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BOR - Papers in Press, published online ahead of print September 13, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054049
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BIOLOGY OF REPRODUCTION 76, 124–129 (2007)
DOI: 10.1095/biolreprod.106.054049
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

The Expression of Prostaglandin Endoperoxide Synthase 2 Messenger RNA and the Proportion of Smooth Muscle and Collagen in the Sheep Cervix During the Estrous Cycle

Claire M. Kershaw 2, Rex J. Scaramuzzi 3, Michael R. McGowan 2, Caroline P.D. Wheeler-Jones 3, and Muhammad Khalid 1 2

Departments of Veterinary Clinical Sciences2 and Veterinary Basic Sciences,3 The Royal Veterinary College, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom

ABSTRACT

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E2 synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.

cervix, estradiol, oxytocin

INTRODUCTION

Within the livestock industry, artificial insemination (AI) is used to maximize the use of superior rams and contain contagious diseases within flocks [1]. These advantages are greatly facilitated by the ability to perform AI with frozen-thawed semen because fresh semen, once collected, is viable for only 24 h [1]. However, the use of AI with frozen-thawed semen in the sheep industry is limited, because fertility rates for cervical AI with frozen-thawed semen are poor, and lambing rates range from 6.7% to 57% [2], which is not commercially acceptable. For fertility rates with frozen-thawed semen to approach those achieved with fresh semen, intrauterine deposition of semen is required. Laparoscopic intrauterine insemination is reliable and achieves fertilization rates of 70% [3]. However, it is also costly and time-consuming, requires technical ability, and is not considered welfare friendly [1, 4]. The alternative technique, transcervical AI, would overcome the welfare, financial, and cost constraints associated with laparoscopic AI as well as the low fertility rates following cervical AI with frozen-thawed semen. Despite these advantages, the use of transcervical AI in ewes is limited because of the anatomy of the sheep cervix, which restricts the passage of an inseminating pipette into the uterine lumen [5]. There is a degree of cervical relaxation during the follicular phase that enables greater penetration of the sheep cervix with an inseminating pipette [5], and this may be regulated by changes in periovulatory hormones [4, 5].

There is evidence to suggest that estradiol regulates changes in the cervical estrogen receptor [6, 7] and oxytocin receptor [8, 9] and that oxytocin stimulates prostaglandin estradiol (PGE2) synthesis via prostaglandin endoperoxide synthase 2 (PTGS2) in cervical segments from cows at proestrus and estrus [10]. PGE2 induces cervical relaxation in late pregnant sheep [11], and the intracervical application of a PGE2 gel induces cervical relaxation and softening in the nonpregnant cow [12]. This suggests that at estrus, the increase in estradiol concentrations, estradiol receptor, and oxytocin receptor stimulates PTGS2 expression and PGE2 synthesis in the cervix to regulate cervical relaxation. PGE2 is likely to mediate cervical relaxation through the rearrangement of collagen bundles within the cervical extracellular matrix [13], because during pregnancy, when PGE2 concentrations are high, collagen bundles separate and become disorganized [14, 15], and PGE2 reduces the tensile strength of collagen fibrils [13].

The present study investigated the expression of PTGS2 mRNA in the sheep cervix and determined the proportion of collagen and smooth muscle within the sheep cervix at four defined stages of the estrus cycle.

MATERIALS AND METHODS

Animals and Tissues

The present study was performed on 17 Welsh Mountain ewes under Home Office authorization in compliance with the Animal (Scientific Procedures) Act (U.K.) 1986. Ewes were synchronized to a common day of estrus with intravaginal Chronogest sponges for 12 days (Intervet UK Ltd., Cambridge, U.K.) and 250 IU of eCG at the time of sponge removal (Intervet UK). All ewes were in estrus within 48 h of sponge withdrawal, which was termed Day 0 of the estrous cycle. On Day 9 of the synchronized cycle (luteal phase), the reproductive tracts of five animals were collected following death by captive bolt and exsanguination. On Day 11, the remaining animals were administered 125 µg of Cloprostenol (prostaglandin F2{alpha} [PGF2{alpha}] analogue, Estrumate; Schering-Plough Animal Health, Welwyn Garden City, U.K.), and the tracts were collected 36 h post-PGF2{alpha} prior to the LH surge (n = 5), 45 h post-PGF2{alpha} during the LH surge (n = 3), and 55 h post-PGF2{alpha} following the LH surge (n = 4). The cervix was cleared of all unwanted tissue, the anterior vagina was removed, and the cervix was separated from the uterine body with a scalpel. The cervix was divided transversely into six equal sections. Alternate sections composed of the uterine region, mid-region, and vaginal region were fixed in neutral-buffered formalin (BDH, Poole, U.K.) for 24 h prior to storage in 70% ethanol. The remaining three sections were stored at –80°C. Fixed cervices were wax embedded, sectioned at 9 µm, and mounted onto Superfrost Plus slides (BDH). Jugular venous blood samples were taken for the duration of the study, and samples were placed in heparinized tubes and centrifuged at 1700 x g for 10 min at 4°C; then, the plasma was decanted and stored at –20°C.

Radioimmunoassay

Plasma estradiol concentrations were determined with a commercial human RIA kit (KE2D; Diagnostic Product Co., Los Angeles, CA) [16]. Plasma progesterone concentrations were determined with a validated RIA for PGE2 [17] that was adapted for progesterone. The sensitivity of the assay was 0.11 ng/ml, the intraassay coefficient of variation was 6.1%, and the interassay coefficient of variation was 6.8%. Plasma LH [18] and FSH [19] concentrations were determined as described previously. The sensitivity of the assay was 0.42 ng/ml for FSH and 0.37 mg/ml for LH, and the intraassay coefficients of variation for FSH and LH were 14.5% and 13.1%, respectively.

Synthesis of Ovine PTGS2 Riboprobes

Total RNA was extracted from sheep chorioallantois with RNA Stat 60 (AMS Biotechnology Ltd., Oxon, U.K.) and then RT-PCR was performed with specific primers on the basis of previously published oligonucleotide sequences [20]. The forward primer corresponded to position 168–189 of the ovine PTGS2 cDNA sequence (accession no. U68486), and the sequence was 5'-AGGTGTATGTATGAGTGTAGGA-3'. The reverse primer was complementary to position 631–651 of the ovine PTGS2 cDNA with the sequence 5'-GTGCTGGGCAAAGAATGCAAA-3'. The PCR conditions were 94°C for 60 sec, 56°C for 60 sec, and 72°C for 60 sec for 40 cycles. The PTGS2 PCR product was cloned into the PGEM-Teasy plasmid (Promega Corporation, Madison, WI), and ovine PTGS2 riboprobes were synthesized following linearization of the plasmid with ApaI and SalI with the SP6 (sense) and T7 (antisense) MEGAscript transcription kits, respectively (Ambion Ltd., Huntingdon, Cambridgeshire, U.K.). During synthesis, riboprobes were labeled with digoxigenin-11-uridine triphosphate (Roche Diagnostics, Mannheim, Germany).

In Situ Hybridization

In situ hybridization protocol. In situ hybridization was performed on eight cervix sections (4x sense and 4x antisense) at the uterine, mid-, and vaginal regions of the cervix for each ewe (n = 17). To prevent variations in staining intensity, in situ hybridization was performed on all cervix sections on the same day with the same reagents. Wax-embedded cervix sections (9 µm thick) were dewaxed in CNP30 clearing agent (TAAB Laboratories Equipment Ltd., Berkshire, U.K.), rehydrated through a graded series of ethanol, and fixed in neutral-buffered formalin (BDH) for 5 min. Sections were washed in PBS, immersed in hot 0.01 M sodium citrate for 30 min, and placed in PBS containing 1 mM EDTA (PBS/1 mM EDTA) for 5 min. Next, sections were permeabilized in PBS/1 mM EDTA containing 1 µg/ml proteinase K (Sigma-Aldrich) at 37°C for 20 min and then immersed in PBS containing 0.2% glycine (Sigma-Aldrich) for 5 min and cold 20% acetic acid in methanol for 10 min; sections were then rinsed in PBS. Riboprobes were diluted in warm (55°C) hybridization buffer (50% deionized formamide, 25% double-strength [2x] saline sodium citrate (SSC), 10% dextran sulfate, 5% 100x Denhardt solution, 1% herring sperm DNA, and 4% water) to a final concentration of 1 ng/µl, and 50 ng (50 µl) of riboprobe was applied to each cervix section. The sections were incubated in a humidified chamber at 55°C for 6 h. After incubation, the sections were washed at room temperature in 2x SSC, immersed in 2x SSC containing 10 µg/ml RNase A (AB Gene, Surrey, U.K.) for 30 min at 37°C, and then rinsed in further SSC washes at 55°C. The sections were placed in alkaline phosphatase buffer 1 (AP1; 100 mM Tris-HCl [pH 7.5], 100 mM sodium chloride, and 2 mM magnesium chloride) for 5 min and blocked in 3% BSA in AP1 for 30 min. Next, 100 µl of diluted anti-digoxigenin-AP antibody (Roche Diagnostics) was applied to each section and incubated at room temperature overnight. Sections were washed in AP1 (3x for 5min), and AP buffer 2 (AP2; 100 mM Tris-HCl [pH 9.5], 100 mM sodium chloride, and 10 mM magnesium chloride) for 5 min. Colorimetric detection of the antibody was performed with 500 µl of AP2 containing 1 drop of levamisole/5 ml AP2 (Vector Laboratories) and 4-nitro blue tetrazolium chloride (3.5 µl/ml AP2) and 5-bromo-4-chloro-3-indolyl-phosphate (3.5 µl/ml AP2) (both Roche Applied Science, Mannheim, Germany). The slides were covered in aluminum foil and left to incubate overnight for 16 h. Sections were rinsed in Tris-buffered saline, counterstained with nuclear fast red (Vector Laboratories), mounted, and stored at room temperature until analysis.

Quantification of in situ hybridization staining. The expression of mRNA staining for PTGS2 was assessed blind so that the reproductive status and region of the cervical tissues were unknown. The cervix was histologically divided into six cell layers on the basis of a classification by More [21]. The cell layers were composed of the luminal epithelium, the subepithelial stroma, the irregular smooth muscle layer, the longitudinal smooth muscle layer, the transverse smooth muscle layer, and the serosa.

The staining intensity and the proportion of positively stained cells were assessed at 100x magnification. The proportion of cells expressing PTGS2 mRNA was assessed subjectively and was defined as the percentage of the area of the cell layer that was stained positive to the nearest 5%. The intensity of staining was graded as 0, 1, 2, 3, or 4, as used previously [22]. An intensity score of 0 indicated that there was no expression, 1 indicated weak staining, 2 indicated moderate purple-lilac staining, 3 represented strong staining with a definite purple color, and 4 represented very strong dark purple staining. The percent expression and staining intensity scores were used to generate an index score by the following calculation: in situ hybridization index score = [% expression x intensity score]/100. An index score was calculated for each of the six cervical cells layers for each individual cervix section (n = 4 per ewe at each region of the cervix). A mean index score for the replicates (n = 4) was calculated that represented the expression of PTGS2 mRNA in each cell layer at each cervical region of the cervix for an individual ewe.

Masson Trichrome Staining for Smooth Muscle and Collagen

Masson trichrome staining protocol. Cervix sections at the uterine, mid-, and vaginal regions of the cervix were dewaxed in CNP30 (TAAB) and rehydrated through a series of graded alcohols. Sections were immersed in warm (58°C) Bouin solution (BDH) for 15 min, rinsed, stained with Weigert Hematoxylin (RA Lamb, East Sussex, U.K.) for 10 min, and then rinsed until only nuclei remained stained. Sections were then stained for smooth muscle with Biebrich Scarlet-Acid Fuschin (RA Lamb) for 3 min, rinsed, and immersed in phosphomolybdic acid for 45 min (BDH). Next, collagen was stained blue with Aniline Blue (RA Lamb) for 3 min, and the tissues were rinsed in distilled water for 2 min, immersed in 1% acetic acid for 2 min, and rinsed in distilled water for 2 x 2 min. Finally, the tissues were rehydrated through increasing concentrations of ethanol, left to air dry, and mounted with DePeX (BDH). To prevent variations in staining, Masson trichrome staining was performed in all cervix sections at the same time.

Quantification of Masson trichrome staining. The percentage of red (smooth muscle) and blue (collagen) staining in the sheep cervix was analyzed with a macro designed by Miss Helen Smith (The Royal Veterinary College, London) in the Leica Q Win Version 3 Quips Programming Software. The macro program thresholds the red staining by a color detection system and creates a binary image that is automatically measured, producing a percentage of the area value for smooth muscle. This is then repeated for the blue staining (collagen). One tissue section per cervical region per ewe was observed at 100x magnification, and the percentage of the area of red and blue staining within the image was calculated. A total of 10–20 readings were taken for each tissue section, covering the whole cervix section. To account for the unstained areas of the cervix section, such as the cervical lumen, all readings were standardized to determine the percentage of smooth muscle or collagen, relative to the total amount of staining, by the following equations. 1) Proportion of smooth muscle = [% red staining/(% red + % blue staining)] x 100. 2) Proportion of collagen = [% blue staining/(% red + % blue staining)] x 100. The mean proportion of smooth muscle and collagen for each cervix section was calculated from the multiple readings from each cervix section (10–20 per section).

Statistical Analysis

In situ hybridization and Masson trichrome data were analyzed in SPSS Version 13.0 Software by repeated-measures ANOVA, by means of a linear mixed model with post hoc tests by the least significant difference test where appropriate. Within the ANOVA, ewes were treated as a random factor.

RESULTS

Radioimmunoassay

Plasma estradiol, progesterone, LH, and FSH profiles were representative of the sheep estrous cycle. Concentrations of estradiol were low throughout the luteal phase and increased following the administration of PGF2{alpha}. Concentrations of estradiol were greatest 32–44 h after PGF2{alpha}, rising to 4.2–4.8 pg/ml. At the time of tissue collection, estradiol concentrations (picograms per milliliter; mean ± SEM) were low in luteal-phase ewes (1.8 ± 0.30), high in pre-LH ewes (4.3 ± 0.20), high in during-LH ewes (4.1 ± 1.77), and low in post-LH ewes (2.1 ± 1.63). Plasma progesterone concentrations were low throughout the follicular and early luteal phases and increased from Days 3 to 11 of the estrous cycle to concentrations greater than 1 ng/ml. Following the administration of PGF2{alpha} on Day 11 of the synchronized cycle, progesterone concentrations declined rapidly to the concentrations observed during the follicular phase. Plasma LH concentrations were low during the luteal phase and early follicular phase and increased 38–44 h after PGF2{alpha}. The LH surge lasted for 7–10 h. Plasma FSH concentrations were low throughout the luteal phase, increasing 40–44 h after the administration of PGF2{alpha} to maximum concentrations of 8.44 ± 3.42 ng/ml and declining within 8–10 h, resulting in an FSH surge that coincided with the LH surge.

PTGS2 mRNA Expression in the Sheep Cervix

The mRNA expression of PTGS2 as determined by the repeated-measures ANOVA (Fig. 1) differed with cervical region (P < 0.001) and cell layer (P < 0.001). However, the variations in the cervical region were not consistent at each cell layer (P < 0.001) or at each stage of the estrous cycle (P < 0.001).


Figure 76010501
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  		FIG. 1. PTGS2 mRNA expression in the sheep cervix prior to the LH surge (a–f) and during the luteal phase (g–l) at the uterine region (a, d, g, j), mid-region (b, e, h, k), and vaginal region (c, f, i, l). Sense (a–c, g–i) and antisense (d–f, j–l). Bar = 850 µm.

Stage of estrous cycle. There was significantly more PTGS2 mRNA expressed in the sheep cervix prior to the LH surge at a time of estradiol dominance than in the cervices at other stages of the estrous cycle. However, there was no effect of the stage of the estrous cycle at the mid-region (P = 0.50) and the vaginal region (P = 0.66) of the cervix where PTGS2 mRNA expression was relatively high at all stages of the cycle. At the uterine region, the mRNA expression of PTGS2 was significantly greater (P < 0.001) in cervices collected prior to the LH surge than in those collected during the luteal phase, during the LH surge, and after the LH surge (Fig. 2). The increase in PTGS2 mRNA expression in cervices prior to the LH surge was observed in the subepithelial stroma (P < 0.01), irregular smooth muscle layer (P < 0.001), longitudinal smooth muscle layer (P < 0.001), and transverse smooth muscle layer (P < 0.001) but not in the luminal epithelium or serosa.


Figure 76010502
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  		FIG. 2. The mean PTGS2 mRNA expression at different regions of the sheep cervix during the luteal phase (solid square: n = 5), prior to the LH surge (open square: n = 5), during the LH surge (solid triangle: n = 3), and after the LH surge (open triangle: n = 4). Significant differences are indicated in the text.

Cervical region. There was significantly more (P < 0.001) PTGS2 mRNA (mean ± SEM) expressed at the vaginal region (1.36 ± 0.277) and mid-region (1.30 ± 0.256) of the cervix than at the uterine region (0.63 ± 0.174). This distribution was observed in cervices collected during the luteal phase (P < 0.001), during the LH surge (P < 0.001), and after the LH surge (P < 0.001) but not prior to the LH surge (P = 0.20) (Fig. 2). The variation in PTGS2 mRNA expression between cervical regions was observed in all of the cell layers (P < 0.01), except for the serosa and luminal epithelium.

Cervical cell layer. The mRNA expression of PTGS2 differed between cervical cell layers (P < 0.001) (Fig. 3). At the uterine region, there was significantly more PTGS2 mRNA expression in the irregular smooth muscle layer than in all other cell layers, except for the subepithelial stroma (P < 0.05), which itself had greater PTGS2 mRNA expression than all cell layers except for the serosa (P < 0.05). At the mid-region of the cervix, the irregular smooth muscle layer expressed significantly more PTGS2 mRNA than any of the other cell layers (P < 0.001). The expression of PTGS2 mRNA in the longitudinal smooth muscle layer was greater than in the transverse smooth muscle layer, serosa, and luminal epithelium (P < 0.01). The luminal epithelium had the least PTGS2 mRNA expression of all the cell layers (P < 0.05). At the vaginal region of the cervix, the irregular smooth muscle layer, subepithelial stroma, and longitudinal smooth muscle layer expressed significantly more PTGS2 mRNA than the transverse smooth muscle layer (P < 0.05), serosa (P < 0.001), and luminal epithelium (P < 0.001). The luminal epithelium expressed less PTGS2 mRNA than any other cell layers (P < 0.05). The pattern of expression between the cervical cell layers at each region was similar at all stages of the estrous cycle.


Figure 76010503
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  		FIG. 3. The mean PTGS2 mRNA expression in each cell layer of the sheep cervix at the uterine region (solid square: n = 17), mid-region (open square: n = 17), and vaginal region (solid triangle: n = 17). Significant differences are indicated in the text.

The Proportion of Smooth Muscle and Collagen in the Sheep Cervix

The proportion of smooth muscle and collagen in the cervix differed with the stage of the estrous cycle (P = 0.001) and cervical region (P < 0.001) (Fig. 4). There was a significant interaction between the stage of cycle and cervical region (P = 0.003). The staining method used determined the relative proportion of smooth muscle and collagen in the sheep cervix; therefore, significant changes in collagen were also observed for smooth muscle but in the opposite direction.


Figure 76010504
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  		FIG. 4. Masson trichrome staining for smooth muscle (red) and collagen (blue) in the sheep cervix prior to the LH surge (a–c) and during the luteal phase (d–f) at the uterine region (a, d) mid-region (b, e), and vaginal region (c, f). Bar = 2100 µm.

Stage of estrous cycle. The proportion of smooth muscle and collagen in the sheep cervix varied at different stages of the estrous cycle (P = 0.001). Prior to the LH surge, the cervices had a higher proportion of collagen, and consequently, a lower proportion of smooth muscle than did those during the luteal phase, during the LH surge, and after the LH surge (Table 1). However, this difference was significant only at the uterine (P < 0.001) and mid- (P < 0.05) regions of the cervix (Fig. 4). At the vaginal region of the cervix, changes in smooth muscle and collagen with the stage of the estrous cycle approached significance (P = 0.059). Post hoc tests showed that at the vaginal region prior to the LH surge, the proportion of collagen in the sheep cervix was greater, and the smooth muscle lower, than in the cervices during the LH surge (P = 0.025) and after the LH surge (P = 0.021) but not during the luteal phase (Table 1).


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  		TABLE 1. The mean ± SEM proportion (%) of collagen at each region of the sheep cervix during the estrous cycle.

Cervical region. The proportion of smooth muscle and collagen differed between cervical regions (P < 0.001). The vaginal region of the cervix had a greater proportion of collagen, and therefore less smooth muscle, than the mid- and uterine regions (P < 0.001), although this was not true at all stages of the estrous cycle (Fig. 4). During the LH surge (P < 0.05) and during the luteal phase (P ≤ 0.001), there was a greater proportion of collagen at the vaginal region than at the mid- and uterine regions of the cervix, whereas after the LH surge, differences were observed only between the vaginal and uterine regions (P = 0.012). Conversely, prior to the LH surge, there was a lower proportion of collagen, and therefore more smooth muscle, at the mid-region of the cervix than at the uterine (P = 0.021) and vaginal (P = 0.002) regions, but no difference was observed between the uterine and vaginal regions of the sheep cervix.

DISCUSSION

PGE2 has been implicated in the cervical dilation of the ewe during pregnancy [11, 23]. However, to our knowledge, no relationship between cervical relaxation in the ewe at estrus and PGE2 has been investigated. The present study examined the changes in PTGS2 mRNA, collagen, and smooth muscle in the sheep cervix during the estrous cycle.

PTGS2 mRNA was expressed and regulated in the sheep cervix during the estrous cycle. To our knowledge, this is the first reported finding of PTGS2 mRNA in the cervix of the nonpregnant ruminant and the first evidence to suggest that cervical PTGS2 mRNA expression is regulated during the estrous cycle. Prior to the LH surge, when E2 concentrations were greatest, cervical PTGS2 mRNA expression was also greatest. High PTGS2 mRNA expression at the time of estrus is consistent with the findings for PTGS2 protein expression in the rat uterus [24], indicating that PTGS2 is increased during a period of estrogen dominance. Estrogen stimulates oxytocin receptor mRNA expression in the cervix of the ovariectomized rat [9], and the estrogen receptor binds to stimulatory protein 1 sites on the sheep oxytocin receptor gene to regulate transcription [8], suggesting that estradiol regulates cervical oxytocin receptor expression in the sheep. Oxytocin receptor mRNA expression in the sheep cervix is greatest during estrus [25], when estradiol concentrations are greatest, and oxytocin stimulates the induction of PTGS2 in cervical segments from proestrus and estrus cows when oxytocin receptor expression is high [10]. We propose that an estradiol-oxytocin-mediated mechanism of PTGS2 regulation occurs in the sheep cervix at estrus in which the increase in estradiol at estrus stimulates oxytocin receptor expression, enabling pituitary oxytocin to bind to its receptor and stimulate PTGS2 expression.

PTGS2 protein expression appears to be regulated at the level of transcription [26, 27] and at a posttranscriptional level via the stability of its mRNA; therefore, the increase in PTGS2 mRNA expression in the sheep cervix prior to the LH surge is likely to increase PTGS2 protein expression. As PTGS2 is the rate-limiting step in prostaglandin synthesis, it can be assumed that increased cervical PTGS2 mRNA will stimulate the production of prostaglandins in the cervix. Oxytocin selectively stimulates PGE2, but not PGF2{alpha} , production in bovine cervical segments via PTGS2 [10], suggesting that the increase in cervical PTGS2 mRNA at the time of estrus will stimulate PGE2 synthesis. PGE2 induces cervical softening in the cervix of late pregnant sheep [11]; therefore, the increase in PTGS2 mRNA expression is likely to induce cervical relaxation at estrus.

The mRNA expression of PTGS2 was greatest in the smooth muscle layers of the cervix and also the subepithelial stroma, suggesting that smooth muscle cells and fibroblasts express PTGS2 mRNA. PTGS2 protein expression has previously been localized to smooth muscle cells in rat cervix [24], and fibroblast cells in human cervical tissue express PTGS2 mRNA [28, 29]. The expression of PTGS2 mRNA in the smooth muscle cell layers as well as in the subepithelial stroma, but not in the luminal epithelium, suggests that during the estrous cycle, the synthesis of PGE2 is primarily associated with cervical relaxation through the relaxation of smooth muscle and changes within the extracellular matrix rather than altering cervical secretions, as may be the case when expressed in epithelium.

The present study determined that there is a gradient of PTGS2 mRNA expression throughout the sheep cervix at all stages of the estrous cycle, except prior to the LH surge. An increase in PTGS2 mRNA expression from the uterine region to the vaginal region of the cervix has been reported previously in the cervix of the pregnant baboon [30], although not in the cervix of nonpregnant animals. The difference in PTGS2 mRNA expression between the regions of the sheep cervix may be caused by cell type and cell density gradients between cervical regions. The proportion of smooth muscle differs throughout the human cervix [31], and differences in progesterone and estrogen receptor expression between cervical regions in the cervix of the nonpregnant cow disappear when cell density is taken into consideration [32].

The rearrangement, degradation, and dissociation of collagen fibers and bundles are associated with cervical relaxation [15, 33, 34]. Therefore, we determined the relative proportions of collagen and smooth muscle in the sheep cervix during the estrous cycle. The increase in the proportion of collagen at the uterine region of the cervix prior to the LH surge in comparison to the other stages of the cycle is associated with high extradiol concentrations and a greater expression of PTGS2 mRNA at this region, suggesting a role for ovarian steroids and PGE2 in the remodeling of collagen fibers and bundles. Estradiol stimulates the separation of collagen bundles in the cervix of pregnant sheep [15], and we suggest that this is mediated by PGE2, because at estrus, when estradiol concentrations were highest, so was cervical PTGS2 mRNA expression. PGE2 decreases the tensile strength of, and increases the interfibrillary distance between, collagen fibrils (which make up collagen fibers) in the cervix of the pregnant rat but does not decrease the fibril length, which suggests that PGE2 separates collagen fibers in the cervix [13]. It is therefore likely that the increase in the proportion of collagen in the sheep cervix prior to the LH surge is caused by the separation of collagen bundles and fibers, which causes a reduction in cervical tensile strength and thereby causes cervical relaxation. The differences observed in the proportion of smooth muscle throughout the cervix have been described previously as declining from 29% of the total cervical stroma at the uterine region to 18% and 6% in the mid- and vaginal regions, respectively [31]. As the technique used in this study determines the relative area of smooth muscle to collagen, a decrease in smooth muscle area is associated with an increase in collagen area, which may explain the increase in the proportion of collagen at the vaginal region of the cervix. The pattern in the proportion of, and the area occupied by, collagen is strongly associated with the pattern of PTGS2 mRNA expression in the sheep cervix during the estrous cycle, suggesting that PTGS2 increases PGE2 synthesis and separates collagen fibers in the sheep cervix.

In conclusion, we propose the following model of cervical relaxation in the ewe at estrus. Prior to the LH surge, estradiol increases PTGS2 mRNA expression in smooth muscle and fibroblast cells, most likely through oxytocin and its receptor. This is likely to stimulate PGE2 synthesis, and PGE2 acts on the cervical extracellular matrix, causing collagen fibers and bundles to separate and disperse throughout the cervix occupying a larger surface area. The separation of collagen may reduce the tensile strength of the cervix, culminating in cervical relaxation.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Andy Pitsillides, Dr. Gabriele Wax, Miss Helen Hunt, Mrs. Lynda Miles, and Mrs. Tanya Hopcroft for technical assistance and Ms. Aviva Petrie for statistical advice.

Correspondence: 1 Muhammad Khalid, Department of Veterinary Clinical Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, U.K. FAX: 44 1707 652090; e-mail: mkhalid{at}rvc.ac.uk

Received: 24 May 2006.

First decision: 6 June 2006.

Accepted: 12 September 2006.

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