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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.006734
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Biology of Reproduction 67, 1846-1852 (2002)
DOI: 10.1095/biolreprod.102.006734 © 2002 Society for the Study of Reproduction, Inc.

Ovary

Changes of Messenger RNA Expression of Angiogenic Factors and Related Receptors During Follicular Development in Gilts1

Takashi Shimizu2,a, Jin-Yi Jianga, Hiroshi Sasadaa, and Eimei Satoa

a Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Aoba-ku,> Sendai 981-8555, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction between angiogenic factors and related receptors is closely associated with follicular angiogenesis. The present study was performed to determine the relationships between the capillary network and mRNA expression of several angiogenic factors and related receptors during porcine follicular development. Ovaries in gilts were collected 72 h after eCG (1250 IU) treatment for histological observation. Granulosa cells and thecal tissues in small (diameter, <4 mm), medium (diameter, 4–5 mm), or large (diameter, >5 mm) individual follicles were collected for detection of mRNA expression of vascular endothelial growth factor (VEGF) 120, VEGF 164, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in granulosa cells and fms-like tyrosine kinase (Flt-1), fetal liver kinase (Flk-1) or the murine homologue of kinase domain region (KDR), bFGF receptor (bFGF-R), and EGF receptor (EGF-R) in thecal tissue by semiquantitative reverse transcription-polymerase chain reaction. The eCG treatment resulted in the emergence of healthy preovulatory follicles (diameter, >6.0 mm) that possessed more capillaries in the thecal cell layer and a significant increase in the percentage of atretic follicles of 1.0–2.9 mm in diameter. The number of capillaries in the thecal cell layer increased significantly in healthy follicles larger than 3 mm in diameter in the eCG group compared with those in controls. The expression of VEGF 120, VEGF 164, and bFGF mRNAs increased in granulosa cells of medium and large follicles from ovaries of prepubertal gilts after eCG treatment. The Flt-1, Flk-1/KDR, and bFGF-R mRNA expression increased in theca cells of medium and large follicles after eCG treatment. The expression of EGF mRNA increased in granulosa cells of small, medium, and large follicles from ovaries after eCG treatment, but the mRNA expression of EGF-R in thecal tissue did not change. These data indicate that preovulatory follicles possessed a larger capillary network and expressed more mRNAs of angiogenic factors in granulosa cells and related receptors in thecal tissue. We concluded that VEGF 120, VEGF 164, bFGF, and EGF may be greatly involved in the angiogenic process of follicular development in prepubertal gilts with eCG treatment.

follicle, follicular development, growth factors, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis plays an important role in follicular development. The capillary network in dominant follicles is both more extensive and more permeable than that in other follicles [1], and such follicles are able to acquire an increased uptake of serum gonadotropins, a variety of hormones, and growth factors [2]. The capillary network limited to the thecal cell layer during follicular development [3] is stimulated by angiogenic factors. A recent study reported that vascular endothelial growth factor (VEGF), known as a potent mitogen for endothelial cells [4] and as a stimulator of vascular permeability [5, 6], was expressed in granulosa cells isolated from pig follicles [7]. Moreover, it has been demonstrated that the short-term inhibition of angiogenesis after anti-VEGF antibody administration during the later growth phase of the dominant follicle interferes with its normal development [8], and the blocking of VEGF action by treatment with a soluble, truncated form of the fms-like tyrosine kinase (Flt-1) receptor resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area, and a marked decline in Flt-1 mRNA expression [9]. These studies suggest that VEGF is associated with follicular development in the mammalian ovary.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) also have angiogenic action in the ovary [10–14]. The bFGF mRNA was predominantly localized to the granulosa cells of the dominant follicles of the rat ovary [15]. In the bovine ovary, bFGF mRNA expression in theca interna increased significantly during the final growth of follicles, whereas its expression in granulosa cells was very weak [16]. Previous studies indicated that EGF is an angiogenic factor [17, 18] and showed it to enhance the proliferation of vascular endothelial cells in vitro [19] and to affect neovascularization in vivo [14]. The EGF is soluble in tissue fluid, can be translocated in tissues, and induces endothelial cells to proliferate and form capillaries [19]. Immunocytochemical studies showed that EGF peptide was localized in the cumulus cells and granulosa cells [20] and in the thecal and interstitial cells around growing follicles [12, 21].

Angiogenic factors such as VEGF, bFGF, and EGF act via their receptors in target cells. Two phosphotyrosine kinase receptors for VEGF, such as Flt-1 and fetal liver kinase (Flk-1) or the murine homologue of kinase domain region (KDR), share 85% sequence identity with human KDR [22–26], bFGF receptor (bFGF-R), and EGF receptor (EGF-R). The Flt-1, Flk-1/KDR, and bFGF-R mRNAs are expressed in theca interna of bovine ovarian follicles [16]. In the porcine ovary, a very strong EGF-R mRNA signal was observed in the cumulus, granulosa, and theca cells [20].

Most of the above studies investigated a single angiogenic factor during follicular development. It would be valuable, in terms of a better understanding of angiogenic regulation in follicles at different developmental stages, to investigate the mRNA expression of several angiogenic factors and their related receptors in the same follicle classes. Furthermore, to our knowledge, the mRNA expression of bFGF, bFGF-R, Flt-1, and Flk-1/KDR has not been studied in porcine follicles. Therefore, we investigated the capillaries in the thecal layer and the mRNA expression of VEGF 120, VEGF 164, bFGF, and EGF in granulosa cells and of Flt-1, Flk-1/KDR, bFGF-R, and EGF-R in the thecal layer in individual follicles isolated from the ovaries of prepubertal gilts after eCG treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Hormone Treatment

Sixteen prepubertal gilts at 3 mo of age with an average weight of 65 kg were used and divided in two groups. One group was injected i.m. with 1250 IU [7] of eCG (Teikoku Zouki Pharmaceutical Co., Tokyo, Japan) to induce follicular development, and the other was injected with saline as the control. After anesthetization by injection of ketamine hydrochloride (6 ml/gilt; Sankyo Co. Ltd., Tokyo, Japan) and atropine sodium salt (0.5 mg/gilt; Tanabe Co. Ltd., Tokyo, Japan), eight animals from each group (16 animals total) were ovariectomized 72 h [27–29] after eCG or saline injection to examine the follicular population (right ovaries of four animals from each group), to observe vascular construction (left ovaries of the above four animals from each group), and to detect mRNA expression (ovaries of the other four animals from each group). The present study was approved by the Ethics Committee for Care and Use of Laboratory Animals for Biomedical Research of the Graduate School of Agricultural Science, Tohoku University.

Histological Examination of Follicles> and Follicular Capillaries

The right ovary from each animal was fixed in 4% (w/v) paraformaldehyde solution. After fixation, the ovary was embedded in paraffin wax and sectioned serially at a thickness of 8 µm. Every 10th section was mounted and stained with hematoxylin-eosin. All antral follicles greater than 0.5 mm in diameter were counted in every section. To avoid counting individual follicles more than once, oocytes with nuclei were used as a mark, and the size of the follicle in which the oocyte was present was measured by means of an ocular micrometer. Each follicle was classified as either healthy or atretic in the absence or presence, respectively, of 10 pyknotic bodies in the granulosa cells of the section. On the basis of their diameter, the follicles were separated into five classes: 0.5–0.9, 1.0–2.9, 3.0–4.9, 5.0–6.0, and >6.0 mm. All capillaries larger than 10 µm in diameter in the section of whole theca interna of follicles larger than 3.0 mm in diameter were counted under 200x magnification (i.e., 20x objective lens and 10x ocular lens). Respectively, 29, 19, and 26 follicles of 3.0–4.9, 5.0–6.0, and >6.0 mm in diameter were counted (one section per follicle) to quantify the average numbers of follicular capillaries.

Scanning Electron Microscopy of Corrosion Casts

The left ovaries were kept in warm, heparinized saline solution until perfusion. Samples of corrosion casts for scanning electron microscopy were prepared according to previously reported methods [30–32]. Briefly, the ovary was perfused with heparinized saline solution followed by a solution of Mercox (Dainippon Ink and Chemicals, Tokyo, Japan) through the ovarian artery. The cast ovary was then warmed in hot water (60°C) for 2 h, corroded in 10% NaOH at 60°C, washed in running warm water, and then dried in a hot oven. The dried samples were glued onto aluminum stubs and coated with platinum. The observation was performed with a scanning electron microscope (S-4200; Hitachi, Tokyo, Japan).

Analysis of mRNA Expression of Angiogenic Factors> and Related Receptors

The ovaries were collected from prepubertal gilts with (n = 4) or without (n = 4) eCG treatment, and follicles were isolated from each ovary and classified as described previously [7]. Briefly, single follicles were isolated in dissection medium (Dulbecco phosphate-buffered medium supplemented with 0.4% BSA). The follicle walls obtained from each follicle were directly transferred to dissection medium to mechanically separate the granulosa cells from the theca shells by gently scraping the follicles with a small spatula. The medium containing dispersed granulosa cells was collected and centrifuged, and the theca shells were then vigorously vortexed and carefully washed to remove any possible granulosa cell contamination. Theca shells and granulosa cells were then stored in liquid nitrogen for analysis of mRNA expression of angiogenic factors and related receptors.

Each total cellular RNA was extracted from the granulosa and thecal tissues of each follicle with the RNeasy Mini Kit (Qiagen K.K., Tokyo, Japan) and quantified using an ultraviolet (UV)-visible recording spectrophotometer (UV-160; Shimadzu Corporation, Tokyo, Japan). Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using Ready To Go RT-PCR Beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) following the method provided by the manufacturer with 500 ng of total RNA. The RT reaction was carried out at 42°C for 15 min, and samples were incubated for reactions at 95°C for 5 min to inactivate the reverse transcriptase and to denature completely the template. The oligonucleotide primers for angiogenic factors and related receptors as well as the amplification profiles (dissociation, annealing, extension, and cycle) are shown in Tables 1 and 2, respectively. The RT-PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. The bands were quantified by densitometry using the NIH Image 1.63 analysis program (National Institutes of Health, Bethesda, MD). ß-Actin mRNA has been found in pig follicle cells with levels that are independent of follicle status and size [33]. Its expression is not affected by growth factors and gonadotropins [34, 35]. Therefore, the gene mRNA levels in the present study were normalized on the basis of ß-actin mRNA content. The follicles were classified on the basis of diameter (small, <4 mm; medium, 4–5 mm; and large, >5mm), and the results were from six follicles in each class.


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  		TABLE 1. Primer pairs used for detection of mRNAs


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  		TABLE 2. Polymerase chain reaction conditions used for detection of mRNA

Data Analysis

All data are presented as the mean ± SEM. Significant differences in the number of follicles at the different developmental stages and in each of the genes among the three developmental stages between control and eCG groups were analyzed by ANOVA followed by the Fisher least significant difference test as a multiple-comparison test. The percentages of atretic follicles at the different developmental stages were analyzed by the chi-square test. Differences were considered to be significant at P < 0.05 or less.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Follicular and Vascular Population Following> eCG Treatment

In the control group, no follicles developed beyond 6.0 mm in diameter, and the number of follicles within each size category decreased as the follicles grew (Fig. 1A). The eCG treatment resulted in the emergence of preovulatory follicles (diameter, >6.0 mm) (Fig. 1A). The number of follicles of 0.5–0.9 mm in diameter was significantly higher in the eCG group than in the controls. Significantly higher percentages of atretic follicles of 1.0–2.9 mm were found, but none larger than 5.0 mm in diameter was observed in the eCG treatment group compared with the controls (Fig. 1B).



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  		FIG. 1. The number of follicles (A) and percentage of atretic follicles (B) at different developmental stages in prepubertal gilts with (n = 4) or without (n = 4) eCG injection. Values with different superscripts in A are significantly different from each other at P < 0.05. An asterisk in B denotes a significant difference from others at P < 0.05

Although the number of capillaries in the theca interna in healthy follicles larger than 3 mm in diameter did not change in the controls, eCG treatment significantly increased the capillary population as the follicles grew (Figs. 2 and 3). The number of capillaries in the atretic follicles did not change in both groups (data not shown).



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  		FIG. 2. Capillaries in the inner plexus of large follicles (diameter, 7 mm) from gilts treated with eCG (a) and of those (diameter, 5 mm) from controls (b) are shown. Note the well-developed capillaries with higher density in a compared with those in b. Bar = 200 µm



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  		FIG. 3. The number of capillaries in theca interna of antral follicles larger than 3 mm in diameter in ovaries of prepubertal gilts with (n = 4) or without (n = 4) eCG injection. Values with different superscripts are significantly different from each other at P < 0.05

Expression of VEGF 120 and VEGF 164 mRNAs> in Granulosa Cells and Flt-1 and Flk-1/KDR mRNAs> in Theca Shells

The mRNA levels of VEGF 120 and VEGF 164 in granulosa cells in the control group remained unchanged at different developmental stages, whereas those in the eCG group increased significantly in medium and large follicles compared with small ones (Fig. 4, A and B). The expression of VEGF 120 and VEGF 164 mRNAs in granulosa cells was significantly greater in medium and large follicles of the eCG group compared with those of controls. The patterns of increased expression for Flt-1 and Flk-1/KDR mRNAs in theca shells in both groups were basically similar to those of VEGF 120 and VEGF 164 in the granulosa cells (Fig. 5, A and B).



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  		FIG. 4. Expression of VEGF 120 (A), VEGF 164 (B), bFGF (C), and EGF (D) mRNAs in granulosa cells of small (diameter, <4 mm), medium (diameter, 4–5 mm), and large (diameter, >5 mm) follicles from prepubertal gilts with (closed bar) or without (open bar) eCG injection. The expressions of each factor were normalized on the basis of ß-actin mRNA content. The data are indicated as the mean ± SEM (n = 6). Different superscripts denote statistically different values (P < 0.05)



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  		FIG. 5. The expression of Flt-1 (A), Flk-1/KDR (B), bFGF-R (C), and EGF-R (D) mRNAs in theca shells of small (diameter, <4 mm), medium (diameter, 4–5 mm), and large (diameter, >5 mm) follicles from prepubertal gilts with (closed bar) or without (open bar) eCG injection. The expressions of each factor were normalized on the basis of ß-actin mRNA content. The data are indicated as the mean ± SEM (n = 6). Different superscripts denote statistically different values (P < 0.05)

Expression of bFGF mRNA in Granulosa Cells> and bFGF-R mRNA in Theca Shells

The expression of bFGF mRNA in granulosa cells in the control group did not significantly change among follicular developmental stages, whereas its expression in the eCG group increased significantly in the medium and large follicles to a greater extent than in the small ones (Fig. 4C). The mRNA levels of bFGF in the medium and large follicles were significantly higher in the eCG group than in controls. The expression of bFGF-R mRNA in theca shells paralleled the expression of bFGF in granulosa cells (Fig. 5C).

Expression of EGF mRNA in Granulosa Cells and EGF-R mRNA in Theca Shells

The mRNA level of EGF in granulosa cells in both groups remained unchanged among the three follicle sizes. However, the expression of EGF mRNA in all developmental stages was significantly higher in the eCG group than in controls (Fig. 4D). The levels of EGF-R mRNA in theca shells were not significantly different between the two groups (Fig. 5D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that preovulatory follicles in eCG-treated gilts possessed more capillaries in theca interna and expressed more mRNAs of VEGF 120, VEGF 164, bFGF, and EGF in granulosa cells and of Flt-1, Flk-1/KDR, and bFGF-R in theca shells. The EGF-R was expressed in theca shells, but it was not increased by eCG. These findings suggested that VEGF, bFGF, and EGF are important factors in angiogenesis during follicular development. The eCG treatment had several effects on the follicles at different developmental stages of prepubertal gilt ovaries. It resulted in the emergence of preovulatory follicles (diameter, >6.0 mm) that have more capillaries in theca interna. In addition, it induced an increase in the number of capillaries in follicles of 3.0–6.0 mm in diameter. Data from the present study support the previous idea that increased vascularity may be an important determinant of follicular dominance [36]. We observed that the rate of atresia in follicles of 1.0–2.9 mm in diameter increased more in the eCG group than in controls. This may be caused by the phenomenon that preovulatory follicles possess more capillaries, dominantly acquire uptake of serum gonadotropins, and have a greater variety of hormones and growth factors compared with other antral follicles. The present study also indicated that the number of follicles of 0.5–0.9 mm in diameter increased more in the eCG group than in controls, suggesting that these increases compensate for the decreased number of follicles of 1.0–2.9 mm in diameter.

The human VEGF gene has five isoforms of 121, 145, 165, 189, and 206 amino acids (VEGF121–206) by alternative splicing of the VEGF mRNA [37, 38]. The porcine VEGF is shorter by one amino acid as compared to the human VEGF, but a potential glycosylation site is present at Asn-74 [39]. Although VEGF 120 and VEGF 164 are known to have different heparin-binding abilities [40] and to be important in the recruitment of endothelial cells into organs, especially during neovascularization [41], the detailed biological differences in these isoforms during follicular development remain unknown. Our results showed that the expression of VEGF 120 and VEGF 164 mRNAs increased in granulosa cells of medium and large follicles after eCG treatment. Gonadotropins have been shown to stimulate the production and expression of VEGF in vitro [42, 43]. We found that eCG induced the expression of VEGF 120 and VEGF 164 mRNAs in granulosa cells in vivo. Especially, the differences between the control and eCG groups in VEGF 120 mRNA expression in medium and large follicles were larger than those of VEGF 164, suggesting that VEGF 120 may be a major mediator in angiogenesis during porcine follicular development. The VEGF mRNA expression is regulated by a variety of factors. Insulin-like growth factor I (IGF-I) has been shown to induce VEGF mRNA in cultured colorectal carcinoma cells [44]. In the ovary, FSH stimulates the production of IGF-I in porcine granulosa cells in vitro [45]. Thus, in the present study, the increased VEGF mRNA expression in granulosa cells may be caused by IGF-I produced by eCG. The VEGF acts via two tyrosine kinase family receptors, namely Flt-1 and Flk-1/KDR [4, 46]. Immunolocalization for Flt-1 was observed in the cytoplasm of theca cells of preovulatory follicles in human [47]. Our results indicated that the expression of Flt-1 and Flk-1/KDR mRNA increased in thecal tissue of medium and large follicles after eCG treatment and paralleled the expression of VEGF 120 and VEGF 164 mRNA, suggesting that expression of Flt-1 and Flk-1/KDR might be activated by VEGF isoforms. The expression of Flt-1 and Flk-1/KDR was reported to be affected by hypoxia, although to a lesser extent than that of VEGF [48]. However, the effect of hypoxia on angiogenic receptors in thecal tissue during follicular development is still unknown.

In the present study, the expression of bFGF mRNA in granulosa cells was observed in medium and large follicles after eCG treatment. The bFGF has been reported to inhibit apoptotic cell death in cultured granulosa cells in vitro [49], and it is involved in mitogenic and angiogenic activity in corpus luteum [50]. We observed that bFGF-R mRNA was expressed in thecal tissue and paralleled the expression of bFGF mRNA in granulosa cells. Therefore, the present results support a previous hypothesis that this factor may be involved in vascularization in the theca interna [51]. In addition, EGF has angiogenic action such as that of VEGF and bFGF. In the present study, the expression of EGF mRNA in granulosa cells increased with eCG treatment at all developmental stages, whereas the expression of EGF-R mRNA in theca shells was unchanged. The EGF causes the suppression of granulosa cell apoptosis [49] and granulosa cell proliferation [52]. The exposure of quiescent human keratinocytes to EGF resulted in a marked induction of VEGF mRNA expression [53]. In addition, EGF stimulated VEGF release by cultured glioblastoma cells [54]. Therefore, our results suggest that EGF stimulates the granulosa cell proliferation and is indirectly associated with formation of the capillary network during porcine follicular development.

The present study found that mRNA expression of angiogenic factors and related receptors changed at different developmental stages. In fact, in prepubertal gilts after eCG treatment, expression of VEGF isoforms and bFGF mRNAs was markedly increased in granulosa cells of medium and large follicle, and EGF mRNA expression was significantly increased in all follicle sizes examined. Moreover, we observed that the number of capillaries increased as the follicles grew. These findings suggest that the active angiogenesis of the capillary network in the thecal cell layer might be initiated in medium follicles (diameter, >4 mm) during porcine follicular development.

In conclusion, our results demonstrated that porcine preovulatory follicles induced by eCG treatment possessed a greater capillary network in the thecal cell layer and expressed mRNAs of VEGF 120, VEGF 164, bFGF, and EGF in granulosa cells and of Flt-1, Flk-1/KDR, and bFGF-R in thecal tissue. These data suggest that interactions between granulosa and theca cells may be mediated through these factors and their receptors and may be involved in perifollicular angiogenesis during porcine follicular development.


   ACKNOWLEDGMENTS
 
We thank the undergraduate students (Nobuyuki Endo and Ikuo Tomioka) and graduate students (Kanako Miyabayashi, Takuya Wakai, Eiji Mizutani, Koji Iijima, and Yasuyuki Abe) in our laboratory for their assistance.


   FOOTNOTES
 
1 Supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences. Back

2 Correspondence: Takashi Shimizu, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.> FAX: 81 22 717 8687; shimizut{at}bios.tohoku.ac.jp Back

Received: 25 April 2002.

First decision: 15 May 2002.

Accepted: 1 July 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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