383 -2 induces oviductal contraction via subtype A in rats

Linah Al-Alem1,*, Phillip J Bridges1,*, Wen Su2, Ming C Gong2, Marc Iglarz3 and CheMyong Ko1,4 1Division of Clinical and Reproductive Sciences and 2Department of Physiology, University of Kentucky, Lexington, Kentucky 40506, USA 3Pharmacology and Preclinical Development, Actelion Pharmaceuticals Ltd, Allschwil CH-4123, Switzerland 4Department of Biology, University of Kentucky, Lexington, Kentucky 40506, USA (Requests for offprints should be addressed to C Ko; Email: [email protected]) *(L Al-Alem and P J Bridges contributed equally to this work)

Abstract Proper function of the oviduct is critical to reproductive relaxation. Microarray and real-time PCR analysis indicated success with regulated contraction and relaxation facilitating that mRNA for both the endothelin receptor subtypes (ETA transportation of the germ cells to the site of fertilization. and ETB) was present in the oviduct, whereas immunohisto- Endothelin-2 (EDN2) is a potent vasoconstrictor produced by chemical examination revealed that ETA protein was the granulosa cells of the preovulatory follicle at the time of dominant isoform, present in the luminal epithelial cells of the ; however, whether this gonadotropin surge-induced oviduct. Real-time PCR analysis demonstrated that mRNA played a role in facilitating germ cell transportation by for EDN2 was expressed in COCs after ovulation. Isometric inducing oviductal contraction was unknown. The objectives tension analysis indicated that EDN2 was a potent oviductal of these experiments were (1) to determine whether the constrictor and that the contractile effect of EDN2 was endothelin receptor system was present in the oviduct, (2) to mediated by the ETA and not the ETB receptor subtype. The test the hypothesis that EDN2 induces oviductal contraction oviductal contraction induced by EDN2 also reversed via a specific endothelin receptor subtype, (3) to determine, as oviductal relaxation induced by PGE2. In summary, ETA a possible alternate source of the , whether mRNA for receptor-specific EDN2-induced contraction as a facilitator of EDN2 was expressed in cumulus–oocyte complexes (COCs) oviductal function suggests a novel pathway involved in germ within the oviduct, and (4) to determine whether EDN2 could cell transport and hence mammalian fertility. overcome prostaglandin E2 (PGE2)-induced oviductal Journal of Endocrinology (2007) 193, 383–391

Introduction We recently reported that endothelin-2 (EDN2), a vasoactive peptide produced by granulosa cells of preovula- For successful fertilization, the female germ cell (the oocyte), tory follicles, was a requisite for successful ovulation (Ko et al. along with its surrounding cumulus cells, must be transported 2006). EDN2 is one of the three 21 endothelin from the to the ampulla region of the oviduct. Initially, isoforms (EDN1, EDN2, and EDN3), which bind to two through the action of beating cilia, the cumulus–oocyte (ETA and ETB) known receptors (Masaki 2004). Upon complex (COC) is transported from the confines of the considering (1) the vasoactive action of EDN2 (Yanagisawa ovarian bursa to the oviduct proper (Garcia-Pascual et al. et al. 1988), (2) that -produced EDN2 may be 1996, Riveles et al. 2004, Wessel et al. 2004). Waves of released with the COC and follicular fluid at the time of oviductal contraction and relaxation then facilitate trans- ovulation, (3) that the COC itself may be a source of EDN2, portation to the site of fertilization, the lower portion of the and (4) the contractile properties of the oviduct, we ampulla (Garcia-Pascual et al. 1996, Talbot et al. 2003). hypothesized that granulosa cell or COC-produced EDN2 Several hormones have been identified that can affect binding to a specific endothelin receptor subtype in the oviductal contractility, including oxytocin, catecholamines oviduct would induce oviductal contraction, which in (Kotwica et al. 2003), angiotensin II, prostaglandins (PGs), conjunction with regulated oviductal relaxation, would and endothelin-1 (Sakamoto et al. 2001, Wijayagunawardane facilitate transportation of the gametes through the oviduct. et al. 2001a,b), however, the precise physiological mechanism To investigate this hypothesis, our first objective was to regulating oviductal function and gamete transportation determine whether the endothelin receptor system was present remains unclear. intheoviductatthetimeofovulation.Following

Journal of Endocrinology (2007) 193, 383–391 DOI: 10.1677/JOE-07-0089 0022–0795/07/0193–383 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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confirmation of specific endothelin binding sites in the animals/time point). Oviducts were dissected from each ovary oviduct, our next objectives were (1) to evaluate COCs as a and processed for later extraction of total RNA. Microarray potential source of post-ovulatory EDN2 production, (2) to analysis was then performed using the Affymetrix Rat 230A determine whether the oviduct was responsive to EDN2 and if and 230B oligonucleotide array sets (Affymetrix; DNA so, (3) to determine through which endothelin receptor Microarray Core Facility, University of Kentucky, Lexington, subtype this response was generated. Since we hypothesized KY, USA) and incorporated into a database, as described that EDN2 would induce oviductal contraction, we utilized an previously (Jo et al. 2004). isometric tension recorder (Guo et al. 2003, Ko et al. 2006) and small sections of the oviduct to directly determine contractility Real-time PCR analysis of this tissue. However, transportation of the gamete to the site of fertilization involves coordinated waves of oviductal Real-time PCR analysis was performed to (1) confirm the contraction and relaxation and hence, in our final objective, microarray expression profiles for ETA and ETB and (2) we evaluated whether the contractile action of EDN2 could to determine whether mRNA for EDN2 was expressed in overcome the dilation or relaxation induced by PGE2.In COCs after ovulation. Rats were killed before follicular summary, the present study was undertaken to determine development was induced with PMSG (0 h), 48 h after whether EDN2 played a role in contraction of the oviduct and PMSG administration, and 12 or 20 h after hCG injection therefore gamete transportation. The identification of a novel (nZ3 animals/time point). Oviducts were dissected from role for EDN2 in oviductal function and hence fertility will each ovary, and granulosa cells and COCs were isolated after provide valuable knowledge that will increase our under- follicular puncture of or dissection of the oviduct standing of the factors regulating reproductive success. (hCGC20 h), as described previously (Jo et al. 2004, Ko et al. 2006). Briefly, with the aid of a dissecting microscope, preovulatory follicles were punctured with a 26 gauge needle to release granulosa cells and COCs. Approximately, 35–50 Materials and Methods COCs per ovary were then collected using a 20 ml pipette and pooled for each animal. Ovulated COCs (10–20 per oviduct) Reagents were obtained by visualizing their location within the Pregnant mare’s serum gonadotropin (PMSG), human oviduct, making a small tear in the oviduct wall and gently chorionic gonadotropin (hCG), and PGE2 were purchased pressing the oviduct to facilitate their release and collection. from Sigma. was a gift from ACTELION Ovulated COCs were also pooled for each animal. All Pharmaceuticals Ltd (Allschwil, Switzerland). EDN2 was samples were snap-frozen and stored at K80 8C for later purchased from the American Peptide Company, Inc. isolation of total RNA. Total RNA was extracted with (Sunnyvale, CA, USA), real-time PCR reagents were TRIzol (Invitrogen Corp.) according to the manufacturer’s purchased from Applied Biosystems (Foster City, CA, USA) directions and the integrity verified by visualization of distinct and the antibodies for immunohistochemistry were purchased 18S and 28S rRNA bands after ethidium bromide staining in from Abcam, Inc. (Cambridge, MA, USA). an agarose gel. cDNA was reverse transcribed from each sample of total RNA as described previously (Ko et al. 2006) and real-time PCR performed using the Applied Biosystems Animals and tissue collection 7300 detection system (Applied Biosystems) to determine The University of Kentucky Animal Care and Use expression levels of mRNA for EDN2, ETA,ETB, and L19 Committee approved all animal procedures. Immature female (as the endogenous control). cDNA for EDN2 was amplified Sprague–Dawley rats were purchased from Harlan Inc. using TaqMan Universal PCR Master Mix (Applied (Harlan, IN, USA). Animals were maintained in a ratio of Biosystems) and the following gene-specific primers and 14 h light:10 h darkness and given a continuous supply of probes: EDN2-F 50-CTC CTG GCT TGA CAA GGA chow and water. Follicular development and ovulation were ATG-3; EDN2-R 50-GCT GTC TGT CCC GCA GTG induced by treatment of 23–25-day old rats with 10 IU TT-30 and EDN2-Probe 50-/56-FAM/TCT GCC ACC PMSG followed 48 h later by 10 IU hCG. TGG ACA TCA TCT GGG T/36-TAMSp/-30. cDNA for ETA,ETB and L19 was amplified using SYBR Green PCR Master Mix (Applied Biosystems) using the following gene- Microarray analysis 0 specific primers: ETA-F 5 -CCC TTC GAC CCC CTA 0 0 To facilitate analysis of gene expression profiles within the ATT TG-3 and ETA-R 5 -TTT TTG TCT GCT GTG 0 0 oviduct during gonadotropin-primed follicular development GGC AAT A-3 ;ETB-F 5 -TGC ATG AGA AAT GGT 0 0 and ovulation, an addition was made to our previously CCC AAT-3 and ETB-R 5 -TGT CGA TGA TGA TGT described rat ovarian gene expression database (Jo et al. 2004). GTA GCA GAT-30; L19-F 50-CCT GAA GGT CAA AGG Briefly, rats were killed before follicular development was GAA TGT G-30 and L19-R 50-GTC TGC CTT CAG CTT induced with PMSG (0 h), 12 or 48 h after PMSG GTG GAT-30. cDNA generated from the appropriate cells administration, and 6 or 12 h after hCG injection (nZ5 from each of the three animals per time point was included in

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C each reaction and the analysis was replicated 2–3 times on base-line depolarization was generated in response to K ,as different days. DEPC-treated water was used to replace described previously (Guo et al. 2003). The response to EDN2 cDNA and act as a negative control for each analysis. The was determined after a 15 min pretreatment of the oviductal relative amount of transcript was calculated by the DDCT sections with the antagonists or vehicle (PBS) and analyzed C method (Livak & Schmittgen 2001) and normalized to L19. relative to the K -induced depolarization of each oviductal section. A preliminary investigation was conducted to determine the minimal effective dose of each compound (data Immunohistochemistry not shown). Treatments included 1 mM EDN2, 100 mg/ml Ovarian sections obtained from rats euthanized at 12 h after tezosentan (a dual endothelin ), 50 nM treatment with hCG were cut to 10 mm on a cryostat BQ123 (an ETA antagonist), 50 nM BQ788 (an ETB (Vibratome, St Louis, MO, USA) and mounted on Super- antagonist), and 300 mMPGE2. frost/Plus Microscope slides (VWR, West Chester, PA, USA). Immediately after mounting, sections were fixed Statistical analysis with 4% paraformaldehyde and immunostaining was per- formed using the VECTASTAIN Elite ABC kit (Vector Data sets were first tested for homogeneity of variance. If Laboratories, Burlingame, CA, USA). Slides were incubated heterogeneity was detected, data were log-(base 10) in 0.3% H2O2 in methanol to destroy endogenous peroxidase activity, then blocked and incubated at 4 8C overnight with an antibody (ab) to the endothelin receptor subtype A (ETA, ab1919) diluted 1:1000 or subtype B (ETB, ab1923) diluted 1:600. The antigen was detected with the peroxidase substrate diaminobenzidine tetrahydrochloride and nickel chloride. Signal was visualized on an Olympus BX51 microscope equipped with a digital camera.

Isometric tension analysis Rats were euthanized at 10 h after treatment with hCG and the oviducts isolated. A section of the oviduct close to the bursal opening was dissected from the oviduct and tied, via silk threads, to tissue holder electrodes attached to a force transducer. The sections of oviduct were maintained in Kreb’s solution and a

Figure 1 Expression of mRNA for ETA and ETB in the oviduct during Figure 2 Pre- and post-ovulatory expression of mRNA for ETA and gonadotropin-primed follicular development and ovulation. ETB in the oviduct. Immature rats were treated with 10 IU PMSG to Immature rats were treated with 10 IU PMSG to induce follicular induce follicular development and with 10 IU hCG 48 h later to development and with 10 IU hCG 48 h later to synchronize synchronize ovulation. Animals were sacrificed for tissue collection ovulation. Animals were killed for tissue collection before PMSG before PMSG treatment, at 48 h after PMSG and at 12 and 20 h after treatment, at 12 and 48 h after PMSG and at 6 and 12 h after hCG hCG (nZ3 per time point). Relative levels of mRNA were obtained (nZ5 per time point). Relative levels of mRNA were obtained by by real-time PCR. Within a panel, values with different superscripts microarray analysis as described by Jo et al. (2004). differ (P!0.05). www.endocrinology-journals.org Journal of Endocrinology (2007) 193, 383–391

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Figure 3 Immunohistochemical expression of ETA (A and B) and ETB (C and D) in the oviduct at ovulation. Immature 23–25 day old rats were treated with 10 IU PMSG to induce follicular development and with 10 IU hCG 48 h later to synchronize ovulation. Animals were killed at 12 h after hCG for tissue collection. Asterisks indicate the absence of ETA protein expression in the ampulla region of the oviduct. SM, ; Epi, epithelial cells.

transformed before statistical analysis. Non-transformed data are depicted in all the figures. One-way ANOVA using SigmaStat 3.5 (Systat Software, Inc. Point Richmond, CA, USA) was used to determine differences in expression of mRNA levels. If differences were detected, Tukey’s test was used to determine which means differed. The Student’s t-test was used to determine the differences in the isometric tension analysis.

Results

Expression of mRNA and protein for ETA and ETB in the oviduct Microarray analysis was performed to determine the relative level of expression of mRNA for the two endothelin receptor Figure 4 Expression of mRNA for EDN1 and EDN2 in the ovary and subtypes in the oviduct during the ovulatory cascade. Analysis oviduct during gonadotropin-primed follicular development and revealed that mRNA encoding both of the endothelin receptor ovulation. Immature rats were treated with 10 IU PMSG to induce follicular development and with 10 IU hCG 48 h later to subtypes (ETA and ETB) was detectable in the oviduct (Fig. 1). synchronize ovulation. Animals were killed for tissue collection Levels of mRNA encoding ETA remained stable throughout before PMSG treatment, at 12 and 48 h after PMSG and at 6 and gonadotropin-primed follicular development, whereas levels of 12 h after hCG (nZ5 per time point). Relative levels of mRNA were mRNA for ETB gradually increased after treatment with hCG obtained by microarray analysis as described by Jo et al. (2004).

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PMSG (P!0.05) and levels of mRNA for ETB were increased w1.75-fold at 12 and 20 h after hCG (P!0.05) when compared with earlier time points. Analysis of mRNA data was therefore inconclusive in establishing whether a specific endothelin receptor subtype mediated the hypothesized EDN2-induced oviductal contraction. In contrast, immunohistochemical analysis of ETA and ETB protein in sections of oviduct obtained at 12 h after hCG revealed both spatial and endothelin subtype-specific differences in endothelin receptor expression (Fig. 3). Strong staining of ETA protein was observed in the luminal epithelium of the isthmus but not in the ampulla portion of the oviduct at 12 h after hCG (Fig. 3A and B). In contrast, ETB protein was weak or absent in both the ampulla and Figure 5 Expression of mRNA for EDN2 in granulosa cells during isthmus of the oviduct at 12 h after hCG. gonadotropin-primed follicular development and ovulation. Imma- ture rats were treated with 10 IU PMSG to induce follicular development and with 10 IU hCG 48 h later to synchronize Expression of mRNA for EDN2 in granulosa cells and COCs ovulation. Animals were killed for tissue collection before PMSG treatment, at 48 h after PMSG and at 12 h after hCG (nZ3 per time Analysis of mRNA expression patterns obtained by micro- point). Relative levels of mRNA were obtained by real-time PCR. array analysis for EDN1 and EDN2 in the oviduct versus Values with different superscripts differ (P!0.05). granulosa cells of the ovary support our hypothesis that granulosa cell-derived EDN2 induces oviductal contraction. Expression of mRNA for EDN1 remains at basal levels in to induce the ovulatory cascade. However, by 12 h after hCG, both the oviduct and granulosa cells throughout the ovulatory the relative level of expression of mRNA for ETA and ETB cascade, whereas mRNA for EDN2 is rapidly induced in within the oviduct appeared similar. granulosa cells by hCG as the ovulatory stimulus (Fig. 4). Real-time PCR was then performed to (1) confirm the Real-time PCR analysis for EDN2 mRNA in granulosa cells microarray expression profiles and (2) extend the analysis to confirmed the results obtained by microarray analysis (Fig. 5). encompass a time when COCs were present within the Real-time PCR was then performed to determine whether oviduct. Consistent with the results from microarray analysis, mRNA for EDN2 was detectable in COCs at 12 h after hCG, no dramatic changes in expression of mRNA for ETA or ETB when mRNA for EDN2 in granulosa cells is abundant, and were observed (Fig. 2). A small, transient decrease in 8 h later when ovulation is complete and the COCs are expression of mRNA for ETA was observed at 48 h after within the oviduct proper. Consistent with the expression of mRNA for EDN2 in granulosa cells collected at 12 h after hCG, expression of mRNA for EDN2 in COCs was EDN2, COCs increased dramatically at this time point (P!0.05; Fig. 6). 3000 b Interestingly, mRNA for EDN2 in COCs collected at 20 h 2500 after hCG was w11-fold higher than in COCs collected at 2000 48 h after PMSG, suggesting that the COC may be a source of EDN2 when within the oviduct itself. 1500 1000 Effect of EDN2 on contractility of the oviduct 500 We previously reported that granulosa cell-produced EDN2 20 expression of mRNA expression Fold change in relative Fold c was a requisite for successful ovulation (Ko et al. 2006); a however, whether EDN2 produced by granulosa cells at 0 ovulation or by COCs within the oviduct could affect PMSG 48 60 68 oviductal constriction remained to be fully investigated. hCG 0 12 20 Hence, isometric tension analysis was performed to Figure 6 Pre- and post-ovulatory expression of mRNA for EDN2 in determine whether EDN2 induced a functional contraction cumulus–oocyte complexes. Immature rats were treated with 10 IU of the oviduct. After pretreatment with PBS (vehicle) for PMSG to induce follicular development and with 10 IU hCG 48 h 15 min, treatment with 1 mM EDN2 induced a potent later to synchronize ovulation. Animals were sacrificed for tissue Z contraction of the oviductal sections and almost abolished collection at 48 h after PMSG and at 12 and 20 h after hCG (n 3per ! . time point). Relative levels of mRNA were obtained by real-time PCR. spontaneous contractions (P 0 05; Fig. 7A and B). The Within a panel, values with different superscripts differ (P!0.05). contractile effect of EDN2 was inhibited by pretreatment of www.endocrinology-journals.org Journal of Endocrinology (2007) 193, 383–391

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Figure 7 Effect of tezosentan on EDN2-induced contraction of the oviduct. Immature 23–25 days old rats were treated with 10 IU PMSG to induce follicular development and with 10 IU hCG 48 h later to synchronize ovulation. Animals were killed at 10 h after hCG for tissue collection. (A) Representative isometric tension recording after treatment of oviductal sections with PBS then 1 mM EDN2. (B) Force percentage of EDN2 C induced oviductal contraction after pretreatment of the tissue with PBS, relative to K -induced depolarization. (C) Representative isometric tension recording after treatment of oviductal sections with 100 mg/ml tezosentan then 1 mM EDN2. (D) Force percentage of EDN2-induced oviductal contraction after pretreatment of the tissue C with tezosentan (Tezo), relative to K -induced depolarization. Data are the meansGS.E.M. of five isometric recordings per treatment group. Within a panel, values with different superscripts differ significantly (P!0.05).

the ovarian sections with 100 mg/ml of the dual endothelin not effective in inducing oviductal contraction (PO0.05; receptor antagonist, tezosentan (Fig. 7C and D). Fig. 8C and D). The ability of BQ123, but not BQ788 to inhibit EDN2-induced oviductal contraction indicates signal- ing through ET , consistent with the abundant expression of Receptor-specific effect of EDN2 on contractility of the oviduct A ETA protein in the luminal epithelium that was observed by Isometric measurement was then performed to determine the immunohistochemical analysis of sections of oviduct col- specific endothelin receptor subtype mediating EDN2- lected at 12 h after hCG (Fig. 3). induced oviductal contraction. When oviductal sections were pretreated with 50 nM of the ET antagonist BQ788, B Effect of EDN2 on PGE -induced dilation of the oviduct treatment with 1 mM EDN2 induced a potent contraction 2 (P!0.05; Fig. 8A and B). In contrast, when oviductal Coordinated waves of oviductal contraction and relaxation sections were pretreated with an equimolar concentration of facilitates transportation of the germ cells within the oviduct the ETA antagonist BQ123, treatment with 1 mM EDN2 was and isometric tension analysis was performed to determine

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Figure 8 Effect of BQ123 and BQ788 on EDN2-induced contraction of the oviduct. Immature 23–25 days old rats were treated with 10 IU PMSG to induce follicular development and with 10 IU hCG 48 h later to synchronize ovulation. Animals were killed at 10 h after hCG for tissue collection. (A) Representative isometric tension recording after treatment of oviductal sections with 50 nM BQ788 then 1 mM EDN2. (B) Force percentage of C EDN2-induced oviductal contraction after pretreatment of the tissue with BQ788, relative to K -induced depolarization. (C) Representative isometric tension recording after treatment of oviductal sections with 50 nM BQ123 then 1 mM EDN2. (D) Force percentage of EDN2 induced oviductal contraction after pretreatment of the C tissue with BQ123, relative to K -induced depolarization. Data are the meansGS.E.M. of 3–4 isometric recordings per treatment group. Within a panel, values with different superscripts differ significantly (P!0.05). whether EDN2 could overcome the oviductal relaxation provide experimental evidence that the contractile response induced in response to PGE2. Treatment of oviductal sections is mediated via the receptor subtype ETA. Levels of mRNA with 300 mM PGE2 reduced the basal tension of the sections for EDN2 are increased dramatically in granulosa cells (Ko and almost abolished the spontaneous contractions (P!0.05; et al. 2006) and COCs of the periovulatory follicle prior to Fig. 9A and B). However, subsequent treatment with EDN2 ovulation and are maintained above preovulatory levels in overcame PGE2-induced relaxation (P!0.05). COCs within the oviduct. It is therefore likely that EDN2- induced contraction plays a role in the regulation of gamete transport within the oviduct; however, the relative contri- Discussion bution of granulosa cell and/or COC produced EDN2 in oviductal function remains to be determined. The experiments described herein demonstrate that the The concept of ovarian hormones regulating or modifying oviduct is responsive to EDN2 as a contractile stimulus. oviductal transportation is not new. For example, Immunohistochemical and isometric contraction data and are well established as antagonistic www.endocrinology-journals.org Journal of Endocrinology (2007) 193, 383–391

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Figure 9 Effect of EDN2 on PGE2-induced relaxation of the oviduct. Immature 23–25 day old rats were treated with 10 IU PMSG to induce follicular development and with 10 IU hCG 48 h later to synchronize ovulation. Animals were killed at 10 h after hCG for tissue collection. (A) Representative isometric tension recording after treatment of oviductal sections with 300 mM PGE then 1 mM EDN2. (B) Force percentage of EDN2 induced 2 C oviductal contraction after pretreatment of the tissue with PGE2, relative to K -induced depolarization. Data are the meansGS.E.M. of five isometric recordings. Values with different superscripts differ significantly (P!0.05).

regulators of ova movement toward the uterus (Ortiz et al. However, this may be a non-specific effect as endothelin 1979, Fuentealba et al. 1988). However, EDN2 appears novel receptors bind to both EDN1 and EDN2 (reviewed in as a follicular produced peptide that is critical for ovulation, (Masaki et al. 1999)). has its mRNA induced by hCG within hours of oocyte In this study, analysis of mRNA expression levels for ETA release (Ko et al. 2006) and is therefore presumably released and ETB throughout gonadotropin-primed follicular with the follicular fluid at the time of ovulation. On the other development and ovulation did not identify ETA as the hand, COCs within the oviduct expressed levels of mRNA functional endothelin receptor subtype present in the oviduct. for EDN2 that were greater than those observed in This was somewhat surprising considering the strong protein preovulatory follicles not exposed to hCG as the ovulatory signal for ETA when sections of oviduct collected at 12 h after stimulus. The large difference in relative levels of mRNA hCG were examined by immunohistochemistry. Previously, observed in granulosa cells at 12 h after hCG and in COCs at Iwai et al. (1993) analyzed mRNA levels of ETA and ETB by 20 h after hCG suggests the possibility of more than one role in situ hybridization in the ovaries and oviducts of mature rats for EDN2 in the regulation of oviductal contractility. at random stages of the estrous cycle. However, differences The role(s) of gonadotropin surge-induced EDN2 at appear in the regional and cellular location of mRNA versus ovulation appear, however, to be independent of the actions protein for ETA between their study and ours. We observed of during the earlier stages of follicular growth strong staining of ETA protein in the luminal epithelium in and development. In the rat, expression of mRNA for EDN1 regions of the oviduct with a thick musculature, whereas remains at a basal level throughout the ovulatory cascade, staining was weak or absent in the ampulla, the thinner whereas mRNA for EDN2 remains at a basal level until 2–4 h muscled section of the oviduct where fertilization occurs. prior to ovulation, at which time it is increased in a dramatic, This is consistent with the expected contractile nature of these yet transient manner (Ko et al. 2006). Immunoreactive EDN1 different regions of the oviduct; however, the precise signaling and EDN2 are also detectable within the preovulatory follicle pathway between ETA-expressing luminal epithelial cells and of women (Kamada et al. 1993, Haq et al. 1996, Sudik et al. the adjacent musculature remains to be fully investigated. 1996); however, whether the gonadotropin surge induces a Although analysis of regional differences in mRNA levels was similar preovulatory increase in EDN2 that was not detected not the focus of the study by Iwai et al. (1993), they detected a with the sampling schedules utilized in these studies is low level of expression of mRNA for ETA in the smooth unknown. However, the dramatic induction of mRNA for muscle layer rather than the epithelium of a thinly muscled only EDN2 immediately prior to ovulation in the rat is section, presumably the ampulla. Expression of mRNA in suggestive of an independent function of this isoform. other areas of the oviduct was not reported which may have However, a role for EDN1 in oviductal contractility cannot influenced their conclusion regarding overall cellular distri- be discounted, as treatment of oviductal sections collected bution. Taken together with the isometric tension results, it from women in the follicular phase of the appears clear that EDN2-induced oviductal contraction is with EDN1 will induce contractions (Sakamoto et al. 2001). mediated by activation of ETA. Since both contractile and

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Downloaded from Bioscientifica.com at 10/01/2021 11:21:45AM via free access Endothelin-2 induced oviductal contraction . LAL-ALEM, P J BRIDGES and others 391 relaxant forces are required to facilitate gamete transportation Haq A, Kayali M, Hammami MM, Jaroudi K & Al-Sedairy ST 1996 through the oviduct (Garcia-Pascual et al. 1996, Talbot et al. Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in-vitro 2003), it was not unexpected that the oviductal relaxation fertilization. Human Reproduction 11 269–273. induced by treatment with PGE2 was completely overcome Iwai M, Hori S, Shigemoto R, Kanzaki H, Mori T & Nakanishi S 1993 by subsequent treatment with EDN2. Overall, this is Localization of endothelin receptor messenger ribonucleic acid in the rat consistent with the physiology regulating oocyte trans- ovary and fallopian tube by in situ hybridization. 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