Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 24, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034710
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
72/3/736    most recent
biolreprod.104.034710v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sayasith, K.
Right arrow Articles by Sirois, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sayasith, K.
Right arrow Articles by Sirois, J.
Agricola
Right arrow Articles by Sayasith, K.
Right arrow Articles by Sirois, J.
BIOLOGY OF REPRODUCTION 72, 736–744 (2005)
DOI: 10.1095/biolreprod.104.034710
© 2005 by the Society for the Study of Reproduction, Inc.

Molecular Characterization of Equine P-Selectin (CD62P) and Its Regulation in Ovarian Follicles During the Ovulatory Process1

Khampoune Sayasith3, Nadine Bouchard3, Derek Boerboom3, Kristy A. Brown3, Monique Doré4, and Jean Sirois2,3

Centre de recherche en reproduction animale and Département de biomédecine vétérinaire3 Département de pathologie et microbiologie,4 Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes an 829-amino acid protein that is highly conserved when compared to the human protein (80% identity). Semiquantitative RT-PCR/Southern blot analyses were performed to study the regulation of P-selectin transcript in preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (ovulation occurs between 39 and 42 h post-hCG in this model). Results showed that levels of P-selectin mRNA remained very low or undetectable throughout the ovulatory process in extracts prepared from the granulosa cell layer. In contrast, a significant increase in P-selectin transcript was observed between 30 and 39 h post-hCG in extracts obtained from thecal layers (P < 0.05). Likewise, immunohistochemistry revealed an increase of immunoreactive P-selectin protein in the vascular endothelium present in thecal layers of follicles isolated 36 and 39 h post-hCG. Thus, the present study describes, to our knowledge for the first time, the primary structure of equine P-selectin and the regulation of P-selectin transcript and protein in follicular thecal endothelial cells before ovulation.

follicle, human chorionic gonadotropin, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The adhesion molecule P-selectin, which is encoded by the selectin P (SELP) gene, is a glycoprotein that belongs to a family of selectins known to play a critical role in the initial steps of leukocyte emigration from blood vessels into extravascular inflammatory sites [1–4]. Originally, P-selectin was localized in {alpha}-granules of human platelets [5, 6] and later in Weibel-Palade bodies of endothelial cells [7]. Its entire primary structure ultimately was revealed following its initial cloning from a human endothelial cell cDNA library [8]. P-selectin is a type 1 membrane-spanning protein containing an amino-terminal lectin domain, a single transmembrane region, and a short carboxyl-terminal cytoplasmic tail. Generally, P-selectin expression is absent or very low on the surface of resting endothelial cells but is markedly increased when the endothelium is exposed to inflammatory mediators [2]. Once expressed, P-selectin, along with E-selectin, mediates the initial tethering and rolling of leukocytes on the surface of endothelial cells, a first step in their recruitment from the bloodstream [1]. These reversible, transient adhesive interactions result from low-affinity binding between endothelial P-selectin and its physiological ligand on leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1, also known as SELPLG) [9–12]. The role of these molecules in leukocyte rolling was confirmed by genetic studies of mice in which SELP and SELPLG were inactivated [13–15].

The process of ovulation, which is triggered by the LH preovulatory surge, involves a series of biophysical and biochemical events that ultimately lead to rupture of the preovulatory follicle and release of the oocyte [16, 17]. Because some of these events include hyperemia, edema, leukocyte extravasation, and tissue damage and repair, the process has been recognized as a self-controlled, recurrent inflammatory reaction occurring at each estrual or menstrual cycle [18–21]. Numerous investigations have provided convincing evidence for a marked infiltration of leukocytes into thecal layers after the gonadotropin surge [22–28]. Interestingly, species-specific differences were observed in the predominant leukocyte subtypes in the preovulatory follicle, with macrophages and neutrophils prevailing in rats and humans [23, 24], neutrophils and eosinophils dominating in sheep [25], and eosinophils being particularly prominent in pigs and horses [26, 27]. Although the precise roles of leukocytes in ovulation remain to be determined, their phagocytic properties and capacity to produce cytokines, chemokines, and proteolytic enzymes are thought to contribute to the physiological inflammation necessary for follicular rupture [29–33]. These processes imply that leukocytes must first migrate from the general circulation into ovarian tissues, a multistep cascade that begins with the selectin-dependent attachment and rolling of leukocytes on endothelial cells [1–4]. To our knowledge, however, no publication regarding the potential regulation of selectins in preovulatory follicles after the gonadotropin surge has appeared.

Considering its large size (diameter, 40–45 mm) and relatively protracted ovulatory process (39–42 h post-hCG), the equine preovulatory follicle provides some unique advantages for studying the cascade of events leading to follicular rupture [34]. As observed in other species, the cellular and vascular changes that occur in equine follicles during ovulation are similar to those present during an acute inflammatory reaction [27]. Several mediators of inflammation, such as cyclooxygenase-2, interleukin-1ß, and a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS1) also were present and regulated in equine preovulatory follicles [34–37]. Interestingly, a massive infiltration of leukocytes, primarily eosinophils, into thecal layers has been observed just before ovulation [27]. To identify some of the fine mechanisms involved in leukocyte recruitment during this key physiological process, the general objective of the present study was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The specific objectives were to clone and characterize equine P-selectin and to describe the expression of P-selectin transcript and protein in equine follicles during hCG-induced ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The Prime-a-Gene labeling system and pGEM-T Easy Vector System I were obtained from Promega Corp. (Madison, WI). The [{alpha}-32P]dCTP was purchased from PerkinElmer Canada, Inc. (Woodbridge, ON, Canada), and the QuickHyb hybridization solution was obtained from Stratagene Cloning Systems (La Jolla, CA). The TRIzol total RNA isolation reagent, SuperScript II reverse transcriptase, 1-kilobase DNA ladder, synthetic oligonucleotides, and 5'-rapid amplification of cDNA ends (RACE) system (Version 2.0) were purchased from Invitrogen Life Technologies (Burlington, ON, Canada). The Qiagen OneStep Reverse Transcription-Polymerase Chain Reaction (RT-PCR) System was obtained from Qiagen, Inc. (Mississauga, ON, Canada). The Expand High Fidelity DNA Polymerase was purchased from Roche Diagnostics (Laval, PQ, Canada). Biotrans nylon membranes (pore size, 0.2 µm) were obtained from ICN Pharmaceuticals, Inc. (Montréal, PQ, Canada), and all electrophoretic reagents were purchased from Bio-Rad Laboratories (Richmond, CA). The hCG was obtained from The Buttler Co. (Columbus, OH). The Vectastain ABC kit was purchased from Vector Laboratories (Burlingame, CA). The purified rabbit anti-human P-selectin polyclonal antibody was obtained from BD Biosciences (San Jose, CA). The diaminobenzidine tetrahydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO). The Antigen Retrieval Citra Plus Solution was obtained from BioGenex Laboratories, Inc. (San Ramon, CA).

Cloning of the Equine P-Selectin cDNA

The isolation and characterization of the full-length equine P-selectin cDNA was performed by a combination of RT-PCR and 5'- and 3'-RACE. A short cDNA fragment was first isolated by RT-PCR using sense and antisense primers designed by sequence alignments of known P-selectin species homologues, 500 ng of pooled equine ovarian RNA, and the Access RT-PCR kit (Promega) as directed by the manufacturer (RT-PCR 1; Fig. 1Aa). Pooled ovarian RNA consisted of equal amounts of RNA prepared from a preovulatory follicle isolated before hCG, another follicle isolated 36 h after hCG, and a corpus luteum obtained on Day 8 of the cycle (Day 0 = day of ovulation) as previously described [38].



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 1. Cloning strategy for equine P-selectin. A) The open reading frame (ORF) of equine P-selectin is represented as an open box, whereas the 5'- and 3'-untranslated regions (UTRs) are shown as solid lines. Numbers in parentheses indicate the size in base pairs of each element. Equine P-selectin was characterized by a combination of RT-PCR (RT-PCR 1 [a] and RT-PCR 2 [b]), 3'-RACE (c), and 5'-RACE (d) as described in Materials and Methods; arrows and numbers indicate the orientation, relative position, and identity of oligonucleotides used in each cloning procedure. B) List of oligonucleotides used for equine P-selectin cloning. The abridged anchor primer (13) and abridged universal amplification primer (15) are components of the 5'-RACE system

A second RT-PCR was designed to obtain a large internal cDNA fragment (RT-PCR 2; Fig. 1Ab). The RT reaction was done using 5 µg of pooled ovarian RNA, a poly-dT oligonucleotide (primer 7), and SuperScript II reverse transcriptase as directed by the manufacturer. Then, nested PCR reactions involved a first reaction that used sense primer 3 and antisense primer 4 and a second nested reaction that employed sense primer 5 and antisense primer 6 (Fig. 1Ab). Sense primers 3 and 5 were designed from sequences obtained from RT-PCR 1 and, thus, were specific for equine P-selectin, and anti-sense primers 4 and 6 were designed by sequence alignments of other P-selectin homologues. In all cases, cDNA fragments were recovered and subcloned into the pGEM-T Easy plasmid vector (Promega) and sequenced by the Service de Séquençage de l'Université Laval (PQ, Canada).

To characterize the missing 3'-end of equine P-selectin, a 3'-RACE was performed as previously described [38] using 5 µg of total RNA isolated from a preovulatory follicle. Briefly, the 3'-RACE/RT reaction was performed using a poly-dT oligonucleotide with anchor sequences at its 5'-end (primer 7) (Fig. 1Ac). The first 3'-RACE/PCR reaction was performed with sense primer 8 and antisense primer 9, whereas the second 3'-RACE/PCR was performed with sense primer 10 and antisense primer 11. To obtain the 5'-end of equine P-selectin, the 5'-RACE system Version 2.0 (Invitrogen Life Technologies) was used according to the manufacturer's instructions. Reverse transcription was performed as directed using antisense primer 12 (Fig. 1Ad) and 5 µg of RNA from a preovulatory follicle. The first 5'-RACE/PCR reaction was performed with sense-abridged anchor primer 13 (Invitrogen Life Technologies) and antisense primer 14, whereas the second 5'-RACE/PCR reaction employed the sense-abridged universal amplification primer 15 (Invitrogen Life Technologies) and antisense primer 16 (Fig. 1Ad). The PCR reactions consisted of 35 cycles of 94°C for 30 sec, 56°C for 60 sec, and 72°C for 1 min. Final products from 3'- and 5'-RACE were subcloned into pGEM-T Easy (Promega) and sequenced as described above.

Equine Tissues and RNA Extraction

Equine preovulatory follicles were isolated at specific stages of the estrous cycle from standardbred and thoroughbred mares as previously described [27]. Briefly, when preovulatory follicles reached 35 mm in diameter during estrus, the ovulatory process was induced by injection of hCG (2500 IU i.v.), and ovariectomies were performed via colpotomy using an ovariotome at 0, 12, 24, 30, 33, 36, or 39 h post-hCG (n = 4– 6 mares/time point). Ovulation occurs at 39–42 h post-hCG in this model [34]. Each preovulatory follicle was dissected from the surrounding ovarian tissue with a scalpel. A small piece of the follicle was cut with fine scissors, fixed in formalin, and used for immunohistochemistry. Isolated preparations of granulosa cells and of thecal layers were obtained as previously described [39]. The relative purity of each preparation has been validated by the selective expression of cell-specific markers (P450 aromatase [CYP19A1] mRNA for the granulosa cell preparation and P450 17{alpha}-hydroxylase/C17–20 lyase [CYP17A1] mRNA for the thecal preparation [40]). Testicular tissues were obtained from the Large Animal Hospital of the Faculté de médecine vétérinaire (Université de Montréal) following a routine castration, whereas other nonovarian tissues were collected at a local slaughterhouse. All animal procedures were approved by the institutional animal use and care committee. Total RNA was isolated from tissues with TRIzol reagent (Invitrogen), according to manufacturer's instructions using a Kinematica PT 1200C Polytron Homogenizer (Fisher Scientific, Montréal, PQ, Canada).

Semiquantitative RT-PCR and Southern Blot Analysis

The Access RT-PCR system was used for semiquantitative analysis of P-selectin and RPL7A mRNA levels (control gene) in equine tissues. Reactions were performed as directed by the manufacturer using sense (5'-ACACAGCCTCCTGTCAGGACAC-3') and antisense (5'-CAGGCGCTGACACTGCACAGC-3') primers specific for equine P-selectin. Sense (5'-ACAGGACATCCAGCCCAAACG-3') and antisense (5'-GCTCCTTTGTCTTCCGAGTTG-3') primers specific for equine RPL7A were designed from a published sequence deposited in GenBank (accession no. AF508309). These reactions resulted in the production of P-selectin and RPL7A DNA fragments of 506 and 516 base pairs (bp), respectively. Each reaction was performed using 100 ng of total RNA, and cycling conditions were one cycle of 48°C for 45 min and 94°C for 2 min, followed by a variable number of cycles of 94°C for 30 sec, 60°C for 1 min, and 68°C for 2 min. The number of cycles used was optimized for each gene to fall within the linear range of PCR amplification and were 27 and 18 cycles for P-selectin and RPL7A, respectively. Following PCR amplification, samples were electrophoresed on 2% Tris-acetate-EDTA-agarose gels, transferred to nylon membranes, and hybridized with corresponding radiolabeled P-selectin and RPL7A cDNA fragments using QuickHyb hybridization solution (Stratagene). Membranes were exposed to a phosphor screen, and signals were quantified on a Storm imaging system using the ImageQuant software version 1.1 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA).

Immunohistochemical Localization of P-Selectin in Equine Follicles

Immunohistochemical staining was performed using the Vectastain ABC kit (Vector Laboratories) as previously described [34]. Briefly, formalin-fixed tissues were paraffin-embedded, and sections (thickness, 3 µm) were prepared and deparaffinized through a graded alcohol series and treated with boiling Antigen Retrieval Citra Plus Solution (BioGenex Laboratories) in a microwave oven as directed by the manufacturer. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing in PBS for 15 min, sections were incubated with diluted normal goat serum (1:75 dilution in PBS) for 20 min at room temperature. The anti-human P-selectin antibody (BD Biosciences, San Jose, CA) was diluted in PBS (1:1000 dilution) and applied, and sections were incubated overnight at 4°C. Control sections were incubated with PBS. After rinsing in PBS for 10 min, a biotinylated goat anti-rabbit antibody (1:222 dilution; Vector Laboratories) was applied, and sections were incubated for 45 min at room temperature. Sections were washed in PBS for 10 min and incubated with the avidin DH-biotinylated horseradish peroxidase H reagents for 45 min at room temperature. After washing with PBS for 10 min, the reaction was revealed using diaminobenzidine tetrahydrochloride as the chromogen. Sections were counterstained with Gill hematoxylin stain and then mounted. The anti-human P-selectin antibody has been shown to cross-react specifically with P-selectin of other species (BD Biosciences), and preliminary immunohistochemistry studies performed on equine gastric tissues revealed a positive staining localized exclusively to the vascular endothelium, in keeping with the expected localization of the protein (data not shown).

Statistical Analysis

One-way ANOVA was used to test the effect of time after hCG on levels of P-selectin mRNA in samples of theca and granulosa cells. Levels of P-selectin mRNA were normalized with the control gene RPL7A before analysis. When ANOVAs indicated significant differences (P < 0.05), the Dunnett test was used for multiple comparisons of individual means. Statistical analyses were performed using JMP software (SAS Institute, Inc., Carry, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the Equine P-Selectin cDNA

A short, 148-bp, equine P-selectin cDNA fragment was first obtained by RT-PCR with primers designed by sequence alignments of P-selectin from other species (Fig. 1Aa). This equine cDNA sequence was then used to generate homologous sense primers that help obtain a large, 2046-bp, internal equine cDNA fragment (Fig. 1Ab). From the latter, primers were designed for 3'- and 5'-RACE reactions, and overlapping fragments corresponding to the 3'- and 5'-ends were isolated (fragments of 802 and 401 bp, respectively) (Fig. 1, Ac and Ad). Results showed that the equine P-selectin cDNA was composed of a 5'-untranslated region of 25 bp, an open reading frame of 2490 bp (including the stop codon), and a 3'-untranslated region of 729 bp containing three 5'-AATAAA-3' polyadenylation signals. The nucleotide sequence was submitted to GenBank with accession number AY509881.

The coding region of equine P-selectin encodes a 829-amino acid protein, which is one amino acid shorter than the human region [8]; 61–62 amino acids longer than the mouse [41], rat [42], and ovine region (GenBank accession number P98109); and 180–185 amino acids longer than bovine [43], rabbit (GenBank accession number AAA81385, and porcine region [44] (Fig. 2). The equine P-selectin sequence is highly conserved compared to the human orthologue, with an 80% identity observed at the amino acid level and an 85% identity at the nucleic acid level. Moreover, all structural domains putatively involved in P-selectin function are conserved in the equine protein, including an N-terminal lectin domain, an epidermal growth factor (EGF)-like domain, nine consensus repeats homologous to complement regulatory proteins (CRPs) motifs, a transmembrane domain, and a short C-terminal cytoplasmic domain (Fig. 2). The equine protein was shown to be rich in cysteine residues (n = 65, 8% of total amino acids) and to contain numerous (n = 15) potential asparagine-linked glycosylation sites, as observed in other species (Fig. 2).



View larger version (78K):
[in this window]
[in a new window]
  		FIG. 2. Predicted amino acid sequence of equine P-selectin and comparisons with other mammalian homologues. The amino acid sequence of equine (equ) P-selectin is aligned with the human (hum), mouse (mou), rat, ovine (ovi), bovine (bov), rabbit (rab), and porcine (por) homologues. Identical residues are marked with a printed period, and hyphens indicate gaps in protein sequences created to optimize alignment. The putative signal peptide cleavage site is indicated by an arrowhead, and the numbers on the right refer to the last amino acid on that line. Box regions include a lectin domain (Lectin), an EGF-like domain (EGF), nine CRP domains (CRP-1 to CRP-9), and a transmembrane domain. Potential asparagine (N)-linked glycosylation sites are marked with an asterisk, and the putative cytoplasmic domain is underlined

Tissue Distribution of Equine P-Selectin mRNA

Analyses with RT-PCR and Southern blotting were used to study the relative expression of equine P-selectin transcript in various tissues. Although P-selectin appeared to be ubiquitously expressed, marked variations were observed across tissues (Fig. 3). Very high levels of P-selectin mRNA were observed in the skin; moderate to high levels in skeletal muscle, lung, testis, and thecal layers from a preovulatory follicle isolated 36 h after hCG; and very low to low levels in the other tissues tested (Fig. 3).



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 3. Expression of P-selectin mRNA in equine tissues. The RNA was extracted from various equine tissues, and samples (100 ng) were analyzed for P-selectin and RPL7A (control gene) content by a semiquantitative RT-PCR/Southern blot analysis as described in Materials and Methods. A) Expression of P-selectin mRNA in equine tissues. B) Expression of RPL7A mRNA in equine tissues. The number of PCR cycles for each gene was within the linear range of amplification, and they represented 27 and 18 cycles for P-selectin and RPL7A, respectively. Numbers on the right indicate the size of the PCR fragment

Regulation of P-Selectin mRNA in Preovulatory Follicles

To study the regulation of P-selectin mRNA during the ovulatory process, RNA extracts were prepared from granulosa cell and thecal layers of equine preovulatory follicles isolated between 0 and 39 h after hCG treatment, and RT-PCR/Southern blot analyses were performed. Results showed that levels of P-selectin transcript in the granulosa cell layer were low or undetectable (Fig. 4A). When results from several follicles were expressed as ratios of P-selectin to RPL7A, no significant change was observed following hCG administration (n = 4–5 follicles [i.e., mares] per time point) (Fig. 4B). In contrast, high levels of P-selectin mRNA were observed in the thecal layer, and the transcript increased after hCG (Fig. 4C). Results from multiple follicles revealed a significant increase in P-selectin after gonadotropin treatment, with levels being significantly higher in thecal layers isolated between 30 and 39 h post-hCG than in those obtained before hCG (0 h, P < 0.05, n = 4– 5 follicles [i.e., mares] per time point) (Fig. 4D).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4. Regulation of P-selectin mRNA in equine granulosa cells and theca cells. Preparations of granulosa and theca cells were isolated from equine preovulatory follicles between 0–39 h post-hCG, and samples (100 ng) of total RNA were analyzed for P-selectin and RPL7A content by a semiquantitative RT-PCR/Southern blot analysis as described in Materials and Methods. Representative results of P-selectin and RPL7A mRNA levels are presented from one granulosa (A) and one theca (C) sample per time point. The P-selectin signal was normalized with the control gene RPL7A (B and D), and results are presented as a ratio of P-selectin to RPL7A (mean ± SEM, n = 4 samples [i.e. mares] per time point except for t = 0 h, at which n = 5 samples [i.e. mares]). Bars marked with an asterisk are significantly different from 0 h post-hCG (P < 0.05)

Immunohistochemical Localization of P-Selectin Protein in Preovulatory Follicles

To characterize the cellular localization of P-selectin protein in equine preovulatory follicles, immunohistochemistry was performed on sections of equine follicles isolated at 0, 12, 24, 30, 33, 36, and 39 h after hCG treatment (n = 5– 6 follicles [i.e., mares] per time point). Results showed that the P-selectin immunoreactive signal was localized exclusively to the vascular endothelium in thecal layers. P-selectin immunoreactivity was either low or undetectable in follicles obtained before hCG treatment (0 h) or at 12, 24, 30, and 33 h post-hCG (Fig. 5, A [0 h] and B [24 h]). In contrast, moderate to strong P-selectin staining was observed in the thecal vascular endothelium of preovulatory follicles obtained 36 and 39 h post-hCG (Fig. 5, C–F) [36 h]. The expression of P-selectin protein coincided with the presence of eosinophils adhering to the endothelium of blood vessels and emigrating into the extravascular compartment (Fig. 5G). A marked loosening of the granulosa cell layer and severe edema and vascular changes in thecal layers were induced by the hCG treatment, as previously characterized [27].



View larger version (153K):
[in this window]
[in a new window]
  		FIG. 5. Immunohistochemical localization of P-selectin protein in equine preovulatory follicles. Because of its large size (diameter, 4.0–4.5 cm), each preovulatory follicle was first dissected from the surrounding ovarian tissue, and a small piece of the follicle wall was fixed in formalin. Immunohistochemistry was performed on sections of follicles isolated between 0 and 39 h after hCG treatment as described in Materials and Methods. Results show no P-selectin staining in follicles obtained at 0 h (A) and 24 h (B) post-hCG, but a marked immunoreactivity (arrows) was observed in blood vessels of follicles isolated at 36 h post-hCG (C–F). A section of a follicle obtained at 36 h post-hCG and stained with hematoxylin-eosin-saffran (G) shows the presence of numerous eosinophils (arrowheads) migrating along the endothelium in a small vessel or in the extravascular compartment. Control staining (H) from the follicular tissue presented in F was negative when the primary antibody was replaced with PBS. The limits of the granulosa cell (gc) layer are indicated. The region above the granulosa cell layer corresponds to the antrum, whereas the region immediately beneath includes thecal layers. Considering the aim of the present study, selected thecal areas containing blood vessels are presented. A complete characterization of the histological changes associated with the equine ovulatory process has been described previously [27]. Tissue contraction during the fixation process is responsible for wrinkles observed in sections of follicle wall. Magnification x200 (A–C, E, G, and H) and x400 (D and F)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates, to our knowledge for the first time, that expression of the adhesion molecule P-selectin is regulated in ovarian follicles during the ovulatory process, with an induction occurring before follicular rupture in endothelial cells of blood vessels located in thecal layers. Studies on the expression of P-selectin in the ovary have been very limited, with only one reported publication on the expression of selectins in the bovine ovary that primarily focused on the corpus luteum [45]. The latter study, which used pairs of ovaries collected from an abattoir and staged according to different macroscopic and microscopic criteria, also included a group of five dominant follicles (diameter, 1–2 cm) putatively identified as preovulatory. In contrast to the results of the present study, P-selectin was not observed in any of the bovine preovulatory follicles, but it remains unknown if they had been exposed to an endogenous gonadotropin surge. Considering the central role played by P-selectin during the inflammatory response, its induction during the equine ovulatory process further strengthens the parallel that has been established between this key physiological process and inflammation [18–21].

Previous investigations have shown that the induction of P-selectin on the surface of endothelial cells can be either very rapid (i.e., within minutes of stimulation) when it involves the mobilization/translocation of preformed P-selectin stored in Weibel-Palade bodies or can require hours when it results from transcription and new protein synthesis [2, 46]. Several inflammatory regulators, such as tumor necrosis factor (TNF) {alpha}, lipopolysaccharides, interleukin-3, interleukin-4, and oncostatin M have been recognized as inducers of P-selectin synthesis in endothelial cells, although important differences exist among species [47–50]. In the present study, the hCG-dependent induction of P-selectin transcript and protein in thecal endothelial cells now identifies the gonadotropin as another regulator of selectin expression. However, the absence of evidence of LH/ hCG receptors on thecal endothelial cells favors an indirect mechanism of action. The gonadotropin could stimulate granulosa cells to produce mediators that would then act on thecal layers to regulate P-selectin expression. The protracted period of time (~30 h post-hCG) needed for induction of P-selectin transcript in equine preovulatory follicles certainly would allow time for the expression of intermediary mediators. Moreover, because it induces an inflammatory-like reaction in preovulatory follicles, hCG likely generates molecules that could act on the endothelium to induce P-selectin. For example, TNF, a known activator of endothelial P-selectin expression, has been localized in human and ovine preovulatory follicles [51, 52]. However, further work is needed to unravel the precise mechanisms involved.

The induction of P-selectin expression in equine follicles before ovulation raises questions regarding its precise role during this process. Marked vascular changes, such as hyperemia, edema, and hemorrhages, have been observed in equine follicles during the ovulatory process, all of which were shown to occur more than 30 h after hCG administration (i.e., within 9–12 h before follicular rupture) [27]. Interestingly, an extensive emigration of eosinophilic leukocytes into the equine follicle wall has been reported during hCG-induced ovulation [27]. The precise timing of eosinophil extravasation, which occurred between 33 and 39 h post-hCG [27], suggests that it could, indeed, result from endothelial P-selectin induction in thecal layers. The physiological ligand for P-selectin, PSGL-1, is expressed by a variety of leukocytes, including eosinophils [53–55]. In fact, eosinophils are thought to express more PSGL-1 than neutrophils express, which may explain their greater ability to bind P-selectin, particularly in conditions where endothelial P-selectin is expressed at low levels [53, 55]. However, the prominent recruitment of eosinophils into equine preovulatory follicles is unlikely to be explained solely by the expression of P-selectin. Studies of asthma, an allergic condition during which eosinophils are selectively recruited into the bronchial mucosa, revealed that eosinophil recruitment involves not only P-selectin but also several other factors that include specific chemotactic cytokines (i.e., chemokines), such as eotaxins, RANTES (regulated on activation, normal T cell expressed and secreted; also known as CCL5), and monocyte chemoattractant proteins [56]. In contrast, the precise molecular basis for the cyclic infiltration of eosinophils into equine, as well as porcine [26] and ovine [25], preovulatory follicles remains uncharacterized. Of interest, the marked vascular emigration of eosinophils into the developing bovine and human corpus luteum recently has been related to P-selectin and RANTES expression [45, 57]. The presence of leukocyte chemoattractant activities and the hormonal regulation of a number of chemokines have been described in preovulatory follicles [58– 63]. Whereas none has been related directly to the selective eosinophil infiltration into ovarian follicles, the potential involvement of eotaxins and RANTES deserves further attention.

A presumptive role for leukocytes during the ovulatory process has been inferred from the literature in a number of species demonstrating leukocyte cyclic influx into follicles before ovulation [22–28]. Indeed, it is thought that infiltrated leukocytes may help to propagate the local inflammatory reaction and participate in the degradation of extracellular matrix and the changes in follicular vasculature that are required for follicular rupture [30, 31]. Several studies have provided evidence that a number of mediators produced by different leukocyte subtypes participate in ovulation, including histamine, interleukin-1, reactive oxygen species, plasminogen activator, and matrix metalloproteinases [30, 31, 64–70]. Furthermore, the number of LH-induced ovulations was shown to increase within in vitro-perfused rat ovaries after the addition of peripheral blood leukocytes [71], whereas the depletion of circulating neutrophils in rats decreased the ovulatory response in vivo [72]. Genetic and pharmacological studies producing mice with reduced or functionally defective macrophages also led to an ovulation defect [73–76]. In contrast, other investigations revealed that leukocyte depletion had no effect on ovulation rate [77, 78] but led to defects in development and function of the corpus luteum [59, 79]. More studies will be needed to determine whether the marked infiltration of eosinophils into the equine preovulatory follicle wall is required for ovulation or corpus luteum formation.

Studies on equine P-selectin thus far have been limited and focused essentially on expression levels in equine platelets and its role in platelet-neutrophil interactions [80, 81]. The present report documents, to our knowledge for the first time, the cloning and characterization of equine P-selectin, with sequencing results further highlighting the relatively conserved primary structure of the protein across species [8, 42–44]. The equine protein begins with a signal peptide of 41 amino acid residues that, after cleavage, results in a predicted, mature protein of 788 amino acid residues. The mature equine protein consists of a very large extracytoplasmic portion representing more than 92% (729 of 788 amino acid residues) of the protein. This extracytoplasmic portion contains three distinct domains: an amino-terminal, calcium-dependent lectin domain involved in carbohydrate binding, an EGF-like domain whose function remains unclear; and a large domain containing nine repeats of a CRP motif and thought to play a role in spacing the lectin domain from the plasma membrane [82]. The length of this latter domain is variable among species, mainly because of differences in the number of CRP motifs. The equine domain is the only one other than the human domain to contain nine CRPs, whereas the mouse, rat, and ovine domain has eight CRPs [41, 42] (GenBank accession number P98109). The bovine, rabbit, and porcine domain has six CRPs [43, 44] (GenBank accession number AAA81385. The remaining domains of equine P-selectin include a single, 24-amino acid transmembrane region and a short, carboxyl-terminal intracytoplasmic tail (35 amino acid residues), as observed in other species [8, 41–44]. Although all the precise functions of the cytoplasmic tail have not been elucidated, evidence suggests that it may play a role in the internalization and intracellular sorting of the molecule [83, 84]. Moreover, whereas P-selectin often has been recognized as an adhesion receptor primarily involved in leukocyte capture, the constitutive association of pp60src (also known as CSK) with P-selectin [85] and the increase in intracellular calcium induced after P-selectin cross-linking [86] suggest that the cytoplasmic tail has signaling function as well.

In summary, the present study is, to our knowledge, the first to characterize the primary structure of equine P-selectin, to demonstrate the regulation of P-selectin transcript and protein in follicular thecal endothelial cells before ovulation, and to unravel part of the molecular basis for the infiltration of leukocytes into preovulatory follicles before rupture. However, the complexity of the mechanisms responsible for leukocyte recruitment to extravascular sites should not be overlooked, and whereas the role of P-selectin in the initial tethering and rolling of leukocytes on the surface of endothelial cells has been recognized clearly, the multiple molecular interactions involved in leukocyte firm attachment and transendothelial migration remain uncharacterized in the follicle. Likewise, the precise chemotactic signals behind the species-specific recruitment of leukocyte subsets in preovulatory follicles remain largely unknown, especially for those attracting eosinophils into follicles. With its relatively large size and protracted ovulatory process, the equine preovulatory follicle should provide a valuable model to address these issues.


   FOOTNOTES
 
1 Supported by the Natural Sciences and Engineering Research Council of Canada Grant OPG0171135 (to J.S.). D.B. was supported by a Canadian Institutes of Health Research (CIHR) Research Fellowship. J.S. is supported by CIHR Investigator Award. Back

2 Correspondence: Jean Sirois, Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec J2S 7C6, Canada. FAX: 450 778 8103; jean.sirois{at}umontreal.ca Back

Received: 27 July 2004.

First decision: 9 September 2004.

Accepted: 4 November 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Springer TA, Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994 76:301-314[CrossRef][Medline]
  2. Vestweber D, Blanks JE, Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 1999 79:181-213[Abstract/Free Full Text]
  3. Patel KD, Cuvelier SL, Wiehler S, Selectins: critical mediators of leukocyte recruitment. Semin Immunol 2002 14:73-81[CrossRef][Medline]
  4. Ley K, The role of selectins in inflammation and disease. Trends Mol 2003 9:263-268
  5. Hsu-Lin S, Berman CL, Furie BC, August D, Furie B, A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. J Biol Chem 1984 259:9121-9126[Abstract/Free Full Text]
  6. McEver RP, Martin MN, A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984 259:9799-9804[Abstract/Free Full Text]
  7. McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF, GMP-140, a platelet {alpha}-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest 1989 84:92-99[Medline]
  8. Johnston GI, Cook RG, McEver RP, Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell 1989 56:1033-1044[CrossRef][Medline]
  9. Setiadi H, Disdier M, Green SA, Canfield WM, McEver RP, Residues throughout the cytoplasmic domain affect the internalization efficiency of P-selectin. J Biol Chem 1995 270:26818-26826[Abstract/Free Full Text]
  10. Sako D, Chang XJ, Barone KM, Vachino G, White HM, Shaw G, Veldman GM, Bean KM, Ahern TJ, Furie B, Cumming DA, Shaw GD, Expression cloning of a functional glycoprotein ligand for P-selectin. Cell 1993 75:1179-1186[CrossRef][Medline]
  11. Yang J, Galipeau J, Kozak CA, Furie BC, Furie B, Mouse P-selectin glycoprotein ligand-1: molecular cloning, chromosomal localization, and expression of a functional P-selectin receptor. Blood 1996 87:4176-4186[Abstract/Free Full Text]
  12. Borges E, Eytner R, Moll T, Steegmaier M, Campbell MA, Ley K, Mossmann H, Vestweber D, The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum. Blood 1997 90:1934-1942[Abstract/Free Full Text]
  13. Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD, Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 1993 74:541-554[CrossRef][Medline]
  14. Ley K, Bullard DC, Arbones ML, Bosse R, Vestweber D, Tedder TF, Beaudet AL, Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 1995 181:669-675[Abstract/Free Full Text]
  15. Yang J, Hirata T, Croce K, Merrill-Skoloff G, Tchernychev B, Williams E, Flaumenhaft R, Furie BC, Furie B, Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration. J Exp Med 1999 190:1769-1782[Abstract/Free Full Text]
  16. LeMaire WJ, Mechanism of mammalian ovulation. Steroids 1989 54:455-69
  17. Espey LL, Lipner H, Ovulation. In: Knobil E, Neil JD (eds.), The Physiology of Reproduction. New York; Raven Press; 1994:725–780
  18. Espey LL, Ovulation as an inflammatory reaction-a hypothesis. Biol Reprod 1980 22:73-106[CrossRef][Medline]
  19. Thibault C, Ovulation. Contracept Fertil Sex 1999 27:605-613[Medline]
  20. Tsafriri A, Reich R, Molecular aspects of mammalian ovulation. Exp Clin Endocrinol Diabetes 1999 107:1-11[Medline]
  21. Richards JS, Russell DL, Ochsner S, Espey LL, Ovulation: new dimensions and new regulators of the inflammatory-like response. Annu Rev Physiol 2002 64:69-92[CrossRef][Medline]
  22. Gerdes U, Gafvels M, Bergh A, Cajander S, Localized increases in ovarian vascular permeability and leukocyte accumulation after induced ovulation in rabbits. J Reprod Fertil 1992 95:539-550[Abstract]
  23. Brannstrom M, Mayrhofer G, Robertson SA, Localization of leukocyte subsets in the rat ovary during the periovulatory period. Biol Reprod 1993 48:277-286[Abstract]
  24. Brannstrom M, Pascoe V, Norman RJ, McClure N, Localization of leukocyte subsets in the follicle wall and in the corpus luteum throughout the human menstrual cycle. Fertil Steril 1994 61:488-495[Medline]
  25. Cavender JL, Murdoch WJ, Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 1988 39:989-997[Abstract]
  26. Standaert FE, Zamora CS, Chew BP, Quantitative and qualitative changes in blood leukocytes in the porcine ovary. Am J Reprod Immunol 1991 25:163-168[Medline]
  27. Kerban A, Dore M, Sirois J, Characterization of cellular and vascular changes in equine follicles during hCG-induced ovulation. J Reprod Fertil 1999 117:115-123[Abstract]
  28. Gaytan F, Morales C, Bellido C, Tarradas E, Sanchez-Criado JE, Effects of indomethacin on ovarian leukocytes during the periovulatory period in the rat. Reprod Biol Endocrinol 2003 1:26[CrossRef][Medline]
  29. Adashi EY, The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr Rev 1990 11:454-464[Medline]
  30. Bukulmez O, Arici A, Leukocytes in ovarian function. Hum Reprod Update 2000 6:1-15[Abstract/Free Full Text]
  31. Brannstrom M, Enskog A, Leukocyte networks and ovulation. J Reprod Immunol 2002 57:47-60[CrossRef][Medline]
  32. Machelon V, Emilie D, Production of ovarian cytokines and their role in ovulation in the mammalian ovary. Eur Cytokine Netw 1997 8:137-143[Medline]
  33. Townson DH, Liptak AR, Chemokines in the corpus luteum: implications of leukocyte chemotaxis. Reprod Biol Endocrinol 2003 1:94[CrossRef][Medline]
  34. Sirois J, Dore M, The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process. Endocrinology 1997 138:4427-4434[Abstract/Free Full Text]
  35. Boerboom D, Sirois J, Molecular characterization of equine prostaglandin G/H synthase-2 and regulation of its messenger ribonucleic acid in preovulatory follicles. Endocrinology 1998 139:1662-1670[Abstract/Free Full Text]
  36. Martoriati A, Gerard N, Interleukin-1 (IL-1) system gene expression in granulosa cells: kinetics during terminal preovulatory follicle maturation in the mare. Reprod Biol Endocrinol 2003 1:42[CrossRef][Medline]
  37. Boerboom D, Russell DL, Richards JS, Sirois J, Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles. J Mol Endocrinol 2003 31:473-485[Abstract]
  38. Boerboom D, Pilon N, Behdjani R, Silversides DW, Sirois J, Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology 2000 141:4647-4656[Abstract/Free Full Text]
  39. Sirois J, Kimmich TL, Fortune JE, Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells. Endocrinology 1991 128:1159-1166[Abstract]
  40. Boerboom D, Kerban A, Sirois J, Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts in equine granulosa cells during human chorionic gonadotropin-induced ovulation: a novel model for the study of aromatase promoter switching. Endocrinology 1999 140:4133-4141[Abstract/Free Full Text]
  41. Sanders WE, Wilson RW, Ballantyne CM, Beaudet AL, Molecular cloning and analysis of in vivo expression of murine P-selectin. Blood 1992 80:795-800[Abstract/Free Full Text]
  42. Auchampach JA, Oliver MG, Anderson DC, Manning AM, Cloning, sequence comparison, and in vivo expression of the gene encoding rat P-selectin. Gene 1994 145:251-255[CrossRef][Medline]
  43. Strubel NA, Nguyen M, Kansas GS, Tedder TF, Bischoff J, Isolation and characterization of a bovine cDNA encoding a functional homolog of human P-selectin. Biochem Biophys Res Commun 1993 192:338-344[CrossRef][Medline]
  44. Rollins SA, Johnson KK, Li L, Birks C, Matis LA, Rother RP, Role of porcine P-selectin in complement-dependent adhesion of human leukocytes to porcine endothelial cells. Transplantation 2000 69:1659-1667[Medline]
  45. Rohm F, Spanel-Borowski K, Eichler W, Aust G, Correlation between expression of selectins and migration of eosinophils into the bovine ovary during the periovulatory period. Cell Tissue Res 2002 309:313-322[CrossRef][Medline]
  46. Blann AD, Nadar SK, Lip GY, The adhesion molecule P-selectin and cardiovascular disease. Eur Heart J 2003 24:2166-2179[Abstract/Free Full Text]
  47. Gotsch U, Jager U, Dominis M, Vestweber D, Expression of P-selectin on endothelial cells is up-regulated by LPS and TNF-{alpha} in vivo. Cell Adhes Commun 1994 2:7-14[Medline]
  48. Dore M, Sirois J, Regulation of P-selectin expression by inflammatory mediators in canine jugular endothelial cells. Vet Pathol 1996 33:662-671[Abstract]
  49. Khew-Goodall Y, Butcher CM, Litwin MS, Newlands S, Korpelainen EI, Noack LM, Berndt MC, Lopez AF, Gamble JR, Vadas MA, Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3. Blood 1996 87:1432-1438[Abstract/Free Full Text]
  50. Yao L, Pan J, Setiadi H, Patel KD, McEver RP, Interleukin-4 or oncostatin M induces a prolonged increase in P-selectin mRNA and protein in human endothelial cells. J Exp Med 1996 184:81-92[Abstract/Free Full Text]
  51. Loret de Mola JR, Goldfarb JM, Hecht BR, Baumgardner GP, Babbo CJ, Friedlander MA, Gonadotropins induce the release of interleukin-1ß, interleukin-6, and tumor necrosis factor {alpha} from the human preovulatory follicle. Am J Reprod Immunol 1998 39:387-390[Medline]
  52. Murdoch WJ, Colgin DC, Ellis JA, Role of tumor necrosis factor {alpha} in the ovulatory mechanism of ewes. J Anim Sci 1997 75:1601-1605[Abstract/Free Full Text]
  53. Symon FA, Lawrence MB, Williamson ML, Walsh GM, Watson SR, Wardlaw AJ, Functional and structural characterization of the eosinophil P-selectin ligand. J Immunol 1996 157:1711-1719[Abstract]
  54. Woltmann G, McNulty CA, Dewson G, Symon FA, Wardlaw AJ, Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow. Blood 2000 95:3146-3152[Abstract/Free Full Text]
  55. Edwards BS, Curry MS, Tsuji H, Brown D, Larson RS, Sklar LA, Expression of P-selectin at low site density promotes selective attachment of eosinophils over neutrophils. J Immunol 2000 165:404-410[Abstract/Free Full Text]
  56. Wardlaw AJ, Eosinophil trafficking in asthma. Clin Med 2001 1:214-218[Medline]
  57. Aust G, Simchen C, Heider U, Hmeidan FA, Blumenauer V, Spanel-Borowski K, Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures. Mol Hum Reprod 2000 6:1085-1091[Abstract/Free Full Text]
  58. Watson ED, Sertich PL, Zanecosky HG, Detection of chemotactic factors in preovulatory follicular fluid from mares. Am J Vet Res 1991 52:1412-1415[Medline]
  59. Murdoch WJ, McCormick RJ, Mechanisms and physiological implications of leukocyte chemoattraction into periovulatory ovine follicles. J Reprod Fertil 1993 97:375-380[Abstract]
  60. Sirotkin AV, Luck MR, Potential leukocyte attractants in the bovine periovulatory ovary. Reprod Nutr Dev 1995 35:675-683[Medline]
  61. Simon C, Caballero-Campo P, Garcia-Velasco JA, Pellicer A, Potential implications of chemokines in reproductive function: an attractive idea. J Reprod Immunol 1998 38:169-193[CrossRef][Medline]
  62. Wong KH, Negishi H, Adashi EY, Expression, hormonal regulation, and cyclic variation of chemokines in the rat ovary: key determinants of the intraovarian residence of representatives of the white blood cell series. Endocrinology 2002 143:784-791[Abstract/Free Full Text]
  63. Yanagi K, Makinoda S, Fujii R, Miyazaki S, Fujita S, Tomizawa H, Yoshida K, Iura T, Takegami T, Nojima T, Cyclic changes of granulocyte colony-stimulating factor (G-CSF) mRNA in the human follicle during the normal menstrual cycle and immunolocalization of G-CSF protein. Hum Reprod 2002 17:3046-3052[Abstract/Free Full Text]
  64. Tamura K, Kogo H, Granulocyte-macrophage colony-stimulating factor enhances interleukin-1ß stimulated histamine release in the preovulatory rat ovary. Eur J Pharmacol 1999 373:207-213[CrossRef][Medline]
  65. Schmidt G, Owman C, Sjoberg NO, Cellular localization of ovarian histamine, its cyclic variations, and histaminergic effects on ovulation in the rat ovary perfused in vitro. J Reprod Fertil 1988 82:409-417[Abstract]
  66. Brannstrom M, Wang L, Norman RJ, Ovulatory effect of interleukin-1ß on the perfused rat ovary. Endocrinology 1993 132:399-404[Abstract]
  67. Adashi EY, The potential role of interleukin-1 in the ovulatory process: an evolving hypothesis. Mol Cell Endocrinol 1998 140:77-781[CrossRef][Medline]
  68. Miyazaki T, Sueoka K, Dharmarajan AM, Atlas SJ, Bulkley GB, Wallach EE, Effect of inhibition of oxygen free radical on ovulation and progesterone production by the in-vitro perfused rabbit ovary. J Reprod Fertil 1991 91:207-212[Abstract]
  69. Shukovski L, Tsafriri A, The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology 1994 135:2287-2290[Abstract]
  70. Pinto CR, Paccamonti DL, Eilts BE, Venugopal CS, Short CR, Gentry LR, Thompson DL Jr, Godke RA, Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin. Theriogenology 2003 60:819-827[CrossRef][Medline]
  71. Hellberg P, Thomsen P, Janson PO, Brannstrom M, Leukocyte supplementation increases the luteinizing hormone-induced ovulation rate in the in vitro-perfused rat ovary. Biol Reprod 1991 44:791-797[Abstract]
  72. Brannstrom M, Bonello N, Norman RJ, Robertson SA, Reduction of ovulation rate in the rat by administration of a neutrophil-depleting monoclonal antibody. J Reprod Immunol 1995 29:265-270[CrossRef][Medline]
  73. Araki M, Fukumatsu Y, Katabuchi H, Shultz LD, Takahashi K, Okamura H, Follicular development and ovulation in macrophage colony-stimulating factor-deficient mice homozygous for the osteopetrosis (op) mutation. Biol Reprod 1996 54:478-484[Abstract]
  74. Cohen PE, Zhu L, Pollard JW, Absence of colony stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice disrupts estrous cycles and ovulation. Biol Reprod 1997 56:110-118[Abstract]
  75. Watanabe H, Tatsumi K, Yokoi H, Higuchi T, Iwai M, Fukuoka M, Fujiwara H, Fujita K, Nakayama H, Mori T, Fujita J, Ovulation defect and its restoration by bone marrow transplantation in osteopetrotic mutant mice of Mitfmi/Mitfmi genotype. Biol Reprod 1997 57:1394-1400[Abstract]
  76. Van der Hoek KH, Maddocks S, Woodhouse CM, van Rooijen N, Robertson SA, Norman RJ, Intrabursal injection of clodronate liposomes causes macrophage depletion and inhibits ovulation in the mouse ovary. Biol Reprod 2000 62:1059-1066[Abstract/Free Full Text]
  77. Murdoch WJ, Steadman LE, Investigations concerning the relationship of ovarian eosinophilia to ovulation and luteal function in the sheep. Am J Reprod Immunol 1991 25:81-87[Medline]
  78. Chun SY, Daphna-Iken D, Calman D, Tsafriri A, Severe leukocyte depletion does not affect follicular rupture in the rat. Biol Reprod 1993 48:905-909[Abstract]
  79. Murdoch WJ, Van Kirk EA, Etiology of attenuated luteal development in prednisolone-induced eosinopenic ewes. Reprod Fertil Dev 2000 12:127-132[CrossRef][Medline]
  80. Weiss DJ, Evanson OA, McClenahan D, Fagliari J, Walcheck B, Shear-induced platelet activation and platelet-neutrophil aggregate formation by equine platelets. Am J Vet Res 1998 59:1243-1246[Medline]
  81. Lalko CC, Deppe E, Ulatowski D, Lutgen A, Hart AP, Patton EA, Lunn DP, Suresh M, Darien BJ, Equine platelet CD62P (P-selectin) expression: a phenotypic and morphologic study. Vet Immunol Immunopathol 2003 91:119-134[CrossRef][Medline]
  82. Patel KD, Nollert MU, McEver RP, P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils. J Cell Biol 1995 131:1893-1902[Abstract/Free Full Text]
  83. Disdier M, Morrissey JH, Fugate RD, Bainton DF, McEver RP, Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. Mol Biol Cell 1992 3:309-321[Abstract]
  84. Green SA, Setiadi H, McEver RP, Kelly RB, The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes. J Cell Biol 1994 124:435-448[Abstract/Free Full Text]
  85. Modderman PW, von dem Borne AE, Sonnenberg A, Tyrosine phosphorylation of P-selectin in intact platelets and in a disulfide-linked complex with immunoprecipitated pp60c-src. Biochem J 1994 299:613-621[Medline]
  86. Lorenzon P, Vecile E, Nardon E, Ferrero E, Harlan JM, Tedesco F, Dobrina A, Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors. J Cell Biol 1998 142:1381-1391[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. K. Banu, J. Lee, M. C. Satterfield, T. E. Spencer, F. W. Bazer, and J. A. Arosh
Molecular Cloning and Characterization of Prostaglandin (PG) Transporter in Ovine Endometrium: Role for Multiple Cell Signaling Pathways in Transport of PGF2{alpha}
Endocrinology, January 1, 2008; 149(1): 219 - 231.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
K. Sayasith, N. Bouchard, M. Dore, and J. Sirois
Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process
Reproduction, February 1, 2007; 133(2): 455 - 466.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
K. Sayasith, M. Dore, and J. Sirois
Molecular characterization of tumor necrosis {alpha}-induced protein 6 and its human chorionic gonadotropin-dependent induction in theca and mural granulosa cells of equine preovulatory follicles
Reproduction, January 1, 2007; 133(1): 135 - 145.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
72/3/736    most recent
biolreprod.104.034710v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow