater at the mid (P = 0.029) and vaginal (P < 0.001)

regions of the cervix compared with that at the uterine

region, and also at the vaginal region compared with the

mid-region (P = 0.017).

4. Discussion

The current study examined the expression of EP2

and EP4 mRNA and the composition of glycosami-

noglycans in the sheep cervix during the estrous cycle to

aid the understanding of cervical relaxation at estrus.

Both EP4 and EP2 mRNAs were expressed in the

sheep cervix throughout the estrous cycle, suggesting

that PGE2 was able to stimulate cervical relaxation via

these receptors. The expression of EP4 mRNA was not

regulated during the estrous cycle, suggesting that EP4

mRNA was not regulated by ovarian steroids, as

reported for EP4 protein in the nonpregnant ovariecto-

mized ewe [15]. The expression of EP2 mRNA was

increased at the uterine region of the cervix prior to the

LH surge, when plasma estradiol concentrations were

greatest and progesterone concentrations low. The

mRNA expression of EP2 was regulated by changes in

estrogen and progesterone in the mouse uterus [16]. We

propose that, in the sheep cervix, EP2 mRNA expression

is stimulated by the periovulatory changes in proges-

terone and estradiol, which causes an increase in

expression prior to the LH surge. The increase in EP2

mRNA expression at estrus suggested that PGE2 may

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261 259

mediate the relaxation of smooth muscle via EP2 to

stimulate cervical relaxation.

The current study determined, for the first time, that

there was a gradient of EP4 and EP2 mRNA expression

along the sheep cervix, with the greatest expression at

the vaginal region and the lowest expression at the

uterine region. It is likely that this gradient was caused

by differences in cell type and cell density among

cervical regions. If the proportion or abundance of

particular cell types changes between cervical regions,

and EP2 and EP4 mRNA is expressed by these cells, then

changes in EP2 and EP4 mRNA between regions are not

unexpected. The proportion of smooth muscle and

collagen differs throughout the sheep cervix [6].

Furthermore, differences in steroid receptor expression

between cervical regions in the cow cervix are

eliminated when cell density is considered [17]. This

suggests that an increase in EP2 or EP4 mRNA at the

vaginal region of the cervix may be explained by an

increase in the proportion of cells that express EP2 and

EP4 mRNA, or an increase in cell density at this region.

The physiologic significance of this gradient is

unknown, but it may be that the vaginal region of the

cervix is more responsive to changes in PGE2 synthesis

during the estrous cycle.

The smooth muscle layers of the sheep cervix

predominately expressed EP2 and EP4 mRNA, as

described previously for EP2 and EP4 protein [18],

supporting our hypothesis that PGE2 induces cervical

relaxation via the relaxation of smooth muscle.

However, EP2 and EP4 mRNA were also expressed in

the subepithelial stroma that contains predominately

fibroblasts, suggesting that EP2 and EP4 were expressed

within fibroblast cells of the sheep cervix. Prostaglandin

E2 acts through EP4 receptors on cervical fibroblasts to

stimulate glycosaminoglycan synthesis [9], suggesting

that the mechanism by which PGE2 induces cervical

relaxation is complex and does not only involve smooth

muscle, but also extracellular matrix components

secreted by fibroblasts, such as glycosaminoglycans.

The current study has shown that there were changes

in hyaluronan glycosaminoglycan-like content (as

determined by Alcian blue staining), but not hyaluronan

concentration (as determined by ELISA), in the sheep

cervix during the estrous cycle. During cervical

relaxation, there is extracellular matrix remodeling

[19], increased water content [20], and an increase in

cervical weight [21–23], which may mask changes in

hyaluronan concentration but not content, as observed

in the cervix of the pregnant rat [7]. In the current study,

hyaluronan concentration was expressed as mg/mg wet

weight of cervix, and the potential increase in weight

during cervical relaxation may explain why hyaluronan

concentration did not differ throughout the estrous

cycle. The pattern of hyaluronan concentration between

cervical regions was not different from the pattern

observed for hyaluronan-like glycosaminoglycan con-

tent; this was probably because any changes in cervical

weight were proportional at each region of the cervix for

each animal.

Hyaluronan-like glycosaminoglycan content in the

cervix was greatest prior to the LH surge; this increase

may be induced by estradiol, because estradiol

stimulates hyaluronan synthesis [24]. Conversely,

progesterone inhibits hyaluronan synthesis [25] and

converts hyaluronan metabolism from the synthesis

phase to the degradation phase [24], and may therefore

decrease hyaluronan-like glycosaminoglycan content in

the sheep cervix during the luteal phase. It is likely that

the regulation of hyaluronan by estradiol and proges-

terone is mediated through PGE2, because estradiol

stimulates cervical oxytocin receptor expression [26]

and oxytocin stimulates COX-2 synthesis through the

oxytocin receptor, leading to an increase in PGE2 [27].

Prostaglandin E2 binds to EP receptors and stimulates

the synthesis of both sulfated and nonsulfated glyco-

saminoglycans in fibroblast cells of the human cervix

[9,28]. In addition cervical COX-2 mRNA expression is

greatest at estrus, particularly at the uterine region [6],

paralleling the pattern of hyaluronan-like glycosami-

noglycan in the cervix during the estrous cycle and

supporting the hypothesis that hyaluronan synthesis is

regulated by PGE2.

Hyaluronan-like glycosaminoglycan was most abun-

dant in the luminal epithelium of the cervix, as

described previously [20]. This suggests that hyalur-

onan is secreted into the cervical lumen, particularly at

estrus when hyaluronan-like glycosaminoglycan con-

tent increases. Hyaluronan has been reported in cervical

mucus [29] and, as hyaluronan has rheological proper-

ties, the increase in hyaluronan-like glycosaminoglycan

prior to the LH surge may be associated with changes in

the viscosity of cervical mucus at estrus.

An increase in hyaluronan-like glycosaminoglycan

at estrus is likely to induce cervical relaxation via

extracellular matrix remodeling. Hyaluronan disperses

and separates collagen bundles in rabbit cervix [30], and

high hyaluronan concentrations in connective tissues

are associated with small-diameter collagen fibrils [31].

The sulfated glycosaminoglycan dermatan sulfate

forms cross-links between collagen fibres and strength-

ens collagen bundles. There was little or no expression

of keratan sulfate and heparan sulfate in the sheep

cervix (current study), and previous evidence suggests

C.M. Kershaw-Young et al. / Theriogenology 72 (2009) 251–261260

that chondroitin sulfate is not present in the sheep cervix

[20]. Consequently, it can be assumed that the sulfated

glycosaminoglycan staining in the current study was

dermatan sulfate. Hyaluronan has rheological proper-

ties and we propose that, at estrus, when hyaluronan

content increases, water is absorbed into the cervix and

the relative concentration of dermatan sulfate (the

content of which we have determined does not change)

decreases. This will reduce cross-links between

collagen fibres causing them to separate and become

disorganized [32]. The dissociation and degradation of

collagen bundles is associated with softening of the

sheep cervix [33]. The increase in hyaluronan-like

glycosaminoglycan content in the sheep cervix prior to

the LH surge was consistent with the changes in

collagen [6], further supporting the hypothesis that

hyaluronan induces cervical relaxation through the

remodeling of collagen bundles.

The current study suggested that cervical relaxation in

the ewe was maximal prior to the LH surge. However, the

optimal timing for cervical AI in ewes is 55 h after

progesterone sponge removal, following the LH surge

[1]. Although the natural mechanism of cervical

relaxation was not at the appropriate time to perform

transcervical AI in ewes, the current study determined the

time at which prostaglandin receptor expression was

greatest during the estrous cycle and may aid the

development of cervical relaxation techniques. We have

recently demonstrated that misoprostol, a PGE1 analogue

that binds to EP receptors, stimulates cervical relaxation

in the ewewhen administered prior to the LH surge (when

EP2 receptor mRNA is greatest and EP4 receptor mRNA

is expressed) and enhances cervical penetration [34].

In conclusion, EP2 and EP4 receptor mRNAs were

expressed in the sheep cervix during the estrous cycle,

and EP2 mRNA expression and hyaluronan glycosa-

minoglycan content were greatest in the sheep cervix

prior to the LH surge. We propose that, at estrus, PGE2

binds to EP2 and EP4 receptors on smooth muscle and

fibroblast cells in the sheep cervix to stimulate the

relaxation of smooth muscle and hyaluronan-like

glycosaminoglycan synthesis. Although not determined

in the current study, perhaps this increase in cervical

hyaluronan will increase water absorption and hence

decrease the relative concentration of dermatan sulfate,

causing collagen fibres and bundles to separate and

disperse thereby culminating in cervical relaxation.

Acknowledgments

The authors thank Mrs. Tanya Hopcroft, Dr. Edward

Bastow, Dr. Gabriele Wax, and Dr. Claire Clarkin for

technical advice. This research was funded by The

Royal Veterinary College, UK Internal Grants Scheme.

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