Ovine Cervix Research Articles

Lipidomic profiling of cervical mucus reveals the potential role of pro-inflammatory derived metabolites on sperm transport across the ovine cervix

Internationally, cervical artificial insemination (AI) in sheep yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway where vaginal AI of frozen-thawed semen to a natural oestrus yields non-return rates in excess of 60%, which has been attributed to the ewe breed used in Norway. This study used both metabolomics and an RNA-sequencing approach to assess the lipid production and composition from cervical mucus and tissue of four European ewe breeds (n = 28–30 ewes per breed) with previously reported differences in pregnancy rates following cervical AI with frozen-thawed semen. These breeds included Suffolk (exhibiting low fertility), Belclare (medium fertility) as well as Norwegian White Sheep and Fur (both with high fertility and pregnancy rates > 60%) at both a synchronised and natural oestrous cycle. The aim was to explore the differences between ewe breeds in the lipidomic profile and to identify candidate biomarkers associated with an optimal environment for cervical sperm transport. The results revealed the identification of 255 lipids, of which 170, 102 and 83 were different between ewe breeds, types of cycle and affected by their interaction, respectively (P < 0.05). Reduced levels of lipids involved in the resolution of inflammation (i.e. 14-HDoHE,17-HDoHE, 15-HETE) were identified in the low-fertility Suffolk breed compared to high-fertility ewe breeds. However, there was an up-regulation of the COX pathway accompanied by increased levels of prostaglandins in the Suffolk breed. These findings indicated a sub-optimal and pro-inflammatory environment that could have a negative effect on cervical sperm transport.

The biological mechanisms regulating sperm selection by the ovine cervix.

In species where semen is deposited in the vagina, the cervix has the unique function of facilitating progress of spermatozoa towards the site of fertilisation while also preventing the ascending influx of pathogens from the vagina. For the majority of species, advances in assisted reproduction techniques facilitate the bypassing of the cervix and therefore its effect on the transit of processed spermatozoa has been largely overlooked. The exception is in sheep, as it is currently not possible to traverse the ovine cervix with an inseminating catheter due to its complex anatomy, and semen must be deposited at the external cervical os. This results in unacceptably low pregnancy rates when frozen-thawed or liquid stored (>24 h) semen is inseminated. The objective of this review is to discuss the biological mechanisms which regulate cervical sperm selection. We assess the effects of endogenous and exogenous hormones on cervical mucus composition and discuss how increased mucus production and flow during oestrus stimulates sperm rheotaxis along the crypts and folds of the cervix. Emerging results shedding light on the sperm-cervical mucus interaction as well as the dialogue between spermatozoa and the innate immune system are outlined. Finally, ewe breed differences in cervical function and the impact of semen processing on the success of fertilisation, as well as the most fruitful avenues of further investigation in this area are proposed.

Collagen, glycosaminoglycans and matrix metalloproteinase-2 and metalloproteinase-9 in the cervix of the ewe during prepubertal development.

The tortuous nature of the ovine cervix restricts the transcervical passage of the cannula, and many studies have aimed to understand the endocrine mechanism of the remodelling of cervical tissue in adult ewe. However, little is known about the remodelling of the cervical tissue during the prepubertal development of the lambs. To obtain histochemical and biochemical evidence about the nature of the prepubertal development of the cervix of the ewe, cervices of Corriedale lambs obtained at 0, 1, 2, 4, 6 and 8months of age (n=5 to 6 in each) were processed. Neutral and acidic glycosaminoglycans (by PAS-Alcian stain) were weakly in the cervical stroma and not shown change during the development, whereas the percentage volume of fibrillar collagen (by van Gieson stain) increases throughout the experimental period in the superficial fold stroma and deep wall stroma (p<0.05). The relative cervical weight (g/kg of body weight) and the collagen concentration (by spectrophotometry, mg/mg wet tissue) showed an early decreasing phase from months 0 to 4 and a later increasing phase from months 4 to 8 (p<0.05). The latent form of matrix metalloproteinase-2 (MMP-2) detected by gelatin zymography (ng/mg protein) decreased from months 0 to 2 and increased from months 4 to 8, whereas the activated form decreased from months 0 to 2, remained low until month 6 and then recovered on month 8 (p<0.0001). Data suggest that the relative cervical weight biphasic pattern during the development is related to MMP-2-dependent changes in the collagen content.

Changes to sperm surface carbohydrates following exposure to seminal plasma and freezing

Epididymal, ejaculated and frozen–thawed spermatozoa are exceptionally different sperm types. When placed directly into the uterus all sperm types display high fertility. However, when forced to traverse the ovine cervix (following vaginal or cervical AI), epididymal and frozen–thawed ram spermatozoa display dramatically lower fertility than fresh ejaculated spermatozoa. We suggest that changes to the outer carbohydrate-rich glycoprotein coat of spermatozoa represent a potential reason for altered cervical transit ability, as this is the primary interface with the female environment. As such, we used lectin binding to investigate modifications of the carbohydrate portion of sperm glycoconjugates in response to seminal plasma exposure and freezing. Fresh and frozen ejaculated spermatozoa and fresh epididymal spermatozoa were subjected to a swim up to remove loosely associated glycoproteins and remnants of freezing diluent. Spermatozoa were stained with fluorescein isothiocyanate (FITC) conjugated lectins (LPA, PNA, WGA and ConA), specific for the sugars of interest (sialic acid, galactose, N-acetylglucosamine andmannose, respectively) and counter-stained with propidium iodide (PI). Median FITC fluorescence of the PI negative (live) population was compared amongst treatments using a restricted maximum likelihood approach. The results provide strong evidence that both exposure to seminal plasma and freezing are processes which alter the outermost surface of spermatozoa. Seminal plasma exposure significantly increased N-acetylglucosamine (WGA; p<0.001) on the sperm membrane. Conversely, freezing led to a significant decrease in available galactose (PNA; p<0.01) and N-acetylglucosamine (WGA; p<0.001) as well as increased availability of mannose (ConA; p<0.001), typically a carbohydrate located proximal to the membrane in glycoconjugates. The observed changes lend support to the theory that while exposure to seminal plasmamay orchestrate meaningful alteration of the sperm surface, the freezing process further modifies this important region, possibly to its detriment. This is the first evidence that industry standard freezing of ram semen leads to large and significant changes in selected sperm surface carbohydrates. Further research is required to fully elucidate the consequenceof these changesandestablishpossiblemeans to prevent or repair the damage to the glycoprotein coat of ram spermatozoa. Research support by AWI and NSWSMBA Trust.

Role of NO–cGMP pathway in ovine cervical relaxation induced by Erythroxylum caatingae Plowman

Erythroxylum caatingae Plowman has a myorelaxing effect on smooth muscle tissue. We investigated the effect of the crude ethanolic extract of E. caatingae Plowman (Ec-EtOH) on the contractility of the ovine cervix. In an isometric system, circular strips were subjected to 90mM potassium (K+) or 30μM carbamylcholine (CCh)-induced contraction. We then exposed the tissue to cumulative concentrations of Ec-EtOH (1–729μg/ml). In other bath solutions, the tissues were exposed to l-NG-nitroarginine methyl ester (l-NAME; 100μM), l-NAME (100μM)+l-arginine (300μM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ; 5μM), 4-aminopyridine (4-AP; 3mM), tetraethylammonium (TEA; 0.3mM), glybenclamide (1μM), atosiban (10μM) or verapamil (3μM), followed by the addition of Ec-EtOH (1–729μg/ml). We also evaluated the effect of cervical Ec-EtOH infusion (2mg) on cervical contractility in vivo. Ec-EtOH decreased cervical contractility induced by K+ or CCh, and 729μg/ml Ec-EtOH decreased 85.4±5.1% the amplitude of basal contractility in vitro, with an EC50 of 17.9±3.7μg/ml. This effect of Ec-EtOH was prevented by l-NAME or ODQ. l-arginine impaired the blunting effect of l-NAME on cervical relaxation caused by Ec-EtOH. However, the potassium channel blockers 4-AP, TEA, and glybenclamide did not modify this myorelaxation triggered by Ec-EtOH. Ec-EtOH also decreased acetylcholine-induced contractions in tissue preincubated with verapamil. In addition, Ec-EtOH decreased ovine cervical contractions in vivo. Thus, Ec-EtOH had a relaxant effect on ovine cervical contractions. This may involve the nitric oxide signal, mediated by cGMP cellular transduction, and be related to intracellular calcium sequestration.

Assessing the usefulness of prostaglandin E2 (Cervidil) for transcervical artificial insemination in ewes

The underlying theme of this study involved the evaluation of the dilatory effects of prostaglandin E2 on the ovine cervix and thus the assessment of its potential applicability to transcervical artificial insemination (TCAI) in ewes. A novel method of prostaglandin E2 administration (controlled slow-release vaginal inserts) was examined, and the practical implications of this approach including cervical penetrability and posttreatment pregnancy rates were evaluated. The Guelph method of TCAI was performed during the seasonal anestrus (n = 40) and the breeding season (n = 40) on multiparous Rideau Arcott × Polled Dorset ewes, with or without the pretreatment with Cervidil (for a duration of 12 hours or 24 hours before TCAI). Cervical penetration rates averaged 82.5% (66 of 80), and they varied neither (P > 0.05) between the two seasons nor between Cervidil-treated ewes and their respective controls. Cervidil priming significantly reduced the total time required for TCAI during the breeding season in comparison with controls (54 vs. 98 seconds), especially after the 24-hour exposure (38 vs. 108 seconds). The time taken to traverse the uterine cervix was negatively correlated (P < 0.05) with the breed (percentage of Rideau Arcott genotype) and lifetime lamb production in seasonally anestrous ewes. Four out of 36 (11%) successfully penetrated ewes in the breeding season (three ewes allocated to the 12-hour control group and one ewe that had received Cervidil for 12 hours) became pregnant and carried the lambs to term. Vaginal mucus impedance at TCAI was significantly and positively correlated with the total time required to complete the procedure in cyclic ewes, and the negative correlation between vaginal mucus impedance and total time values at the time of controlled intravaginal drug release device removal approached to significance in anestrous ewes. The present results indicate a moderate benefit of using Cervidil for inducing cervical dilation before TCAI in ewes, mainly in the breeding season. The specific reason(s) for impaired fertility after the TCAI using frozen–thawed ram semen remains to be elucidated.

Morfologia da cérvice de ovelhas Santa Inês adultas nas fases luteínica e folicular

A cérvice ovina tem formato irregular, tornando um desafio transpassá-la durante a inseminação artificial. Logo, este trabalho teve por objetivo descrever as características morfológicas da cérvice de ovelhas da raça Santa Inês, comparando-se as fases do ciclo estral. Foram coletados 112 úteros de ovelhas adultas não gestantes e silicone foi injetado no lúmen da cérvice obtendo-se um molde do canal cervical. O comprimento médio foi de 41,33± 16,38 mm e o tipo de óstio cervical mais frequente foi o bico-de-pato (46%). O número mínimo de anéis encontrados nas cérvices foi de dois e o máximo de sete, com média de 4,70 ± 1,05 anéis. A morfometria de cada anel cervical indica que a circunferência interna se afunila na porção média e em seguida torna a aumentar. Não houve diferença significativa (p&gt;0,05) quando se compararam as fases luteínica e folicular e entre os tipos de cérvice quanto ao diâmetro das dobras, altura das dobras, circunferência interna, ponto médio entre as dobras e distância do orifício para as dobras. Nas condições estudadas, concluiu-se que o desenvolvimento de um aplicador de sêmen deve levar em consideração os limites morfométricos encontrados, embora a morfologia e morfometria da cérvice da espécie ovina seja bastante variada, não havendo uma forma padrão para a espécie, inclusive quando são comparadas as fases luteínica e folicular.

The expression of ERα, OTR, cPLA2, COX-2, and PPARγ in the cervix of the ewe during the estrous cycle

The ovine cervix relaxes at estrus allowing easier entry of spermatozoa into the uterus. The mechanism responsible for this relaxation is not fully elucidated and we hypothesized that cervical relaxation at estrus is induced by ovarian and pituitary hormones stimulating the local production of prostaglandin E2 via a biosynthetic pathway involving a number of mediators including oxytocin, phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPARγ). The aim of this study was to investigate the cervical expression of estradiol receptor alpha (ERα), oxytocin receptor (OTR), cPLA2, COX-2, and PPARγ at three stages of the estrous cycle (the luteal phase and two times during the follicular phase, just before and just after the LH surge). An experiment was conducted during the breeding season, in 25 ewes to test this hypothesis. Samples of cervical tissue were collected from groups of ewes at three stages of the estrous cycle: the luteal (N = 8), “pre-LH surge” (N = 8), and “post-LH surge” (N = 9) stages. Cervical tissue from uterine, mid, and vaginal regions of the cervix were analyzed by Western immunoblot analysis for ERα, OTR, cPLA2, COX-2, and PPARγ. The results showed that the levels of all five proteins were lowest during the luteal phase of the estrous cycle in all regions of the cervix. The levels of all except cPLA2, increased significantly during the “pre-LH surge” stage. The levels of cPLA2 and ERα increased in the “post-LH surge” stage and those for OTR and PPARγ were unchanged and those for COX-2 were lower. These data show that the cervical levels of all five of the intermediates in the synthesis of prostaglandin E2 that were examined in this study were higher in the “pre-” and “post-LH surge” stages compared with the luteal phase of the estrous cycle and these findings are consistent with our hypothesis.

Relaxant effect of the essential oil of Croton nepetifolius on ovine cervix

We investigated the in vitro effect of the essential oil of Croton nepetifolius Baill., Euphorbiaceae (EOCN), on spontaneous or induced contractions of circular and longitudinal muscles from the ovine cervix during the luteal phase of the estrous cycle. The relaxant effect of EOCN was expressed as a percentage of the contraction recorded before the addition of the oil and calculated relative to the preparations exposed only to the vehicle. The IC50 (concentration of oil required to produce a 50% maximal reduction in muscle contraction) for relaxation of spontaneous contractions in circular and longitudinal muscles was significantly lower than the IC50 for blockade of K+-induced contraction (27.19 µg mL-1 versus 262.72 µg mL-1 and 40.92 µg mL-1 versus 222.47 µg mL-1, respectively). Interestingly, there was a high degree of selectivity in the action of EOCN on cervix layers concerning the inhibition of acetylcholine-induced contraction in circular (IC50 277.10 µg mL-1) and longitudinal (IC50 52.56 µg mL-1) muscles. In conclusion, EOCN is able to relax ovine cervix during the luteal phase. This work opens the perspective of applying EOCN in ovine embryo transfer.

Cervical expression of hyaluronan synthases varies with the stage of the estrous cycle in the ewe

The natural cervical relaxation which occurs at estrus in the ewe may be initiated by binding of hyaluronan (HA) to its receptor CD44. Indeed, we have previously shown that HA content and fragment size in the ovine cervix varies with the stage of the estrous cycle. Despite the importance of cervical relaxation in promoting sperm transport and facilitating the possible development of transcervical artificial insemination (AI), the mechanisms coordinating these changes in HA content remain to be defined. Hyaluronan synthases (HAS) 1, 2, and 3 regulate HA biosynthesis and herein, we describe the changing pattern of HAS isoform expression during the estrous cycle to determine whether this may underpin HA-mediated changes in relaxation of the ovine cervix. Accordingly, cervices were collected from 24 cyclic sheep (n = 8 / group) at the luteal, pre-luteinizing hormone (LH) and post-LH surge stages. Protein and mRNA expression for HAS 1, 2 and 3 was determined in five different tissue layers (epithelium, subepithelial stroma, and longitudinal, circular and transverse muscle) of the vaginal, mid and uterine regions of each cervix by immunohistochemistry and in situ hybridization, respectively. HA synthases were expressed in all the tissue layers and regions of the cervix, and the pattern of expression was similar for mRNA and protein. HAS1 protein and mRNA expression was significantly (P ≤ 0.05) higher at the pre-LH surge stage, while HAS 2 and 3 protein and mRNA expression was significantly (P ≤ 0.001) higher at the luteal stage. Overall, both HAS protein and mRNA expression was significantly (P ≤ 0.001) higher in the epithelial layer and the vaginal region. These findings are in accordance with our previous results and explain the differences observed in the HA content and differing HA fragment size at different stages of the estrous cycle.

A Review of Advances in Artificial Insemination (AI) and Embryo Transfer (ET) in Sheep, with the Special Reference to Hormonal Induction of Cervical Dilation and its Implications for Controlled Animal Reproduction and Surgical Techniques

Artificial insemination (AI) and embryo transfer (ET) play an important role in the genetic improvement of sheep herds. This review primarily describes the methods that have recently been researched and/or adapted to circumvent the problematic anatomy of the ovine cervix, which prevents the widespread application of transcervical AI and ET in this species. A mechanism of cervical ripening at parturition and the endocrine influences on cervical tone are described as a backdrop to understanding the action of various pharmacological agents used to induce cervical dilation. Although several catheters have been designed to penetrate the ovine cervix, and a specific protocol has been developed for TCAI in sheep (Guelph Method), penetration rates and resulting pregnancy/lambing rates are still inconsistent and unpredictable. Hormonal dilation of the cervix may significantly improve the ease with which the ovine cervix can be traversed. Treatment with Cervidil ® , a dinoprostone-containing vaginal insert with a slow release mechanism, currently used to induce labor in women, could significantly improve cervical penetration in ewes without adverse effects on uterine contractility. This mode of drug delivery holds promise to result in the development of a safe method to induce cervical dilation for a broad range of transcervical reproductive manipulations in mammalian species including, but not limited to, AI, ET, embryo and oocyte recovery, treatment of ringwomb and transcervical surgical procedures. However, more studies are needed to evaluate the effects of hormonal methods on cervical morphology/pathomorphology, sperm/embryo transport and viability, and fertility in both cyclic and seasonally anovular ewes.

Traversing the ovine cervix – a challenge for cryopreserved semen and creative science

This review brings together research findings on cervical relaxation in the ewe and its pharmacological stimulation for enhancement of the penetration needed for transcervical insemination and embryo transfer. On the basis that the success of artificial insemination is the percentage of ewes lambing, a review is made of recent research aimed at understanding and minimising the sub-lethal effects of freezing and thawing on the viability of spermatozoa, their membrane integrity and their ability to migrate through cervical mucus, as these characteristics have a major influence on fertility, particularly when semen is deposited, artificially, in the os cervix. Milestones of achievement are given for transcervical intrauterine insemination, embryo recovery and transfer and the birth of lambs of pre-determined sex, firstly following intracytoplasmic sperm injection, then laparoscopic intrauterine insemination using highly diluted flow-cytometrically sorted fresh semen and subsequently by os cervix insemination using sexed semen that had been frozen and thawed. Diversity of research endeavour (applied, cellular, molecular), research discipline (anatomy, histology, immunology, endocrinology) and research focus (cell, tissue, organ, whole animal) is embraced within the review as each has significant contributions to make in advancing recent scientific findings from the laboratory into robust on-farm transcervical insemination and embryo transfer techniques.

Intracervical application of hyaluronan improves cervical relaxation in the ewe

Artificial insemination (AI) using frozen semen is a key method to enable rapid genetic improvement but its use in the sheep industry is currently limited by poor fertility. Laparoscropic AI is most effective but has not gained popularity due to cost and welfare considerations. Transcervical intrauterine AI (TCAI) may offer a practical alternative but the complex anatomy of the ovine cervix limits adequate penetration of the inseminating pipette. Hyaluronan (HA) is a glycoaminoglycan whose content in the cervix increases at oestrus and which may contribute to the degree of natural relaxation observed at this time. This study investigated the effect of intracervical application of HA on the depth of cervical penetration in sheep. Oestrus was synchronised on three occasions in 48 Welsh mountain ewes with progesterone sponges and PMSG. Each animal initially served as its own untreated control. Ewes were subsequently treated intracervically with 25 mg of: (2i) low molecular weight (MW) HA; (2ii) high MW HA or (2iii) both low and high MW HA (n = 16/group) at 52 h after sponge removal or with low MW HA at: (3i) 50 h; (3ii) 52 h or (3iii) both 50 h and 52 h. Depth of cervical penetration measured at 54 h was increased from 1.22 cm to 3.66 cm by treatment with low MW HA (P ≤ 0.001), with no differences between the number of treatments (1 or 2) or the time at which the HA was administered (50 or 52 h). High MW HA alone or together with low MW HA had no effect. In conclusion, intracervical application of low MW HA 52 h after sponge removal increases cervical penetration up to 3.4cm to allow TCAI in sheep.

Hyaluronan (HA) content, the ratio of HA fragments and the expression of CD44 in the ovine cervix vary with the stage of the oestrous cycle

The complex anatomy of the ovine cervix limits the success of trans-cervical artificial insemination in sheep. However, there is a degree of natural relaxation of cervix at oestrus that is accompanied by an increase in the water content. As hyaluronan (HA) has a high affinity for water molecules, in this study, we tested the hypothesis that the HA content of the cervix, the proportion of different size fragments of HA and expression of its receptor CD44 vary with the stage of the oestrous cycle. Oestrous was synchronized in 25 Welsh mountain ewes, and their cervices were collected either during luteal phase (n=8) or pre-LH (n=8) or post-LH (n=9) surge stage of the oestrous cycle. The pre-LH surge group had the highest HA content (2.96 ng/mg of cervical tissue), which was significantly (P<or=0.05) higher than that observed for the post-LH surge (2.04 ng/mg) group. The luteal phase group had a mean HA content intermediate between the pre- and post-LH surge groups, and was significantly different from either. The frequency of cervical samples containing both sizes of HA fragments (small and large) was significantly higher (P<or=0.05) in the pre-LH surge group compared with the luteal and the post-LH surge groups, whereas that in post-LH surge group was significantly (P<or=0.05) higher than that in the luteal group. The number of cervical samples that contained only small HA fragments was significantly (P<or=0.05) higher in the luteal group compared with both the pre- and post-LH surge groups, whereas the number of samples containing only large HA fragments was significantly (P<or=0.05) higher in the post-LH surge group compared with the luteal or pre-LH surge groups. Overall mean expression of CD44 in the vaginal and mid regions was significantly (P<or =0.001) higher than that in the uterine region, with no difference between the vaginal and mid regions of the cervix. Pattern of CD44 expression depended on the stage of the oestrous cycle. At the luteal stage, CD44 expression did not vary among epithelial, sub-epithelial, circular and longitudinal smooth muscle layers, whereas at the pre- and post-LH surge stages, the expression in the epithelial layer was significantly (P<or=0.001) higher than that in the other three layers. In general, CD44 expression in the transverse smooth muscle layer was significantly (P<or=0.05) lower than the expression in all the other layers at all the stages of the oestrous cycle. The results indicated that the HA varied with the steroid status. Higher HA values at a time when cervical relaxation is naturally higher may indicate its involvement in remodelling of the cervix at oestrus.

Intra-cervical application of Misoprostol at estrus alters the content of cervical hyaluronan and the mRNA expression of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and cyclooxygenase-2 in the ewe

The complex anatomy the of ovine cervix limits the success of transcervical artificial insemination in sheep, but Misoprostol (a PGE1 analogue) relaxes the cervix and facilitates transcervical artificial insemination. However, the mechanism by which Misoprostol causes cervical relaxation is not known. This study examined if intra-cervical Misoprostol altered the hyaluronan content and the mRNA expression of COX-2, LHR, or FSHR in the cervix of the estrus ewe. Estrus was synchronized in cyclic ewes with progestagen pessaries and 48h after sponge removal ewes were treated intra-cervically with 0 (controls), 200, or 400μg Misoprostol. Hyaluronan content was determined by ELISA and mRNA expression of LHR, FSHR, and COX-2 was analyzed by in situ hybridization using digoxigenin-11-uridine-5′-triphosphate labeled riboprobes. The hyaluronan content of the cervix was significantly higher in sheep that received 200 (P<0.05) or 400 (P<0.05) μg Misoprostol compared to controls. Moreover, it was significantly (P<0.05) higher in the vaginal region compared to mid and uterine regions. Misoprostol increased (P<0.05) the mRNA expression of LHR and COX-2 but not FSHR. The expression for all three genes was highest in the vaginal region and lowest in uterine region. The luminal epithelium and circular smooth muscle layers had higher (P<0.05) expression for LHR, FSHR, and COX-2 mRNAs, and the sub-epithelial stroma had the lowest (P<0.05). We propose that the intra-cervical application of Misoprostol induces the mRNA expression of LHR, FSHR, and COX-2 through a positive feedback loop. The data suggest that softening of the cervix by Misoprostol is caused by an increase in the hyaluronan content of the cervix.

Oestrogen and progesterone receptor binding capacity and oestrogen receptor alpha expression (ERα mRNA) along the cervix of cycling ewes

The aim of the present work was to study the oestrogen receptor (ER) and progesterone receptor (PR) binding capacity and the oestrogen receptor alpha (ERalpha) mRNA concentration in cranial and caudal cervix during the ovine oestrous cycle. Cervical samples of synchronised Corriedale ewes were obtained on Day 1 (n=7), 6 (n=6) or 13 (n=7) after oestrus detection (Day 0). The ER and PR binding capacity by ligand-binding assay and the ERalpha mRNA concentration by solution hybridisation in both cranial and caudal zones of the cervix were determined. The ER and PR binding capacity were higher (P<0.005) on Day 1 than on Days 6 and 13 in both cranial and caudal zones. The ERalpha mRNA concentrations were higher (P<0.0001) on Day 1 than on Days 6 and 13 only in the caudal zone. The PR binding capacity and ERalpha mRNA concentration were higher (P<0.005) in the caudal than in the cranial zone on Day 1. The ER and PR expression in the ovine cervix varied during the oestrous cycle in agreement with the known upregulation exerted by oestrogen and downregulation exerted by progesterone. Differences in ER and PR expression along the longitudinal axis of the ovine cervix were found, reflecting histological and functional differences between the cranial and caudal zones.

Localization and steroid regulation of prostaglandin E2 receptor protein expression in ovine cervix

Although prostaglandin E2 (PGE2) has been identified as a central mediator of the cervical ripening process, the mechanisms responsible for PGE2 ripening are still poorly understood, partly because of the lack of information concerning the precise cellular localization and regulation of PGE2 (EP) receptors in the cervix. To provide new insights into the mechanisms of cervical ripening, we used indirect immunofluorescence to localize cervical EP receptor protein expression in ovariectomized ewes and examined the effect of administration of progesterone or estradiol. EP receptors were widely distributed in cervical blood vessels, epithelium of the cervical canal, circular and longitudinal muscles, and stroma. Estradiol replacement decreased EP1 and EP3 receptor protein in blood vessel media (by 23 and 31% respectively, P < 0.05) and decreased EP1 receptor protein expression in the longitudinal muscle layer (by 27%, P < 0.05). Stromal EP1 and EP3 receptor protein expression was also reduced by estradiol (by 29 and 20% respectively, P < 0.05). Progesterone replacement had no significant effect on EP receptor protein expression. The arterial changes would favor PGE2-induced vasodilatation, subsequent edema and leukocyte infiltration during the cervical ripening process whereas the muscular alterations would facilitate smooth muscle relaxation and cervical dilatation. Furthermore, estradiol provoked perinuclear localization of EP3 receptor protein in the longitudinal muscle layer. This latter result suggests that cellular EP receptor localization is regulated by estradiol and that PGE2 may also control smooth muscle contraction and regulate ovine cervical dilatation in an intracrine manner via EP3 receptors.

Labor-Associated Regulation of Prostaglandin E and F Synthesis and Action in the Ovine Amnion and Cervix

Prostaglandins (PGs) are key regulators of cervical dilatation and membrane breakdown at the onset of labor. PG synthase and receptor expression has been previously documented in uterine tissues; however, mechanisms governing the changes occurring in the cervix and amnion are less well established. The aim of the current study was to determine the level of expression of PG synthetic enzymes and receptors in these tissues in association with induced labor in sheep. Labor was induced in sheep at 135 days of gestation by continuous fetal dexamethasone infusion. Amnion and cervical tissue was obtained before and after labor for measurement of mRNA encoding enzymes (cytosolic phospholipase A2 [cPLA2], PGH synthase-2 [PGHS-2], PGF synthase [PGFS], and PGE synthase [PGES]) and receptors (FP and EP1-4) by real-time polymerase chain reaction (PCR). cPLA2 expression increased significantly in cervical tissue at labor onset, whereas expression of the other enzymes measured did not change. There was a marked rise in EP3 expression in the cervix, but abundance of this receptor was lower than EP2 and FP expression, which did not change. The amnion exhibited a labor-associated decrease in PGHS-2, PGFS, and FP mRNA expression. The regulation of PG synthesis and action occurring in the amnion and cervix in association with labor appear to differ markedly between the two tissues, indicating tissue-specific roles for PGs. The data support a role for increased PG synthesis and action in the cervix and suggest a decrease in PG production and action in the amnion, in sharp contrast to the pattern reported in human amnion.

Detection of morphological changes of the ovine cervix in response to sex steroids using a fluorescence confocal endomicroscope

This study examined morphological changes of the ovine cervix in response to sex steroids using confocal microscopy. Experimental animals were ovariectomized, and the hormonal status of 4 groups of ewes (n = 5) was manipulated using sex steroids (no replacement, estradiol, progesterone, estradiol, and progesterone). The results were correlated with control ewes (n = 7) in naturally occurring reproductive states (estrus and midluteal). Plasma progesterone concentrations of experimental animals were comparable with those observed during normal reproduction. Confocal microscopy enabled subcellular resolution of the cervical epithelium and the detection of morphological changes associated with alterations in progesterone and estradiol in both artificially manipulated and naturally cycling ewes. Differences in nuclear size, distribution, and density could be differentiated in confocal images and histologic sections of ectocervix from animals in estrus and in the presence of exogenous estradiol. Confocal microscopy has potential diagnostic value for the detection of cellular and subcellular changes of the cervical epithelium.

Proteinase-activated receptors in ovine cervical function

In sheep, inflammation not only functions in cervical dilation at parturition, but also plays an important part in the non-pregnant ewe cervix, as demonstrated by the high level of expression of interleukin (IL)-8 at oestrus. Ewes artificially induced to ovulate have significantly lower levels of IL-8 gene expression at oestrus compared with natural oestrus, indicating an inhibition of inflammation and function, offering an explanation for the low rates of conception in vaginally inseminated synchronised ewes. To identify potential pro-inflammatory agents to combat the anti-inflammatory effects of hormonal synchronisation of oestrus, we have investigated the role of proteinase-activated receptor (PAR)-1 and PAR-2. To localise and measure the level of expression of these receptors, ovine-specific probes were derived for PAR-1 and PAR-2 and used for quantitative in situ hybridisation in the ovine cervix. Both PAR-1 and PAR-2 were expressed in the luminal epithelium of the cervix throughout the oestrous cycle, with expression being highest at oestrus. The gene expression of PAR-2 at oestrus was approximately 30% higher than that of PAR-1. Artificial synchronisation of oestrus by either an intravaginal progesterone sponge or prostaglandin F2+/- injections did not inhibit PAR-1 or PAR-2 expression at oestrus; rather, in the case of PAR-2, progesterone synchronisation increased it. Both synchronising procedures increased the expression of PAR-1 and PAR-2 during the luteal phase of the cycle. Therefore, agonists of PAR-1 and PAR-2 may be potentially useful pro-inflammatory agents countering the inhibition of inflammation by hormonal synchronisation.