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Ovary |
Department of Agricultural and Life Science4
Field Center of Animal Science and Agriculture,5 Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
Departamento de Fisiologia,6 Facultad de Ciencias Veterinarias Universidad Nacional de Asuncion, 1061 San Lorenzo, Paraguay
Institute of Physiology,7 Technical University of Munich, 85350 Freising-Weihenstphan, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicular development, granulosa cells, ovary, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Angiogenesis, the formation of a new network of blood vessels, is essential for follicular development and ovulation [4, 5]. The selected dominant follicle has a high degree of vascularity, which results in preferential delivery of gonadotropins compared with the other follicles [6] and may affect follicular steroidogenesis. Atresia of nonovulatory follicles is accompanied by the regression of the thecal blood vessel network [5, 7, 8]. Thus, there is a close relationship between status of the follicular blood vessel network and the degree of follicular function.
Recent findings have suggested that angiopoietin (ANPT)-1, ANPT-2, and endothelial cell-specific receptor tyrosine kinases Tie1 and Tie2 may play an important role in the modulation of vascular growth and regression in the cyclic ovary [9–14]. Generally, the ANPT-Tie system is thought to act in concert with growth or survival factors such as vascular endothelial growth factor (VEGF). In this system, ANPT-1 is necessary to maintain and stabilize blood vessels [15]. ANPT-2, which acts as a natural antagonist for ANPT-1, appears to cause endothelial cells to undergo active remodeling [15]; thus, ANPT-2 destabilizes blood vessel structure.
The balance between the ANPT-2:ANPT-1 ratio and VEGF expression is important for angiogenesis and blood vessel regression [10, 12]. An increase in the ANPT-2:ANPT-1 ratio is associated with destabilization of blood vessels, which is a prerequisite for new blood vessel formation. The destabilized blood vessels appear to face one of two fates. When VEGF is high, active angiogenesis results in the formation of a new blood vessel network (high ANPT-2:ANPT-1 ratio, high VEGF), whereas a lack of VEGF support results in a regression of blood vessels (high ANPT-2:ANPT-1 ratio, low VEGF). A low ANPT-2:ANPT-1 ratio with low VEGF results in a stabilization of blood vessels.
The ANPT-Tie system seems to affect luteal function by regulating angiogenesis and blood vessel regression in the corpus luteum (CL) in the cow [10]. Other researchers have reported that mRNAs for ANPT and Tie are expressed in the ovarian follicles of the rat and the primate [9, 11, 13]. However, the expression of the ANPT-Tie system has not been examined in bovine follicles. To investigate the role of the ANPT-Tie system during follicular development and atresia, we examined the mRNA expression of ANPT-1, ANPT-2, Tie1, and Tie2 in developing, mature, and atretic bovine follicles. In addition, we used a microdialysis system (MDS) implanted within the thecal layer in vitro to examine whether ANPTs directly influence steroid secretion in the preovulatory follicle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Entire reproductive tracts from German Fleckvieh cows were collected at a local slaughterhouse in Germany within 10–20 min after slaughter and were transported on ice to the laboratory. The stage of the estrous cycle was defined by macroscopic observation of the ovaries (color, consistency, CL stage, and number and size of follicles) and the uterus (color, consistency, and mucus) [16]. Only follicles that appeared healthy (i.e., well vascularized with transparent follicular wall and fluid) and whose diameter was >5 mm were used. Large follicles (>14 mm) were collected only after CL regression, with signs of mucus production in the uterus and cervix, and were assumed to be preovulatory. The follicles were classified according to the estradiol-17ß (E2) content in follicular fluid (FF) as follows: 1) <0.5 ng/ml FF, 2) >0.5–5 ng/ml FF, 3) >5–20 ng/ml FF, 4) >20–180 ng/ml FF, and 5) >180 ng/ml FF. Hereinafter, we refer to follicles in categories 1–4 as developing follicles and to those in category 5 as mature follicles. The corresponding size of follicles were in the range of 1) 5–7 mm, 2) 8–10 mm, 3) 10–13 mm, 4) 12–14 mm, and 5) >14 mm. Because healthy follicles have relatively constant progesterone (P4) levels in FF, only follicles with P4 of <100 ng/ml FF were used for evaluation to exclude atretic follicles. Follicle status was further confirmed by mRNA expression for FSH receptor and aromatase cytochrome P450 in granulosa cells (GCs) and LH receptor in theca interna (TI) and GCs in a companion study already published [17].
Collection and Classification of Atretic Follicles
Ovaries were collected from Holstein cows at a local slaughterhouse in Japan and transported on ice to the laboratory. All follicles whose diameter was >8 mm were separated from ovaries. Collected follicles were classified based on E2 and P4 concentration in FF as E2 dominant (E2 > 100 ng/ml FF) or P4 dominant (E2 < 10 ng/ml FF and P4 > E2). To check the condition of GCs, total RNA was extracted from GCs collected by scraping the inside of the follicular wall. Follicles with an undetectable level of total RNA in GCs were defined as follicles in which there were no intact GCs. P4-dominant follicles were further divided into three groups based on FF P4 concentration and condition of GCs: early atretic follicles (P4 15–70 ng/ml FF with GCs), midatretic follicles (P4 > 100 ng/ml FF with GCs), and late atretic follicles (P4 > 100 ng/ml FF, no GCs detected).
Collection of Preovulatory Follicles for the MDS Experiment
Three ovaries were collected from two Holstein cows. These cows were kept under a normal management program of the Field Center and fed daily with corn silage, hay, and concentrate, with permanent free access to water. Experimental procedures complied with the Guide for Care and Use of Agriculture Animals of Obihiro University. The cows were induced to superovulate using a standard decreasing-dose regimen of twice daily i.m. injections of FSH (Antrin10; Denka Pharmaceutical Co., Kawasaki, Japan) over 4 days (total dose: 28 units). On the third day of the FSH treatment, cows were treated with a luteolytic dose of 500 µg of the prostaglandin (PG) F2
analogue cloprostenol (Estrumate; Sumitomo Pharmaceutical Co., Osaka, Japan). Ovariectomy was performed at 60 h after PGF2 injection. Preovulatory follicles were dissected from ovaries and subjected to MDS study. At the end of the MDS experiment, steroids (P4, E2) and PGE2 concentrations in FF were determined after extraction, confirming that these follicles had experienced an LH surge as previously described [18].
Follicular Tissue Preparation
Follicles were cleared of the surrounding tissues with forceps under a stereomicroscope. After aspiration of FF, the follicles were bisected, GCs were gently scraped and flushed with PBS, and the TI was peeled off from the remaining follicular wall. All procedures were done on ice within 1 h after follicle collection. Follicular tissues were frozen and stored at -80°C until RNA isolation.
Hormone Determinations
Hormone assays were done after extraction with diethyl ether as described previously [18]. The concentrations of the different hormones were determined in duplicate by double-antibody enzyme immunoassay (EIA) using 96-well ELISA plates (Corning Glass Works, Corning, NY). The EIA for P4, E2, androstendione (A4), and PGE2 was performed as previously described [18–20]. The standard curve ranged from 0.05 to 50 ng/ml for P4, from 2 to 2000 pg/ml for E2, from 8 to 8000 pg/ml for A4, and from 0.02 to 20 ng/ml for PGE2. The ED50 values of the assay for P4, E2, A4, and PGE2 were 1.0 ng/ml, 70 pg/ml, 250 pg/ml, and 1.8 ng/ml, respectively. The average intra- and interassay coefficients of variation were 6.5% and 9.7% for P4, 6.3% and 9.5% for E2, 6.8% and 9.1% for A4, and 9.5% and 12.0% for PGE2, respectively. The average postextraction recoveries for P4, E2, A4, and PGE2 were 85%, 80%, 82%, and 74%, respectively.
Semiquantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted by guanidium acid-isothiocynate-phenol-chloroform methods [21]. RNA was dissolved in water and spectroscopically quantified at 260 nm, and its integrity was confirmed by gel electrophoresis. Total RNA (2 µg) was used to generate single-strand cDNA in a 60-µl reaction mixture by use of hexanucleotides as primers according to the protocol for the M-MLV Reverse Transcriptase Kit (Promega, Madison, WI). The optimal amount of total RNA and reaction mixture for reverse transcription (RT) was evaluated by testing different RNA concentrations. The primers for ANPT-1, ANPT-2, Tie1, Tie2, and ß-actin were designed using an online primer design tool, Primer 3 [22], based on the bovine sequences obtained from the Entrez Nucleotides database (National Center for Biotechnology Information, Bethesda, MD). Twenty-six amplification cycles were used, and the primer sequences and resulting fragment sizes for all examined genes are shown in Table 1. The 25-µl polymerase chain reaction (PCR) mixture contained 3 µl cDNA, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.6 µM each primer, and 0.5 units of thermostable polymerase PrimeZyme (Biometra, Götingen, Germany). All amplifications were done as follows: an initial denaturation step at 94°C for 2 min, 26 cycles of 94°C for 30 sec, 55°C for 1 min, and 68°C for 2 min, and a final elongation step at 68°C for 2 min. To determine the optimal quantity of reverse transcripts needed for PCR and to verify that cDNA product was dependent on the amount of transcript used, various quantities of transcript template and different numbers of cycles were tested in the PCR assay. To exclude any contaminating genomic DNA, all experiments included controls lacking the reverse transcriptase. As a negative control, water was used instead of RNA for the RT-PCR to exclude any contamination from buffers and tubes. Aliquots of the PCR products (10 µl) were fractionated by electrophoresis using a 2% agarose gel containing ethidium bromide, and bands were visualized under ultraviolet light. The signal intensity was analyzed by computerized densitometry using the Image Master Program (Luminous Imager version 2.0G; Aisin Cosmos RD Co., Aichi, Japan). Relative abundance of target mRNA was estimated using the standard curve. The value was normalized using ß-actin as an internal standard.
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MDS In Vitro
The MDS for bovine preovulatory follicles has been described previously [18]. Each follicle was dissected from surrounding stromal tissue, and two to four capillary dialysis membranes (Fresenius SPS 900 Hollow Fibers, cutoff molecular size 1000 kDa, 0.2 mm diameter, 5 mm long; Fresenius AG, St. Wendel, Germany) were implanted into the thecal layer with at least 5 mm between capillaries. The capillaries were affixed to the surface of the follicular tissue by Histoacryl blue (B. Braun-Dexon GmbH, Spangenberg, Germany). Both ends of the capillary were glued to silicone elastomer tubing (inside diameter of 0.3 mm). For perfusion, one end of the tube was connected to a peristaltic pump and the other was routed to a fraction collector. The prepared follicles were then placed in organ culture chambers (modified 2070 tube; Falcon, Franklin Lakes, NJ) filled with 50 ml Medium 199 (Sigma Chemical Co., St. Louis, MO) containing Earle salts, 10 mM NaHCO3, 365 mg/L L-glutamine, 25 mM Hepes, 5 g/L BSA, 60 mg/L penicillin, 100 mg/L streptomycin, 56 mg/L ascorbic acid, and 2 mg/L amphotericin B at pH 7.4. The chambers were maintained in a water bath at 38°C throughout the complete period of perfusion. The medium was continuously exchanged at a flow rate of 15 ml/h. During incubation, follicles were perfused with Ringer solution at a flow rate of 3 ml/h. After 2 h of preperfusion, fractions of the perfusate were collected every 2 h up to 12 h. Collected samples were stored at -30°C until hormone determination.
Human recombinant ANPTs (ANPT-1, ANPT-2; Regeneron Pharmaceuticals, Tarrytown, NY) were diluted in Ringer solution to obtain the required final concentrations of 10 and 100 ng/ml. The doses were determined based on the result of a preliminary experiment and on the transfer capacity of the membrane, which was previously estimated to be 0.1% [23]. The solutions that contained ANPT-1 or ANPT-2 were infused into the MDS during the middle 4 h of the 12 h culture period.
Statistical Analyses
The significance of differences in mRNA expression of examined genes was assessed by ANOVA, followed by a Scheffé F test as a multiple comparison test. Differences in ANPT-2:ANPT-1 ratio were analyzed for significance using a Dunnett test.
The mean hormone concentrations in the first two fractions (first 4 h perfusion with Ringer solution only) were used to calculate the individual baseline for each hormone because of a large variation in the absolute amount of hormone released into each of the MDS lines implanted in the various follicles. All values were expressed as a percentage of the corresponding baseline. To simplify the view of figures, the values of the first two perfusates were pooled, and they are shown in a single column as a baseline (100%). The effects of the ANPT-1 and ANPT-2 on the release of steroids were tested by comparison with the individual baselines using ANOVA followed by a Student t-test. The absolute concentrations of the hormones in the MDS fractions (mean ± SEM) are given in the figure captions. All experimental data are shown as mean ± SEM. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The signals for mRNA encoding ANPT-1, ANPT-2, Tie1, and Tie2 were detected in the TI, but signals for only ANPT-1 and ANPT-2 were detected in GCs (Fig. 1). Because the TI expressed both ANPTs and Tie mRNA and because the vascular network is restricted to the TI, the expression of mRNA for the ANPT-Tie system in the TI during follicular development and atresia was analyzed by semiquantitative RT-PCR in the further studies.
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Expression of mRNA for ANPT-1, ANPT-2, Tie1, and Tie2 in TI During Follicular Development
The expression of ANPT-1 mRNA did not change in follicles with different E2 concentrations (Fig. 2B). The expression of ANPT-2 mRNA in mature follicles with E2 > 180 ng/ml FF was lower than that in the other developing follicles (Fig. 2C). The ANPT-2:ANPT-1 ratio in the TI of mature follicles was lower than that in the follicles with E2 > 5–20 ng/ml FF (Fig. 2D). Tie1 and Tie2 mRNA expression in the follicles were unchanged throughout follicular development (data not shown).
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Expression of mRNA for ANPT-1, ANPT-2, Tie1, and Tie2 in TI During Follicular Atresia
ANPT-1 and ANPT-2 mRNA expression in the TI did not change during follicular atresia (Fig. 3, B and C). The differences in the value of ANPT-1 and ANPT-2 mRNA expression were large among each follicle. In individual follicles, however, expression of ANPT-2 mRNA was always higher than that of ANPT-1 mRNA. Therefore, the ANPT-2:ANPT-1 ratio in early atretic follicles was higher than that in the E2-dominant follicles (Fig. 3D). The expression of Tie1 mRNA in early atretic follicles was higher than that in the E2-dominant follicles (Fig. 4B). Tie2 mRNA expression also was greater in early atretic follicles but decreased markedly by the time follicles reached the mid and late stages of atresia (Fig. 4C).
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Effects of ANPT-1 and ANPT-2 on the Release of Steroid Hormones from Bovine Preovulatory Follicles In Vitro
An increase in P4 release was observed during infusion of ANPT-1 (10 and 100 ng/ml, P < 0.01; Fig. 5) and after infusion of ANPT-2 (100 ng/ml, P < 0.01). Both ANPT-1 and ANPT-2 treatments inhibited the release of A4 after infusion with 100 ng/ml (P < 0.05). ANPT-1 (10 and 100 ng/ml) increased E2 secretion during infusion (P < 0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ANPTs act as major modulators of angiogenesis by binding to their receptor, Tie, which is expressed in vascular endothelial cells [15]. The vascular network in the bovine follicle is well developed in the TI [4, 5], suggesting that it is the major site of ANPT action. In fact, the results of the present study indicate that the TI expresses both ANPT and Tie mRNA, whereas GCs express ANPT mRNA but not Tie mRNA. These findings suggest that the ANPT-Tie system in the TI plays a major physiological role in local angiogenesis and/or vascular function in bovine follicle. In addition, it is possible that ANPTs produced in GCs may also bind to their receptor Tie in the TI in a paracrine fashion. In support of this idea, GCs in primate tertiary follicles do not express Tie2 mRNA and receptor protein [13]. Furthermore, the expression of ANPT-1 and ANPT-2 mRNA is restricted to the TI of the rat preovulatory follicle [9]. These findings indicate that TI is the major site of ANPT production and action. Accordingly, the work described here focused on the local expression of the ANPT-Tie system in the TI during bovine follicular growth and atresia.
The decrease in the ANPT-2:ANPT-1 ratio in the mature follicles in the present study indicates that this ratio shifts from high (destabilized vessels) to low (stabilized vessels) as follicular maturation progresses. In a companion study using follicular samples identical to those in the present study, Berisha et al. [17] showed that VEGF and its receptor mRNA is expressed in bovine follicular tissues and that the concentration of VEGF in FF increases throughout follicular development. These observations indicate that a high ANPT-2:ANPT-1 ratio and increasing expression of VEGF occurs in developing follicles and that active angiogenesis occurs in these follicles. These observations are also in accordance with the reports showing the increase in the number of blood vessels during follicular development [5, 24]. In contrast, a reduction of the ANPT-2:ANPT-1 ratio in mature follicles indicates that the blood vessels are relatively stable and that angiogenesis is slowed down despite the high levels of VEGF [17]. Because mature follicles used in the present study were >14 mm and therefore fully grown [1], it is possible that angiogenesis had been completed in these follicles and only maintenance of blood vessels was required. VEGF upregulates vascular permeability [25]; thus, VEGF might enhance vascular permeability rather than promote angiogenesis in the mature follicles.
It has been reported that the number of thecal blood capillaries decreases during follicular atresia in various species, including the cow [5, 7, 8]. In addition, atretic bovine follicles exhibit increased apoptotic structures in blood capillaries and decreased numbers of healthy capillaries compared with healthy follicles [5]. In rat atretic follicles, ANPT-2 mRNA was highly expressed in the granulosa layer [9]. In out study, the ANPT-2:ANPT-1 ratio increased in bovine early atretic follicles. An increase in Tie2 mRNA was also observed at the same stage. These findings indicate that the ANPT-Tie system in early atretic follicles is upregulated and may lead blood vessels toward destabilization. The expression of VEGF and its receptor (Flt-1) mRNA is reduced in primate atretic follicles [13]. Thus, we propose that the activated ANPT-Tie system with low VEGF (i.e., high ANPT-2:ANPT-1 ratio, low VEGF) turns blood vessels toward regression in the early atretic follicles. Importantly, Tie2 mRNA expression dramatically decreased in both mid and late atretic follicles, indicating that the ANPT-Tie system is completely disrupted at later stages of atresia. Consequently, these findings lead us to speculate that the change of the ANPT-Tie system in the bovine follicle may be involved in the induction of follicular atresia.
Using in vitro MDS, we demonstrated that ANPT-1 and ANPT-2 inhibited A4 release and stimulated P4 release from preovulatory follicles obtained from cows after gonadotropin surge. A decrease in A4 secretion and an increase in P4 secretion are also observed in the bovine preovulatory follicle after the LH surge [26–28]. A recent study demonstrated that the ANPT-2:ANPT-1 ratio in preovulatory follicles was increased after hCG treatment, suggesting that blood vessels were destabilized [11]. New capillaries in preovulatory follicles begin to invade from the thecal layer to the granulosa layer after the LH surge [29]. Our observations suggest that the ANPTs may be modulators that affect follicular steroidogenesis in preovulatory follicles, but the mechanism involved has not yet been identified.
In the present study, treatment with ANPTs also affected E2 secretion in the preovulatory follicles; however, the mechanism of this action is not clear. The synthesis of E2 occurs only in GCs in bovine follicles, but the results of the present study clearly showed that the ANPT receptor Tie2 is expressed only in the TI. These results indicate that ANPTs exert their effect in a paracrine manner.
The results of the present study show that mRNA expression for ANPT-1, ANPT-2, Tie1, and Tie2 changes during follicular development, maturation, and atresia in antral bovine follicles and that both ANPT-1 and ANPT-2 affect steroidogenesis in the preovulatory follicle. These results suggest that the ANPT-Tie system is involved in the structural (angiogenesis) and secretory changes that occur during follicular development and atresia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 81 155 49 5593; akiomiya{at}obihiro.ac.jp
3 Current address: Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI 53706
Received: 14 March 2003.
First decision: 2 April 2003.
Accepted: 6 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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denotes significant difference between groups (P < 0.05)
