Abstract
Angiogenesis, changes in blood flow, and extracellular matrix remodeling are the processes associated with the development and demise of the bovine corpus luteum (CL) during the estrous cycle. APJ (putative receptor protein related to angiotensin type 1 receptor) is a G-protein-coupled receptor, and its ligand, apelin, has been identified as a novel regulator of blood pressure and as an angiogenic factor. We hypothesized that the apelin–APJ system is involved in luteal function. This study investigated whether apelin–APJ exists in bovine CL and determined their expression profiles and localization during luteal phase and prostaglandin F2α (PGF2α)-induced luteolysis. During the luteal phase, apelin mRNA expression increased from early to late CL and decreased in regressed CL. APJ mRNA expression increased from early to mid-CL and remained elevated in late and regressed CL. Apelin and APJ proteins were immunohistochemically detected only in the smooth muscle cells of intraluteal arterioles during the luteal phase. PGF2α stimulated apelin and APJ mRNA expression at 0.5–2 and 2 h respectively, and then the mRNA expression of apelin–APJ was inhibited from 4 h during PGF2α-induced luteolysis. Additionally, apelin mRNA and protein were stimulated at 1 h after PGF2α injection only in the periphery of mid- but not early CL. The present study indicated that the apelin–APJ was localized in the smooth muscle cells of intraluteal arterioles, and responded to PGF2α at the periphery of mid-CL in the cow. Thus, the apelin–APJ system may be involved in the maturation of CL and the luteolytic cascade as a regulator of intraluteal arterioles in cow.
Introduction
APJ (putative receptor protein related to angiotensin type 1 receptor), previously designated as an orphan G-protein-coupled receptor, was cloned from a human gene (O'Dowd et al. 1993). APJ most closely resembles the angiotensin type 1 receptor; however, the APJ receptor does not bind to angiotensin II (O'Dowd et al. 1993). APJ mRNA and immunoreactive APJ are abundantly expressed in various human and rat tissues including the ovary, lung, heart, artery, vein, kidney, liver, and brain (O'Carroll et al. 2000, Medhurst et al. 2003, Kleinz et al. 2005). In 1998, Tatemoto et al. (1998) isolated a novel endogenous apelin peptide from the bovine stomach, which was found to be a ligand of the receptor APJ. This peptide is produced through processing from the C-terminal portion of the pre-proprotein consisting of 77 amino acid residues and exists in multiple molecular forms. Apelin mRNA and immunoreactive apelin are detected in various tissues and organs such as stomach, brain, heart, blood vessels, lung, uterus, and ovary (Habata et al. 1999, Kawamata et al. 2001, De Falco et al. 2002). Apelin causes endothelium-dependent vasorelaxation by triggering the release of nitric oxide (NO), and this effect can be almost completely abolished in the presence of endothelial NO synthase (eNOS) inhibitor, suggesting that the apelin–APJ system may exert a vasorelaxation effect via activation of the eNOS pathway (Tatemoto et al. 2001, Ishida et al. 2004, Zhong et al. 2007). Moreover, it is reported that apelin peptide is a potent angiogenic factor inducing endothelial cell (EC) proliferation, EC migration, and the development of blood vessels in vivo (Kasai et al. 2004, Cox et al. 2006).
The bovine corpus luteum (CL) is a transient organ that secretes progesterone (P), a prerequisite for the establishment and maintenance of pregnancy. The CL undergoes drastic changes in its function and structure during the estrous cycle. Active angiogenesis and P synthesis occur during the development, while a drastic decrease in P secretion (functional luteolysis) and disruption of vascular vessels and luteal cells (structural luteolysis) are induced by the luteolytic action of prostaglandin F2α (PGF2α; Nett et al. 1976, McCracken et al. 1981). Luteal vascular ECs represent more than 50% of the total number of cells in the CL and secrete vasoactive substances directly regulating P secretion (O'Shea et al. 1989), suggesting that the blood vessels and the ECs within the CL have an essential role in luteal function in cow. Most recently, we demonstrated that the acute increase in luteal blood flow is the earliest physiological signals to initiate luteolysis in cow, and this phenomena may be induced by vasodilatation due to the action of luteal NO stimulated by PGF2α (Acosta et al. 2002, Miyamoto et al. 2005).
Given their role in angiogenesis and blood vessel dilation, apelin and its receptor APJ are hypothesized to be involved in the luteolytic cascade including acute increase in the luteal blood flow, but the expression of apelin–APJ in the bovine CL has not been described so far. Therefore, in the present study, to determine local production and the possible role of the apelin–APJ system in the bovine CL, we investigated the mRNA expression and the localization of apelin and APJ during formation, maturation, and regression of the CL in cow.
Results
The mRNA expression of apelin and APJ in the bovine CL throughout the luteal phase
The expression of mRNAs in the CL tissue during the luteal phase is shown in Fig. 1. Apelin mRNA expression increased (P<0.05) throughout the luteal stages, reaching the highest level in the late-stage CL before decreasing in the regressed CL (Fig. 1A). APJ mRNA expression increased from the early to mid-luteal stages and maintained high levels toward the regressed luteal stages (Fig. 1B).
Relative mRNA levels of apelin (A) and APJ (B) in bovine CL throughout the estrous cycle (n=6 for each stage of the CL). All values are shown as mean±s.e.m. (relative to GAPDH mRNA levels). Different superscript letters indicate significant differences (P<0.05) as determined by ANOVA followed by Bonferroni's multiple comparison test.
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0409
Immunohistochemical localization of apelin and APJ in the bovine CL
The protein expression of both apelin (Fig. 2A and B) and APJ (Fig. 2D and E) in the mid- and late CL is shown in Fig. 2. Immunostaining of both apelin and APJ was detected in blood vessels (indicated by arrows) but not in luteal parenchyma, and staining intensity and pattern was similar during the luteal phase (data not shown). Protein from both apelin and APJ was immunohistochemically observed in all CL stages during the luteal phase. To determine the cell-specific localization of apelin and APJ, we observed continuous sections stained with the smooth muscle (using smooth muscle actin (SMA) antibody) and ECs (using antibody of von Willebrand Factor (vWF)). In Fig. 2G and H, we showed that apelin and APJ were positively stained in the smooth muscle cells (Fig. 2G–I) of luteal blood vessels but not in the endothelial (Fig. 2J) and luteal cells.
Immunohistochemical localization of apelin and APJ in bovine CL of (A and D) mid- and late (B and E) luteal stages. Apelin (A and B) and APJ (D and E) proteins are selectively expressed in the area of intraluteal arterioles (arrows). The specificity of the staining was monitored in negative control sections (C and F) by replacing the antibody with goat anti-rabbit IgG. Scale bars represent 100 μm (A, B, D and E). Immunohistochemical detailed localization of apelin and APJ in the bovine CL of mid-luteal stages were shown in G–J. Apelin (G) and APJ (H) proteins are selectively expressed in smooth muscle cells of intraluteal arterioles (I). Serial sections (I and J) were stained for anti-a smooth muscle actin antibody (I) and von Willebrand factor (J) respectively. These sections indicate the same blood vessels within the CL (G–J). Scale bars represent 10 μm (G–J).
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0409
Changes in mRNA expression of apelin and APJ during PGF2α-induced luteolysis in cow
The changes in mRNA expression for apelin and APJ are shown in Fig. 3. Apelin mRNA expression significantly increased at 0.5–2 h after PGF2α injection. Thereafter, the mRNA expression of apelin showed a significant decrease at 4 h after PGF2α and maintained low levels compared with control (Fig. 3A). APJ mRNA expression increased at 2 h after PGF2α injection and significantly decreased at 4 h after PGF2α and maintained low levels compared with control (Fig. 3B).
Relative mRNA levels of apelin (A) and APJ (B) in bovine CL during PGF2α-induced luteolysis (n=5 for each time point). The expression of mRNA was expressed as the percentage of the baseline (0 h). All values are shown as mean±s.e.m. (relative to GAPDH mRNA levels). Different superscript letters indicate significant differences (P<0.05) as determined by ANOVA followed by Bonferroni's multiple comparison test.
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0409
Luteal phase-dependent (early CL versus mid-CL) and site-dependent (periphery versus center of the CL) effects of PGF2α
We investigated the effect of PGF2α, which depended on the luteal phase and local area of the CL. PGF2α as treatment and saline as control were injected during the early (day 4) and the mid-CL (days 10–12). The mRNA expression and the index of positive immunostaining area of both apelin and APJ are shown in Fig. 4. In the early CL, PGF2α did not affect apelin mRNA expression (Fig. 4A) and the immunostaining area (Fig. 4C) both at the periphery and in the center of the CL. On the other hand, PGF2α administration drastically increased the apelin-positive area and apelin mRNA expression at the periphery but not in the center of the mid-CL (Fig. 4A and C). The APJ-positive area and mRNA expression in the early CL were not changed after PGF2α treatment (Fig. 4B and D). Although PGF2α administration stimulated APJ mRNA expression (Fig. 4B) in the center but not in the periphery of the mid-CL, APJ-positive staining (Fig. 4D) was increased by PGF2α both in the periphery and in the center of the mid-CL.
Effect of PGF2α injection on apelin (A) and APJ (B) mRNA expression and on apelin (C) and APJ (D) immunostaining-positive area in the early and the mid-CL in cow. The experiments were conducted on day 4 as early CL, days 10–12 as mid-CL, and PGF2α or saline as control was injected respectively (early CL control, n=5; early CL PGF2α treat, n=5; mid-CL control, n=4; mid-CL PGF2α treat; n=4). At 30 min after injection of PGF2α or saline, luteal blood flow was observed using color Doppler ultrasound. After observing, the cows were ovariectomized at 1 h after treatment, and the portions of the CL were fixed for immunohistochemistry and processed for mRNA analysis. Sampling areas within the CL were designated as periphery and center of the CL. All values are shown as mean±s.e.m. Asterisk indicates statistically different values (P<0.05).
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0409
Discussion
In the present study, we evaluated for the first time the expression of apelin–APJ in the bovine CL tissue during the estrous cycle and PGF2α-induced luteolysis. Immunohistochemistry showed that apelin and APJ were localized in the smooth muscle cells of intraluteal arterioles. Luteolytic PGF2α stimulated the expression of apelin–APJ at an early stage of luteolysis. Most recently, we demonstrated that the acute increase in the luteal blood flow is the earliest physiological signal to initiate luteolysis in cow (Acosta et al. 2002, Miyamoto et al. 2005). Thus, the bovine CL has an apelin–APJ system that may be involved in the acute increase of the luteal blood flow as the earliest physiological event in cow.
Apelin–APJ has been already investigated in many organs including the brain, heart, lung, kidney, uterus, and ovary using PCR and immunohistochemistry (Habata et al. 1999, O'Carroll et al. 2000, Kawamata et al. 2001, De Falco et al. 2002, Medhurst et al. 2003, Kleinz et al. 2005). Recent studies have reported that although apelin mRNA and protein are mainly expressed in the ECs of the artery and vein, other cell types including cardiac muscle cells, adipocytes, chondrocytes, and smooth muscle cells also have been observed to show apelin immunoreactivity in rats (Tatemoto et al. 2001, Cheng et al. 2003, Medhurst et al. 2003), mice (Zhong et al. 2007), humans (O'Carroll et al. 2000, Kleinz et al. 2005), and lizards (De Falco et al. 2004). APJ mRNA and protein are also widely expressed such as in the endothelial and smooth muscle cells in most of the peripheral tissues (O'Carroll et al. 2000, Medhurst et al. 2003, De Falco et al. 2004, Kleinz et al. 2005). In the present study, the expression of both apelin–APJ mRNA and protein was strictly detected on the smooth muscle cells of luteal arterioles only, but not on the endothelial and luteal cells in the bovine CL. This result suggests that the apelin–APJ system may not be likely regulate the synthesis of P directly, and that this system may be associated with the vascular function in the CL.
During the luteal phase, the expression of apelin mRNA gradually increased from the early to late CL followed by a decrease in the regressed CL in the cow. Also, APJ mRNA expression was significantly higher in the mid to regressed CL than in the early CL. The co-expression of mRNA for apelin–APJ strongly suggests that apelin may have some function as a local regulator in the bovine CL. Indeed, the presence of a localized source of apelin induced vascular development and angiogenic branching whose effects were abolished in APJ-deficient frog embryos in vivo (Cox et al. 2006). Moreover, apelin can stimulate the proliferation, migration, and angiogenesis of the ECs in mouse (Kasai et al. 2004). Therefore, our findings suggest that the apelin–APJ system localized in smooth muscle cells may be an autocrine and/or paracrine factor, and may have physiological roles in the vascular establishment, maturation, and maintenance in the CL. However, it is still not possible to isolate smooth muscle cells from the bovine CL in our laboratory; thus further investigations on isolated smooth muscle cells in culture is needed to examine the function of apelin–APJ system.
A luteolytic dose of PGF2α induced a very clear and acute increase in the luteal blood flow only in peripheral area within the CL, and this phenomenon was detected within 0.5–2 h after PGF2α only in the mid-CL but not in the early CL (Acosta et al. 2002). Thus, we hypothesized that luteolytic PGF2α stimulates the expression of eNOS followed by vasodilation in the periphery of the CL and induces an acute increase in the luteal blood flow at a very early stage of luteolysis in cow (Miyamoto et al. 2005). In the present study, PGF2α-increased apelin mRNA expression at 0.5–2 h completely coincides with the timing of the luteal blood flow increase. Moreover, both apelin mRNA and protein expression were stimulated by PGF2α injection at 1 h only in the periphery area of the mid-CL. It has been reported that apelin modulates phosphorylation and activation of the eNOS signaling pathway causing NO release, which is completely abolished by the NOS inhibitor treatment (Tatemoto et al. 2001, Ishida et al. 2004, Zhong et al. 2007). In addition, apelin signaling via APJ induces vasodilation due to the stimulation of NO production followed by a decrease in the blood pressure, suggesting that the apelin–APJ system may have a regulatory role for vascular toning by modulating the eNOS–NO signaling pathway (Ishida et al. 2004, Zhong et al. 2007). Although the administration of apelin decreases the blood pressure in wild-type mice, this hypotensive response to apelin is abolished in APJ-deficient mice (Zhong et al. 2007). On the other hand, the expression of APJ mRNA and APJ immunoreactivity were also stimulated by the administration of PGF2α in the bovine mid-CL. These results suggest that the apelin–APJ system may be involved in the acute increase of the luteal blood flow due to the regulation of NO-vasodilation as the earliest physiological event for the luteolytic cascade in the cow. Furthermore, a severe decrease in the luteal blood flow by vasoconstriction occurs from 4 h after PGF2α injection (Acosta et al. 2002), when mRNA expression of apelin and APJ were drastically reduced in the present study. The findings support the hypothesis that the apelin–APJ system regulates vasodilation in the bovine CL.
We considered the possibility that apelin–APJ regulates the eNOS–NO system in the bovine CL because the apelin–APJ system localized in smooth muscle cells in the present study. In general, the interaction between endothelial and smooth muscle cells in the vessel wall is considered to be an important factor in the control of blood vessel growth and function. The gap junction is formed with a tunnel-like structure and enables regulatory molecules, nutrients, and ions of less than about 1 kDa (i.e., Ca2+, cAMP, inositol 1,4,5-triphosphate) to be exchanged between adjacent cells (Yamasaki & Naus 1996). It was reported that apelin immediately increased Ca2+ in smooth muscle cells (Dai et al. 2006). Moreover, eNOS binds to calmodulin in a reversible and Ca2+-dependent manner and releases NO for short periods (Moncada et al. 1991). We therefore speculate that apelin–APJ system stimulated by PGF2α increased Ca2+ in the smooth muscle cells of luteal arterioles, and Ca2+ may be transferred to the neighboring ECs. These findings suggest that the apelin–APJ system might be involved in the regulation of the luteal blood flow and NO-vasodilation in the bovine CL.
In addition to the roles of apelin–APJ as an angiogenic factor and vasorelaxant, apelin–APJ has a role as an anti-apoptotic factor in humans (Xie et al. 2007) and mice (Tang et al. 2007). In fact, apelin induces the expression of Bcl-2 protein, down-regulates the production of Bax protein, and also blocks the release of cytochrome c and activation of caspase-3, resulting in the suppression of apoptosis in osteoblastic cells (Tang et al. 2007, Xie et al. 2007). Moreover, knock-down of APJ with siRNA abolishes the anti-apoptotic effect of apelin in human osteoblastic cells (Xie et al. 2007). During PGF2α-induced luteolysis in this study, the mRNA expression of apelin–APJ was inhibited at low levels from 4 h after PGF2α injection. Indeed, the Bax expression levels and Bax/Bcl-2 ratio increased at 4 h after PGF2α injection in the bovine CL (Yadav et al. 2005). These findings suggest that down-regulation of the apelin–APJ system may be related to structural luteolysis induced by cell apoptosis in the bovine CL. However, further studies are needed to clarify the local roles of apelin–APJ in apoptosis occurring in the regressing CL.
In summary, the present study defines the expression of both apelin–APJ mRNA and immunoreactivity in the bovine CL during the luteal phase and PGF2α-induced luteolysis. The apelin–APJ system was localized in the smooth muscle cells of intraluteal arterioles, and responded to PGF2α at the periphery of the mid-CL. Thus, the apelin–APJ system may be involved in the CL maturation and the luteolytic cascade as a regulator of intraluteal arterioles in cow.
Materials and Methods
All animal experiments were conducted at the Field Center of Animal Science and Agriculture, Obihiro University, and the experimental procedures complied with the Guidelines for the Care and Use of Agricultural Animals of Obihiro University.
Collection of bovine CL during estrous cycle
Ovaries bearing the CL from Holstein cows were collected at a local slaughterhouse. The luteal stages were classified as early, mid, late, or regressed (n=6 for each stage of the CL) by macroscopic observation of the ovary as described previously (Miyamoto et al. 2000). After the stages were determined, the CL were immediately separated from the ovaries. Thereafter, they were minced and ∼0.1 g luteal tissues were placed in a 1.5 ml microcentrifuge tube with 400 μl TRIzol reagent (Gibco BRL), homogenized immediately, and stored at −80 °C until analysis.
RNA extraction and real-time PCR
Total RNA was extracted from the luteal tissues following the protocol of Chomczynski & Sacchi (1987) using TRIzol reagent and treated with DNase using a commercial kit (SV total RNA Isolation System; Promega Co.), and they were frozen at −20 °C in THE RNA Storage Solution (Ambion Inc., Austin, TX, USA). The mRNA expression for apelin, APJ, and GAPDH were quantified by real-time PCR with a LightCycler (Roche Diagnostics Co.) as reported previously (Watanabe et al. 2006). The primers used for real-time PCR were as follows: apelin (106 bp) – forward, 5′-AAGGCACCATCCGATACCTG-3′ and reverse, 5′-ATGGGACCCTTGTGGGAGA-3′; APJ (100 bp) – forward, 5′-TCTGGGCCACCTACACCTAT-3′ and reverse, 5′-ACGCTGGCGTACATGTTG-3′; and GAPDH (160 bp) – forward, 5′-CTCTCAAGGGCATTCTAGGC-3′ and reverse, 5′-TGACAAAGTGGTCGTTGAGG-3′. The PCR products were subjected to electrophoresis, the target band cut out, and purified using a DNA purification kit (SUPRECTM-01; TaKaRa Bio. Inc., Otsu, Japan). Three to five stepwise-diluted DNA standards were included in every PCR run. Primer sets were tested in the luteal tissue samples to confirm amplification of single bands, amplified products were cloned, and sequenced to confirm their identity, prior to the use of primers in the analysis of samples. The values were normalized using GAPDH as the internal standard.
Immunohistochemistry
Ovaries bearing the CL from Holstein cows were collected at a local slaughterhouse. The luteal stages were classified as early, mid, late, or regressed (n=4 for each stage of the CL) by macroscopic observation of the ovary as described previously (Miyamoto et al. 2000). After the stages were determined, the CL were immediately separated from the ovaries. The tissue samples were fixed in Bouin's fixative for 24 h at room temperature and then embedded in paraffin wax. Serial sections of 5 μm were mounted on a glass microscope slide coated with aminopropyltriethoxysilane (APS). The sections were stained with hematoxylin–eosin for general histological observations.
Light microscopic immunohistochemical staining using the avidin–biotin peroxidase complex (ABC) method (Hsu et al. 1981) was used in the present study. The sections were deparaffinized in xylene, rehydrated in a graded series of ethanol, and then washed with distilled water (DW). Subsequently, endogenous peroxidase was inactivated with 0.3% H2O2 in methanol for 10 min at room temperature and washed with 0.01 M PBS (pH 7.4). After treatment with normal goat serum for 30 min at room temperature, the sections were incubated with polyclonal antibodies for vWF (dilution 1:200; Dako, Glostrup, Denmark), which is the marker of the ECs, SMA (M0851, dilution 1:200; Dako), rabbit apelin-12 (dilution 1:100, has cross-reactivity with bovine; Phoenix Pharmaceuticals Inc., Belmont, CA, USA), and rabbit APJ receptor (dilution 1:100, has cross-reactivity with bovine; Phoenix Pharmaceuticals Inc.) overnight at 4 °C. As a negative control, the sections were incubated with goat anti-rabbit IgG overnight at 4 °C. After incubation, the sections were washed with PBS, incubated with biotinylated goat anti-rabbit IgG (1:200, BA-1000; Vector Laboratories Inc., Burlingame, CA, USA) for vWF, apelin, and APJ, and with biotinylated goat anti-mouse IgG (dilution 1:200, BA-9200; Vector Laboratories Inc.) for SMC for 30 min at room temperature, and then washed with PBS. Horseradish peroxidase-conjugated ABC (1:2, PK-6100, Vectastain Elite ABC kit; Vector Laboratories Inc.) was combined with secondary antibody at room temperature for 30 min. The binding sites were visualized with 0.02% 3,3′-diaminobenzidine tetrahydrochloride in 50 mM Tris–HCl (pH 7.4) containing 0.02% H2O2. After immunohistochemical staining, the sections were lightly counterstained with Mayer's hematoxylin. The sections were washed with DW, dehydrated in a graded series of ethanol, cleared in xylene, and a coverslip was added.
Percentage area of apelin and APJ immunostaining
The positive staining area was extracted using PopImaging (Ver. 3.01; Digital Being Kids, Japan) to calculate the percentage area of immunostaining (area of immunostaining divided by the total area measured×100) as consulted previously (Al-zi'abi et al. 2003). The areas were analyzed at a magnification of 200× using one section from each animal and five fields per section. The results were expressed as percentage of means±s.e.m. per unit area.
PGF2α-induced luteolysis in cow
Experimental design 1: effect of luteolytic PGF2α for apelin–APJ mRNA expression in the mid-CL
Forty multiparous, non-lactating Holstein cows were used for this study. The day of estrus was designated as day 0. Cows (n=5 for each time point) at the mid-luteal phase (days 8–12) were given an i.m. injection of 25 mg PGF2α (0 h; cloprostenol, Estrumate; Takeda Pharmaceutical. Co. Ltd, Osaka, Japan) and the ovaries collected by ovariectomy at 0, 0.5, 2, 4, 12, 24, 48, and 64 h. The CL tissue sample was collected and immediately placed in a 1.5 ml microcentrifuge tube containing 0.4 ml TRIzol reagent, homogenized immediately, and stored at −80 °C until analysis.
Experimental design 2: luteal phase-dependent (early CL versus mid-CL) and site-dependent (periphery versus center of the CL) effects of PGF2α
Eighteen multiparous, non-lactating Holstein cows were used for this study. The estrus synchronization protocol followed was an i.m. injection of 500 μg PGF2α analog followed by an i.m. injection of 100 μg GnRH at 48 h after the PGF2α injection. The day of estrus was designated as day 0. The experiments were conducted on day 4 as the early CL and days 10–12 as the mid-CL, and PGF2α or saline as control was injected respectively (early CL control, n=5; early CL PGF2α treatment, n=5; mid-CL control, n=4; mid-CL PGF2α treatment, n=4). At 30 min after injection of PGF2α or saline, the luteal blood flow was observed using color Doppler ultrasound. After observing, the cows were immediately ovariectomized at 1 h after treatment. To examine the spatial localization and the local effect of PGF2α for apelin and APJ in the CL, the sampling area within the CL was designated as the periphery (in the range of 1 mm from the boundary between the luteal tissue and ovarian parenchyma) and the center (in the range of 1.5 mm from center section (crossover point at major and minor axes after halving of the CL) of the CL). A CL tissue sample of both the periphery and the center was collected; thereafter, they were minced and ∼0.1 g luteal tissues placed in a 1.5 ml microcentrifuge tube containing 0.4 ml TRIzol reagent, homogenized immediately, and stored at −80 °C until analysis. For immunohistochemistry, the CL was enucleated from the ovary and dissected free of connective tissue. The tissue samples were fixed in Bouin's fixative.
Statistical analysis
All data were expressed as mean±s.e.m. The time of PGF2α analog injection was defined as 0 h. The expression of mRNA of apelin and APJ in Experimental Design 1 after PGF2α administration was expressed as the percentage of the baseline (0 h). The statistical significance of differences in (1) the amount of apelin and APJ mRNA in the CL during the luteal phase, (2) the change in apelin and APJ mRNA expression during PGF2α-induced luteolysis, and (3) the effect of PGF2α injection on apelin and APJ expression between the early and mid-luteal phases were assessed by ANOVA followed by Bonferroni's multiple comparison test. Probabilities <5% (P<0.05) were considered significant.
Acknowledgements
The authors thank Schering-Plough Animal Health K K, Japan, for Estrumate and Conceral. This study was supported by the Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS) and the 21st Century COE Program (A-1), Ministry of Education, Culture, Science and Technology, Japan. K S is supported by JSPS Research Fellowships for Young Scientists. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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