| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
b Institute of Physiology, Technical University of Munich, D-80350 Freising-Weihenstephan, Germany
c Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
When Ang I was added to the MVE at a concentration of 10-9 M, it was converted to Ang II (21%). The production of Ang II from Ang I time-dependently rose for 24 h. Addition of captopril (an inhibitor of Ang-converting enzyme [ACE]) to the MVE cultures significantly inhibited Ang II production from 6 h to 24 h (P < 0.05). Addition of estradiol-17ß (E2) + vascular endothelial growth factor and E2 + basic fibroblast growth factor to MVE cultures increased Ang II production, whereas E2 or growth factors alone had no effect. Specific transcription for AT1R and AT2R was detected in bovine CL and MVE. There were no significant changes in Ang II tissue concentration or AT1R mRNA expression using reverse transcription-polymerase chain reaction during the estrous cycle. In contrast, AT2R mRNA expression decreased during the midluteal phase (P < 0.05) and increased to the highest level during the late luteal phase (P < 0.05).
Results demonstrated that Ang II is generated from Ang I in MVE isolated from the developing bovine CL, indicating that MVE have ACE activity. In addition, mRNA expression for Ang II receptors was detected in the bovine CL and the luteal MVE. These results suggest that Ang II is produced by actions of the local renin-angiotensin system, at least in part, on MVE in the bovine CL, and that this peptide may be involved in the regulation of luteal function during early development and luteolysis.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
By using an in vitro microdialysis system (MDS) implanted in bovine luteal explants from the midluteal phase of the estrous cycle, we have recently shown that Ang II decreases progesterone (P4) release and that Ang II release is stimulated by prostaglandin (PG) F2
[7]. These results suggest that the luteolytic effect of PGF2 may be partly mediated by Ang II. In addition, it has been also reported that luteal endothelin-1 (ET-1), another vasoactive peptide produced in microvascular endothelial cells (MVE), has a local luteolytic action that is similar to those of Ang II in the bovine corpus luteum (CL) [8,9]. ACE was predominantly localized in bovine pulmonary artery endothelial cells [10], and Ang II receptors were localized in bovine aortic endothelial cells [11], indicating that endothelial cells are the principal sites of the Ang II production and action in cattle. Moreover, Ang II has been shown to stimulate the expression of mRNA for a powerful angiogenic factor, basic fibroblast growth factor (bFGF), in bovine luteal cells [12], and to stimulate the growth of bovine aortic endothelial cells [13]. These findings suggest that Ang II may also regulate angiogenesis in the developing CL. All these results indicate that vascular endothelial cells, which account for up to 50% of the bovine CL [14,15], may play essential roles by releasing vasoactive peptides in the CL function.
The aim of this study was, therefore, to investigate the possible physiological importance of Ang II by studying basic regulation of Ang II in MVE and CL by examining 1) the cell capacity to convert Ang I to Ang II, 2) the effects of endocrine and paracrine/autocrine factors on Ang II production in MVE derived from the developing CL, and 3) the relationship between Ang II peptide concentration and expression of mRNA for angiotensin type 1 and 2 receptors (AT1R and AT2R) in the bovine CL at different stages of the estrous cycle.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture and Ham's F-12 (1:1), amphotericin B, gentamicin, transferrin, sodium selenite, arachidonic acid, P4, estradiol-17ß (E2), PGF2, PGE2, atrial natriuretic peptide, Ang I, and captopril were from Sigma (St. Louis, MO); fetal calf serum was from Biochrom (Berlin, Germany); collagen type I (Vitrogen 100) was from Collagen Corporation (Fremont, CA); ET-1 and oxytocin (OT) were from Peninsula Laboratories (Merseyside, UK); insulin and protease inhibitors (Complete tablets) were from Boehringer Mannheim (Mannheim, Germany); bovine LH (USDA bLH-B-5) was kindly provided by the USDA Animal Hormone Program (Beltsville, MD); bFGF was from Pepro (Frankfurt am Main, Germany); vascular endothelial growth factor (VEGF recombinant bovine) was from Dr. D. Gospodarowicz, Chiron Corp. (Oakland, CA); Trizol reagent was from Gibco BRL (Gaithersburg, MD); and thermostable polymerase PrimeZyme was from Biometra (Göttingen, Germany).
Endothelial Cell Culture
Microvascular endothelial cells (type III) isolated from developing bovine CL (Days 1–10) were kindly provided by Dr. K. Spanel-Borowski (University of Leipzig, Germany). The phenotype of the type III endothelial cells is a spindle-shaped monolayer with vacuoles, and the diameter of these cells is about 30 µm [16]. Cells were grown in DMEM/F-12 medium containing 5% fetal calf serum on plates precoated with 1% collagen type I. Experiments were performed on confluent cultures in 48-well plates (Corning, Corning, NY), and the cells were from passages 14–17. Before addition of the various compounds, cells were transferred to serum-free medium containing 0.1% BSA, insulin (2 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), and arachidonic acid (10 µM). Cells were then incubated in medium only, or in medium containing Ang I (10-9 M) and captopril (an ACE inhibitor, 10-5 M), P4 (10-6 M), E2 (10-11 M), LH (10 ng/ml), PGF2 (10-7 M), PGE2 (10-7 M), atrial natriuretic peptide (10-7 M), ET-1 (10-7 M) OT (10-7 M), VEGF (10 ng/ml), bFGF (10 ng/ml), or a combination of these factors for 24 h at 37°C in a CO2 incubator (5% CO2 and 95% air). At least 3 experiments were performed, with each concentration of agents tested with 3 replications/experiment.
Hormone Determination
Concentration of Ang II in the medium was determined with the second-antibody enzyme immunoassay (EIA). We used undiluted medium for the Ang II EIA. The EIA for Ang II [7] used a rabbit anti-human Ang II antibody provided by Dr. K. Wakabayashi, Gunma University, Japan. The standard curve of Ang II ranged from 2.4 to 2500 pg/ml, and the ED50 of the assay was 100 pg/ml. The intra- and interassay coefficients of variation were on average 6.4% and 8.7%, respectively. The cross-reactivity of Ang II antibody with Ang I in the medium was 1%.
Collection of Bovine CL
Ovaries with CL were collected from German Fleckvieh cows at a local abattoir within 10–20 min after exsanguination. The luteal stage was classified to early, mid, and late stages by macroscopic observation of the ovary as described previously [17]. After determination of the stages, CL were separated immediately from the ovaries, frozen rapidly in liquid nitrogen, and stored at -80°C until processed.
Tissue Extraction of Ang II
Tissue (1 g wet weight) was transferred into 10 ml of an acidic buffer (pH 2.8). This buffer contains 205 µl orthophosphoric acid, 2.264 g NaH2PO4xH2O, 1.86 g EDTA, 7.01 g NaCl, 0.2 g NaN3, 2.0 g BSA, 1 ml Triton X-100, and one Complete tablet per liter. These tablets contain both reversible and irreversible protease inhibitors, and inhibit a broad spectrum of serine, cysteine, and metalloproteases as well as calpains. The mixture was homogenized in an ice bath with Ultra Turrax equipment (Janke and Kunkel, Staufen, Germany). The tissue was homogenized with 15-sec bursts of shredding at maximum speed, separated by 45-sec intervals of cooling. The homogenate was subsequently centrifuged at 2000 x g for 15 min at 4°C. The supernatant (an aliquot of 1 ml) was transferred to a small encapped Octyl column (C8: Ci-C8H17) from Amersham-Pharmacia (Freiburg, Germany). The column was calibrated with 3 ml methanol (MeOH) and 3 ml distilled water, and Ang II was eluted with 4 ml acetonitrile (10% v:v, acidified with 0.1% trichloracetic acid). The eluate was evaporated and diluted with assay buffer for Ang II EIA. The recovery rate of Ang II with this process was 42%.
RNA Isolation
Total RNA from bovine CL was isolated by the single-step method of Chomczynski and Sacchi [18] using Trizol reagent. Total RNA from endothelial cells was isolated using the NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany). Finally, RNA was dissolved in water and spectroscopically quantified at 260 nm. To verify the quantity and quality of RNA, aliquots were electrophoresed on a 1% denaturing agarose gel that was stained with ethidium bromide.
Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)
Four micrograms of total RNA were used to generate single-strand cDNA in a 60-µl reaction mixture as described [19]. Conditions for enzymatic amplification were optimized for each PCR as follows: the AT1R and AT2R PCRs contained 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.6 µM of each primer, and 0.5 U of the thermostable polymerase PrimeZyme to 5 µl cDNA (final volume 25 µl). The ubiquitin PCR was performed under the same conditions as those for AT1R and AT2R, but a higher concentration of primer (1.5 µM) was used. On the basis of a detailed optimization of temperature, amplification of AT1R and AT2R consisted of one denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec and at 64°C (AT1R) or 68°C (AT2R) for 45 sec. One additional elongation step was carried out at 72°C for 2 min. Amplification of the housekeeping gene consisted of one denaturation step at 94°C for 2 min; 22 cycles of 94°C for 45 sec, at 55°C for 45 sec, and at 72°C for 45 sec; and one additional elongation step at 72°C for 2 min.
To determine the optimal quantity of reverse transcript needed for PCR and to verify that the cDNA product was dependent on the amount of transcript used, varying quantities of transcript template were used in the PCR reaction. The RT product from 3 µl was in the linear range for these amounts and produced a visible band. To exclude the possibility of amplification of genomic DNA, all experiments included reactions in which the RT enzyme or cDNA template was omitted. As a negative control, water was used instead of RNA for the RT-PCR to exclude any contamination from buffers and tubes. The primers were designed to encode the bovine sequences by using the EMBL database, or were used as described elsewhere and commercially synthesized (Amersham-Pharmacia). The primers were chosen using the "Husar" online software package in Heidelberg (http://genome.dkfz-heidelberg.de). As a negative control, water was used instead of RNA for the RT-PCR to exclude any contamination from buffers and tubes. The primers were as follows: AT1R forward 5'-GAAGCTGGAAGACAACCA-3' and reverse 5'-TCCCAAAGTAGACCTGCC-3'; AT2R forward 5'-CACCACCACCATCTGCTT-3' and reverse 5'-TCTGAACTGGGGTGCAGA-3' (335 bp; corresponding to bases 138–472 of the ovine mRNA partial sequence: EMBL no. S81979); ubiquitin forward 5'-ATGCAGATCTTTGTGAAGAC-3' and reverse 5'-CTTCTGGATGTTGTAGTC-3' (189 bp) [20].
Aliquots of the PCR reaction products (5 µl) were added to 1 µl bromphenol blue glycerin and fractionated by electrophoresis through a 1.5% agarose gel containing ethidium bromide in a constant 60-V field. To determine the length of the RT-PCR products, a mass ladder and 100-bp marker were used. The resultant band intensities were scanned by a video documentation system (Amersham-Pharmacia) and analyzed with the Image Master 1D program (Amersham-Pharmacia). Each PCR product was confirmed by use of direct PCR product sequencing (TopLab, Munich, Germany).
Statistical Analysis
All experiments were repeated 3–13 times. Data are presented as means ± SEM. Means were analyzed by ANOVA followed by Fisher's protected least-significant difference test. Differences were considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
As shown in Table 1, conversion of Ang I to Ang II increased dose-dependently with the addition of Ang I (P < 0.05). Ang II production was not detected without addition of Ang I, or in the presence of 10-10 and 10-11 M Ang I in the MVE culture. The conversion of 10-9 M Ang I to Ang II was estimated at about 21%. At this Ang I concentration, the cross-reactivity of Ang II antibody with Ang I in the medium was 1%. On the basis of these results, for further experiments, a dose of 10-9 M was selected for Ang I as a precursor of Ang II.
|
Time-Dependent Production of Ang II
The production of Ang II from Ang I (10-9 M) time-dependently rose for 24 h (Fig. 1). Addition of captopril (ACE inhibitor, 10-5 M) to the MVE cultures significantly inhibited the Ang II conversion from Ang I from 6 h to 24 h (P < 0.05; Fig. 1).
|
Effects of VEGF and bFGF on the Production of Ang II
The effects of VEGF (10 ng/ml) and bFGF (10 ng/ml) on Ang II production by bovine MVE are shown in Figure 2. Addition of E2 (10-11 M), P4 (10-6 M), VEGF, or bFGF alone and in combination with P4 to the MVE cultures had no effect on the production of Ang II (Fig. 2). However, addition of E2+VEGF or E2+bFGF to the MVE cultures increased the Ang II production (P < 0.05; Fig. 2). Addition of LH (10 ng/ml), PGF2 (10-7 M), PGE2 (10-7 M), atrial natriuretic peptide (10-7 M), ET-1 (10-7 M), or OT (10-7 M) alone had no effect (data not shown).
|
Ang II Production and mRNA Expression for AT1R and AT2R in Bovine MVE
Addition of captopril (10-5 M) decreased Ang II production by bovine MVE cultured in medium containing Ang I (10-9 M) when compared with basal production (P < 0.05; Fig. 3a). Under these conditions, specific transcription for AT1R and AT2R were detected in bovine MVE. AT1R and AT2R mRNA expression in the MVE did not change with captopril treatment despite its clear effect on Ang II production (Fig. 3, b and c).
|
AT1R and AT2R mRNA Expression and Ang II Peptide Concentration in Bovine CL at Different Luteal Stages of the Estrous Cycle
Specific transcripts for AT1R and AT2R were detected in bovine CL. To confirm the integrity of the mRNA templates and RT-PCR protocol, the housekeeping gene ubiquitin was examined in all samples. A representative sample for the ubiquitin RT-PCR products (189+417 bp) is shown in Figure 4a. The relative signal intensities for AT1R and AT2R PCR products were assessed after correction based on the ubiquitin signal intensities.
|
A typical example of the expressions of AT1R and AT2R mRNAs in the CL is shown in Figure 4b. Ang II tissue levels and the mRNAs for AT1R and AT2R in the early, mid, and late luteal phases in the CL are shown in Figure 5. There were no significant changes in Ang II tissue concentration and AT1R mRNA expression during the estrous cycle (Fig. 5, a and b). In contrast, AT2R mRNA expression decreased during the midluteal phase (P < 0.05) and increased again to the highest level during the late luteal phase (P < 0.05; Fig. 5c).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The combination of E2+VEGF and E2+bFGF in the MVE cultures increased Ang II production in this study. VEGF [21] and bFGF [22] are known to be strong angiogenic factors. Basic FGF mRNA expression is stimulated dose-dependently by Ang II in bovine luteal cells [12]. It has also been demonstrated that Ang II induces neovascularization in the eyes of rabbits [23] and stimulates the growth of bovine aortic endothelial cells [13]. In addition, it should be noted that in the present study these stimulations required the presence of E2. Low concentrations of E2 have been shown to be an important regulator of bovine capillary endothelial cell adhesion, proliferation, and capillary-like tube formation [24]. Furthermore, it has been shown that VEGF may modulate estrogen-mediated capillary endothelial cell proliferation, angiogenic growth, and differentiation [24,25]. Thus, these findings suggest that VEGF and bFGF together with E2 may accelerate Ang II production in MVE, which may ensure the cascade mechanisms of angiogenesis during CL development. In contrast, P4 in combination with VEGF or bFGF is inactive.
In the in vitro MDS of luteal explants, Ang II stimulated P4 release in the early CL [26] but decreased P4 release in the midluteal CL [7]. Additionally, Ang II concentrations in the perfusate fractions were higher during the early-luteal phase (9.2 pg/ml) as compared with those of the midluteal phase (3.9 pg/ml) [26]. These data indicate that intraluteal production of Ang II is more active during the early-luteal phase when angiogenesis is mostly active, and suggest that Ang II may support both angiogenesis and steroidogenesis during CL development.
We previously demonstrated that Ang II regulates release of P4, OT, and ET-1 in the bovine CL using an in vitro MDS [7]. In cultured bovine luteal cells, Stirling et al. [12] have also shown that Ang II decreases P4 production supported by LH, and this effect was blocked by Ang II receptor antagonist. These results suggest that Ang II directly acts on both luteal and endothelial cells via Ang II receptors, because P4 and OT are secreted by luteal cells [27], whereas ET-1 is secreted by endothelial cells [28]. Contrary to this suggestion, however, it has been reported that bovine luteal cells do not possess significant amounts of binding sites for Ang II [6]. In the present series of experiments, we demonstrated AT1R and AT2R mRNA expression in the bovine CL at different stages of the estrous cycle and in the MVE isolated from the bovine developing CL. These results provide the first evidence for the expression of Ang II receptors mRNA in the bovine CL. However, we could not analyze the expression of mRNA for AT1R and AT2R in purified luteal cells apart from endothelial cells in the present experiment. Thus, to evaluate the possible cross-talk between different cell types by Ang II in the CL, the exact localization of ATRs in the CL must be clarified in future studies.
The expression of AT2R mRNA in the CL was lowest in the midluteal CL and highest in the late-luteal CL. However, the amount of Ang II production did not affect the expression of Ang II receptors mRNA in cultured MVE. Thus, the mechanism that down- and up-regulates AT2R in the CL may not be attributable to Ang II itself.
In the MDS study using the midluteal CL in vitro, PGF2 enhanced the secretion of Ang II, and Ang II together with PGF2 showed the maximal effect in P4 suppression [7]. In the present MVE study, however, PGF2 alone had no effect on the Ang II production in culture. As the expression of mRNA for PGF receptor was found in both luteal cells and MVE in the bovine CL [29], the observed action of PGF2 on Ang II production may directly occur on luteal cells, but not on MVE. This possibility that luteal cells also have ACE activity may be supported by the findings that bovine theca cells produce prorennin and release renin, and that this local RAS is stimulated by LH in culture [30]. Therefore, luteinized theca cells (small luteal cells) may also have a local RAS that is regulated differently from the RAS of MVE. Consequently, a complex interaction of RAS in both luteal cells and MVE probably controls local production and action of Ang II in the CL. These mechanisms appear to involve a pronounced vasoconstriction of arterioles, which should be induced by ET-1 [31] and possibly Ang II [32] during the first stage of luteolysis. At the same time, the increased secretion of Ang II may inhibit the P4 secretion from luteal cells that directly relates to functional luteolysis. Clearly, the concept requires further detailed studies on luteal cells and in vivo observations.
In conclusion, we have confirmed that Ang II is generated from Ang I in MVE isolated from the bovine developing CL, indicating that MVE have ACE activity. In addition, mRNA expression for Ang II receptors was detected in bovine CL and in luteal MVE. These results suggest that Ang II is produced, at least in part, by action of a local RAS on MVE in the bovine CL, and that this peptide may be involved in the regulation of luteal function during development and in synergism with ET-1 during luteolysis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This study was supported by a Grant-in-Aid for Scientific Research (C) and the Japan-Germany joint research project of the Japan Society for the Promotion of Science, the Novartis Foundation (Japan) for the Promotion of Science (A.M.), and the German Research Foundation (Scha 257/14-1). K.H. was supported by H. Wilhelm Schaumann-Stiftung, and A.M. was supported by Alexander von Humbolt Stiftung. 
2 Correspondence. FAX: 49 8161 714204; physio{at}pollux.weihenstephan.de
Accepted: September 7, 1999.
Received: February 8, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro. Biol Reprod 1999; 60:1104–1109.-induced antisteroidogenic action. Endocrinology 1996; 137:1306–1312.[Abstract] promotes the inhibitory action of endothelin-1 on the bovine luteal function in vitro. J Endocrinol 1997; 152:R7-R11. and luteinizing hormone receptors in various bovine luteal cell types. Biol Reprod 1998; 58:849–856.This article has been cited by other articles:
|
|
 |
|
  K. Shirasuna, S. Watanabe, T. Asahi, M. P B Wijayagunawardane, K. Sasahara, C. Jiang, M. Matsui, M. Sasaki, T. Shimizu, J. S Davis, et al. Prostaglandin F2{alpha} increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis Reproduction, April 1, 2008; 135(4): 527 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Ferreira, J. F. Oliveira, R. Fernandes, J. F. Moraes, and P. B. Goncalves The role of angiotensin II in the early stages of bovine ovulation Reproduction, November 1, 2007; 134(5): 713 - 719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Shirasuna, T. Shimizu, K.-G. Hayashi, K. Nagai, M. Matsui, and A. Miyamoto Positive Association, in Local Release, of Luteal Oxytocin with Endothelin 1 and Prostaglandin F2alpha During Spontaneous Luteolysis in the Cow: A Possible Intermediatory Role for Luteolytic Cascade Within the Corpus Luteum Biol Reprod, June 1, 2007; 76(6): 965 - 970. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Pepperell, G. Nemeth, Y. Yamada, F. Naftolin, and M. Merino Localized accumulation of angiotensin II and production of angiotensin-(1-7) in rat luteal cells and effects on steroidogenesis Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E221 - E233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Shirasuna, H. Asaoka, T. J. Acosta, M. P.B. Wijayagunawardane, M. Ohtani, M. Hayashi, M. Matsui, and A. Miyamoto Real-Time Relationships in Intraluteal Release among Prostaglandin F2{alpha}, Endothelin-1, and Angiotensin II During Spontaneous Luteolysis in the Cow Biol Reprod, November 1, 2004; 71(5): 1706 - 1711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Mattot, L. Moons, F. Lupu, D. Chernavvsky, R. A. Gomez, D. Collen, and P. Carmeliet Loss of the VEGF164 and VEGF188 Isoforms Impairs Postnatal Glomerular Angiogenesis and Renal Arteriogenesis in Mice J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1548 - 1560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Villablanca, K. A. Lewis, and J. C. Rutledge Time- and dose-dependent differential upregulation of three genes by 17beta -estradiol in endothelial cells J Appl Physiol, March 1, 2002; 92(3): 1064 - 1073. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Acosta, N. Yoshizawa, M. Ohtani, and A. Miyamoto Local Changes in Blood Flow Within the Early and Midcycle Corpus Luteum after Prostaglandin F2{alpha} Injection in the Cow Biol Reprod, March 1, 2002; 66(3): 651 - 658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.H. Schauser, A.H. Nielsen, H. Winther, V. Dantzer, and K. Poulsen Localization of the Renin-Angiotensin System in the Bovine Ovary: Cyclic Variation of the Angiotensin II Receptor Expression Biol Reprod, December 1, 2001; 65(6): 1672 - 1680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.J. Acosta, T. Ozawa, S. Kobayashi, K. Hayashi, M. Ohtani, W.D. Kraetzl, K. Sato, D. Schams, and A. Miyamoto Periovulatory Changes in the Local Release of Vasoactive Peptides, Prostaglandin F2{alpha}, and Steroid Hormones from Bovine Mature Follicles In Vivo Biol Reprod, November 1, 2000; 63(5): 1253 - 1261. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
