The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe

• Published in: Theriogenology Vol. 73; no. 7; pp. 856 - 872
• Main Authors: Chowdhury, M.W.H., Scaramuzzi, R.J., Wheeler-Jones, C.P.D., Khalid, M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-04-2010; [Oxford]: Butterworth-Heinemann; [New York]: Elsevier Science
• Subjects: angiogenesis growth factor; Angiogenic Proteins - analysis; Angiogenic Proteins - genetics; angiopoietin; Angiopoietin-1 - analysis; Angiopoietin-1 - genetics; Angiopoietin-2 - analysis; Angiopoietin-2 - genetics; angiopoietin-Tie systems; Angiopoietins; Animals; blood flow; blood vessels; dimensions; Estrous cycle; Estrous Cycle - metabolism; Ewe; ewes; Female; follicular development; Gene Expression; granulosa cells; Granulosa Cells - chemistry; histology; hormonal regulation; Immunohistochemistry; In Situ Hybridization; messenger RNA; morphogenesis; Ovarian follicle; Ovarian Follicle - anatomy & histology; Ovarian Follicle - chemistry; Ovarian Follicle - metabolism; ovarian follicles; Receptor, TIE-1 - analysis; Receptor, TIE-1 - genetics; Receptor, TIE-2 - analysis; Receptor, TIE-2 - genetics; receptors; RNA, Messenger - analysis; Sheep - metabolism; temporal variation; Theca Cells - chemistry; thecal cells; Vascular endothelial growth factor (VEGF); Vascular Endothelial Growth Factor A - analysis; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor Receptor-2 - analysis; Vascular Endothelial Growth Factor Receptor-2 - genetics; vascular endothelial growth factors; Ewe; Estrous cycle; Ovarian follicle; Vascular endothelial growth factor (VEGF); Angiopoietins
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2009.10.011

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n
=
5) or before (n
=
5), during (n
=
4), and after (n
=
4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by
in situ hybridization using digoxigenin-11-UTP–labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2
mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5
mm) and medium (2.5 to 4.0
mm) follicles, but large follicles (>4.0
mm) expressed higher mRNA and protein levels (all P
<
0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.