Numerous reports suggest a key role of the inflammatory mediator, prostaglandin E2 (PGE2), in the process of follicular growth [1–3]. In mice, targeted disruption of the cyclooxygenase-2 (COX-2) gene results in female infertility [4,5]. In macaque follicles, COX2 mRNA was detected in granulosa cells (GCs) after human chorionic gonadotrophin administration . Follicular PGE2 synthesis is dramatically increased in the hours preceding ovulation in several species [7–11]. PGE2 is involved in the differentiation process of ovarian follicle. The investigators demonstrated the expression of COX2 and the production of PGE2 by the cumulus cells during in-vivo and in-vitro maturation . Moreover, PGE2 was found to induce cumulus expansion in vitro .
The development of new blood vessels in the ovary (angiogenesis) is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth . PGE2 has been strongly implicated as a positive regulator of angiogenesis . Overexpression of COX2 and increased production of PGE2 in colon epithelial cells have been associated with the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), which in turn induces endothelial cell migration and microvascular tube formation . Treatment of endothelial cells with selective COX2 inhibitors has been shown to reduce microvascular tube formation and this effect is partially reversed by cotreatment with PGE2 . COX2 expression was detected in newly formed blood vessels within tumors grown in animals, whereas quiescent vessels under normal conditions express only COX1 . PGE2 has been shown previously to upregulate the expression of VEGF in a number of different cell types including rheumatoid synovial fibroblast cells , cultured rat Muller cells , osteoblast cells , human monocytic cell line, isolated perfused rat lung , and in lung tissue . Mechanisms of angiogenesis in the follicular wall remain to be poorly understood.
Sources of PGE2 are numerous in the ovary. PGE2 can be derived from resident macrophages, follicular GCs, and leukocytes . PGE2 has been proposed as an important mediator of follicle maturation and angiogenesis. To our knowledge, the precise contribution of exogenous PGE2 to both processes was not reported in the literature. Therefore, this study was carried out to investigate the effect of exogenous injection of PGE2 on ovarian follicle growth, maturation, follicular angiogenesis, ovarian expression of VEGF in correlation with the effect of pregnant mare serum gonadotropin (PMSG). Morphologic, morphometric, and immunofluorescent techniques were used in the study.
Materials and methods
The study investigated the effect of exogenous injection of PGE2 on the ovary by means of the following:
- (1) Follicle growth through analysis of follicle (tertiary and ovulatory) count and diameter in comparison with the control and PMSG;
- (2) Follicular angiogenesis through quantitative assessment of the follicular microvessel density (MVD);
- (3) Ovarian expression of VEGF.
Immature rats (22 days postpartum) were used in this study to avoid confusion with the physiological events and the influence of steroid on ovarian follicles across the estrous cycle, which changes ovarian composition.
Animals and drug treatments
Immature female albino rats with approximately 50 g body weight and 22 days old were used in the study. Rats were purchased from the Research Unit and the Bilharzial Research Center of Faculty of Medicine (Ain Shams University, Egypt). Rats were maintained under routine conditions with free access to food, water, and 12 h light:12 h darkness. Animals were divided into three groups (10 animals/group): group 1 (control group) where rats received a single dose of 50 μg of normal saline. Group 2 (PMSG group) where rats received a single dose of 20 IU of PMSG (Sigma-Aldrich, St. Louis, Missouri, USA). Group 3 (PGE2 group) where rats received a single dose of 50 μg of PGE2 (Sigma-Aldrich).
Injections were administrated through the intraperitoneal route. All animals were killed 24 h later using a lethal dose of ether.
Ovarian histology and morphometry
After killing the rats, ovaries were extracted and immediately fixed in 10% formalin in water, processed, and embedded in paraffin. The embedded ovaries were serially sectioned (5 μm). Paraffin sections were stained with hematoxylin and eosin. For morphologic and morphometric analysis, every 10 of 100 sections/ovary were examined under a Vanox light microscope (Olympus, Tokyo, Japan) as described previously and validated [24,25]. Tertiary and ovulatory follicles were counted per ovarian section using an image analyzer. The longest diameter of the follicle was measured using the image analyzer [24,25].
- (1) Anti-CD34 antibody (Serotec Ltd, Melborne, Victoria, Australia; 1 : 20);
- (2) Anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA; 1 : 100);
- (3) Fluorescein-conjugated rabbit antibody as the secondary antibody (Dako, Botany, NSW, Australia, Inc.; 1 : 40).
Ovarian sections were deparaffinized in xylene baths, rehydrated through descending grades of alcohol, and rinsed in phosphate-buffered saline (PBS, pH 7.2). Sections were then fixed in cold acetone for 10 min at room temperature. For immunofluorescent staining of the CD34 antigen, antigen retrieval was performed by heating the slides at 100°C in 0.01 mol/l citrate buffer (pH 6.0) for 15 min . Slides were left to cool and then washed in cold running tap water. Thereafter, the slides were preincubated with normal rabbit serum in a 1 : 50 dilution in PBS with 1% bovine serum albumin (Sigma, Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min to block nonspecific reactions. Subsequently, the slides were incubated with the primary antibodies prepared in 2% bovine serum albumin in PBS at their appropriate dilutions for 2 h at room temperature. After washing in PBS (3×1; 5 min each), slides were incubated with Fluorescein isothiocyanate-conjugated secondary antibody for 30 min at room temperature. After the final wash, glass cover slips were mounted on glass slides with mounting fluid and sealed with clear nail varnish. Slides were examined under a fluorescence microscope equipped with filters for discrimination of fluorescein.
A computer-assisted image analysis system was used to analyze the stained slides. Light microscopic images were captured and then transformed into 32-bit color images. For transformation, a digital camera (Fujix HC-2000; Fuji Photo Film, Tokyo, Japan) attached to a light microscope (Vanox AHBS3; Olympus) was used, which was connected to a computer.
Assessment of immunofluorescent staining
Vascular endothelial growth factor staining
The number of immunoreactive follicles (for anti-VEGF antibody) in each stained ovarian section was counted and the mean of these readings in serial stained sections was calculated.
Single or small cluster of positive immune-stained endothelial cells (using anti-CD34 antibody) away from large vessels were considered as representing a new single microvessel. The sections were first examined at a low power (×100) to identify the area with the highest density of microvessels, after which all follicular microvessels in three ×200 power fields (0.7 mm2) were counted and the average of the readings was taken as the MVD.
The data were analyzed using the program statistical package for social sciences (SPSS, IBM SPSS-The Analytic Professional, USA). Student's t-test was used to evaluate the differences with regard to follicle count, diameter measurements, and also the number of positive stained follicles or cells in the immune-stained sections. The probability level of P value less than 0.05 was considered statistically significant.
Group 1 (control)
Examination of hematoxylin and eosin-stained ovarian sections of the control group showed that the surface of the ovary was covered with fibrous tissue capsule (tunica albuginea) and it was divided into outer cortex and inner medulla. The cortex revealed cellular connective tissue stroma that contained the ovarian follicles, whereas the medulla showed abundance of loose connective tissue (Fig. 1). Ovarian follicles observed were in different stages of maturation: primordial, primary, or secondary follicles. Primordial follicles showed an oocyte surrounded by a single layer of flattened follicular cells. In primary follicles, the oocyte was surrounded by a single layer of cuboidal GCs, whereas in secondary follicles the oocyte was surrounded by multiple layers of cuboidal GCs without an antrum (Figs 2 and 3). Healthy follicles were recognized by having a normal-shaped oocyte surrounded by GCs that were regularly apposed on an intact basement membrane, with a normal appearance of GC nuclei without signs of pyknosis. Tertiary (antral) follicles were follicles containing an antrum, and ovulatory (mature) follicles characterized by large continuous antral space were also seen (Figs 4 and 5).
Groups 2 and 3
PMSG (Fig. 6) and PGE2 (Fig. 7) groups demonstrated a remarkable reduction in ovarian interstitial connective tissue and an obvious increase in the number of growing follicles at different stages of maturation.
Under the light microscope and using serial ovarian sections from the three study groups, counting of tertiary and ovulatory follicles revealed that PGE2 demonstrated a highly statistical significant increase in follicle count (28.75±1.10, P<0.001) compared with the control (12.75±1.70). Similarly, PMSG group revealed a mean follicle count of 34.75±1.10 (P=0.004). Histogram 1 demonstrates follicle counts for all groups.
Measurement of diameters of tertiary and ovulatory follicles in ovarian sections of the three groups revealed a highly statistical significant increase of follicle diameter in PGE2 group (5.28±0.17, P<0.03) and the PMSG group (5.52±0.21, P<0.005) versus the control group (3.3±0.09). Histogram 2 illustrates follicle diameter measurements.
Localization of positive immune-stained endothelial cells representing newly formed follicular microvessels using anti-CD34 antibody was detected primarily inside the theca cell layer of both secondary and preovulatory follicles. Figures 8–10 illustrate micrographs for immunofluorescent staining with CD34 antibody in the control (Fig. 8), PGE2 (Fig. 9), and PMSG (Fig. 10) groups. An obvious increase in the number of positive immune-stained cells is detected in PGE2 and PMSG groups compared with the control group.
Assessment of follicular MVD through enumeration of positive immune-stained cells revealed that PGE2 and PMSG groups demonstrated intense follicular neovascularization compared with the control group (P<0.03) (Histogram 3). Microvessel count was 22.6±1.66 in the PGE2 group, 21.3±0.9 in the PMSG group, and 7.2±0.58 in the control group.
Expression of vascular endothelial growth factor
Positive immune staining was observed in ovarian sections of the three groups. The density of immune labeling was nearly similar in the control (Fig. 11), PGE2 (Fig. 12), and PMSG (Fig. 13) groups.
Enumeration of the total number of positively stained follicles in ovarian sections of the three study groups was 35±0.8 in the PMSG group, 31±1.3 in the PGE2 group, and 30±0.9 in the control group with no statistically significant difference (Histogram 3).
This study demonstrated the effects of exogenous PGE2 administration on the processes of follicular growth and angiogenesis in the immature rat in vivo using morphologic, morphometric, and immunofluorescent techniques. In addition, the study investigated ovarian VEGF expression after PGE2 administration.
Extensive studies in the literature suggest a crucial role for PGE2 in follicle growth [1–12]. However, the impact of exogenous PGE2 treatment on follicle growth was not the subject of investigation. In this study, exogenous treatment with PGE2 resulted in follicle growth manifested by an increase in the number and size of growing follicles. PGE2 increased follicle count and diameter measurements in a comparable manner with PMSG.
Many factors contribute to follicular development, such as gonadotrophic hormones. Several studies demonstrated that gonadotrophic hormone administration induced the expression of COX2 and the production of PGE2 by the follicular cells of ovarian follicles [27–31]. These investigations delineated that gonadotrophic hormones affect the ovary through PGE2. In contrast, exogenous PG injections were found to coordinate the preovulatory hormone surge [32,33]. In the hypothalamus, PGs have been shown to stimulate the secretion of luteinizing hormone releasing hormone , which leads to the release of the gonadotropins . In addition, Ojeda et al.  have reported that indomethacin inhibits the secretion of gonadotrophin-releasing hormone from the hypothalamus. Lau and Saksena  have observed that a single injection of 1 mg of PGE2 stimulates the release of LH in overiectomized rats.
Another mechanism that may be involved in PGE2-induced follicular growth is the crucial role of PGE2 in cellular mitogenesis and survival [38,39], a process that is essential for follicular growth and maturation. In colon epithelial cells, PGE2 has been shown to promote proliferation and survival of cells through inhibition of apoptosis. It is reasonable that in the rat ovary, PGE2 may be utilizing similar mechanisms that promote follicular cell proliferation.
Vascular density is quantitatively assessed by counting vessels labeled with antibodies to different endothelial cell markers on both frozen and paraffin-embedded immune-stained sections. In this study, CD34 antibody, being a highly sensitive and specific marker for endothelial cells of blood vessels, was used . In this study, exogenous PGE2 treatment resulted in intensive staining for the CD34 marker compared with the control. Quantitative estimation of new microvessel formation in ovarian follicles, termed as MVD (follicular MVD), revealed a significant increase versus the control. This study documented follicular neovascularization as a result of exogenous PGE2 injection in the rat ovary. Recently, COX2 expression and PGE2 production have been linked directly with endothelial cell function and angiogenesis . COX2 induced the production of angiogenic factors by colon cancer cells, leading to tumor angiogenesis . Masferrer et al.  demonstrated that COX2 inhibitors have antiangiogenic activity where they effectively inhibited mouse corneal angiogenesis, whereas COX1 inhibitors did not. Angiogenesis in wound healing is regulated by COX2 and PGE2 . Preretinal neovascularization was found to be PGE2-mediated in vivo .
Some studies suggest that PGE2 induces angiogenesis through VEGF upregulation. However, conflicting results have been reported with regard to the correlation of the proangiogenic protein, VEGF, and the process of angiogenesis. In both benign and malignant gastrointestinal tumors, upregulation of VEGF was associated with tumor angiogenesis [42,43]. In contrast, other investigators showed an absence of a significant positive correlation between MVD and VEGF expressions [44,45], suggesting that other factors may play a more important role in the process of angiogenesis.
In this study, exogenous PGE2 injection did not result in overexpression of ovarian VEGF compared with the control. The data are consistent with the abovementioned studies. PGE2 might induce follicular angiogenesis through other mechanisms. Some studies suggest that PGE2 plays a key role in the release and activity of proangiogenic proteins (other than VEGF), which directly stimulates endothelial cell migration and angiogenesis in vivo, and enhances vascular endothelial cell survival by upregulation of the antiapoptotic proteins [15,46]. Exogenous PGE2 treatment might utilize similar mechanisms to induce follicular angiogenesis. Future studies are needed to explore the precise mechanism of action of PGE2.
In summary, data presented in this study clarified that exogenous PGE2 induced ovarian follicle growth and angiogenesis. Expression of VEGF was not enhanced after PGE2 administration. Exogenous PGE2 treatment may prove to be a successful approach for the treatment of infertility cases in clinical practice.
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