JSAR Young Investigator Award. Studies of Follicular Vascularity Associated with Follicle Selection and Ovulation in Cattle

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1 Journal of Reproduction and Development, Vol. 53, No. 1, 2007 JSAR Young Investigator Award Studies of Follicular Vascularity Associated with Follicle Selection and Ovulation in Cattle Tomas J. ACOSTA 1) 1) Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Okayama , Japan Abstract. We reviewed recent in vivo studies of the real-time changes in the vasculature of the follicle wall during selection of the dominant follicle as well as during ovulation in cows. Changes in follicle diameter and vascularity were determined by transrectal ultrasonography. Blood flow within the walls of the two largest follicles was detected at the time of wave emergence (largest follicle=5 mm in diameter). Before selection of a follicle (largest follicle <8.5 mm in diameter), the degrees of vascularity of the two largest follicles were not significantly different. After the largest follicle reached a diameter of 10 mm, the vascularity of the largest (dominant) follicle was higher than that of the second largest (subordinate) follicle. In the preovulatory follicle, follicular vascularity gradually increased, and as ovulation approached, the LH-surge induced an increase in blood flow within the follicle wall. The above results suggest that maintenance of follicular vasculature and appropriate blood supplies to follicles are essential for establishment of follicular dominance. Consequently, only a dominant follicle with high vascularity may have a chance to reach final maturation and acquire ovulatory capacity. Key words: Blood flow, Cattle, Follicle, Ovulation, Selection (J. Reprod. Dev. 53: 39 44, 2007) n the mammalian ovary, follicular growth is considered to be under the control of pituitary gonadotropins. In the cow, at least three cycles (63 days) are required for a preantral follicle (approximately 0.15 mm in diameter) to attain ovulatory size (approximately 18 mm in diameter) [1, 2]. During the growing phase, follicular responsiveness to follicle stimulating hormone (FSH) and luteinizing hormone (LH) increases drastically. Follicles larger than 2 mm in diameter become responsive to FSH and constitute the population of recruitable follicles. During the process of follicular selection, the future ovulatory follicle becomes more sensitive first to FSH and then to LH. All gonadotropin-induced functions are expressed during maturation of preovulatory Accepted for publication: November 20, 2006 Correspondence: TJ Acosta ( acosta@cc.okayama-u.ac.jp) follicles [3]. Vascular changes are involved in the cyclic remodeling of ovarian tissue that occurs during follicular growth and ovulation. Increase in the blood supply to individual follicles appears to be associated with follicular growth rates and the ability to become the dominant follicle, while reduced thecal vascularity appears to be closely associated with follicular atresia [4]. Therefore, a series of in vivo experiments was conducted to evaluate the changes in diameter and vasculature of the three largest follicles during follicular selection as well as during the last stages of follicular maturation and ovulation. From these in vivo studies, we propose that follicular vasculature and blood supply to individual follicles are critical factors for follicular development allowing follicles to acquire ovulatory capacity.

2 40 ACOSTA and OKUDA Fig. 1. Development of the three largest follicles and surges of FSH during an estrous cycle in a cow with two follicular waves. The data for each wave was normalized to the day of wave emergence (Emer) when the largest follicle reached 5.0 mm. Fig. 2. Images of a preovulatory (PO) and atretic follicle (AT) of similar size showing clear differences in vascularization of the follicular wall. The scale bar represents 1 cm. Adapted from Acosta et al. (2003). Fig. 3. Images of a growing future dominant follicle (8 mm) showing a vascular area in which blood flow was detected by color Doppler ultrasonography (left panel) and depiction of the Doppler power spectrum (right panel). The red color indicates blood flow toward the transducer s face, and the blue color indicates blood flow away from the transducer s face. The color gain for flow mode is set to detect movement of at least 1 cm/sec. The spectrum is used to determine blood flow velocities (peak systolic velocity, peak diastolic velocity, and timeaveraged maximum velocity) and vascular indices (pulsatility index; resistance index). A, B, and C indicate the part of the spectrum used to calculate the above end points. Follicular Growth and Selection Selection of the dominant follicle in cattle occurs from a cohort of growing antral follicles, termed a follicular wave [5 7]. Two or three successive follicular waves occur during the estrous cycle. The first wave (Wave 1) appears around the time of ovulation, and the second appears during middiestrus (Wave 2) (Fig. 1). Following emergence of a group of follicles 4 or 5 mm in diameter, the follicles develop in a common-growth phase for about 3 days. When the largest follicle reaches a mean diameter of 8.5 mm, the common-growth phase ends and a gradual difference in diameters becomes apparent. The largest follicle continues to grow and becomes dominant, and the smaller (subordinate) follicles begin to regress or temporarily grow at a reduced rate and then regress [7]. This important event in follicle selection during a follicular wave in monovular

3 ROLE OF VASCULATURE IN FOLLICLE DEVELOPMENT AND OVULATION 41 species is called diameter deviation [7]. In cattle, estradiol 17β and LH receptors are involved in the intrafollicular events associated with deviation [7]. However, whether the change in follicular vascularity is associated with follicle selection remains unclear. A close interrelationship between follicular diameter and follicular vascularity suggests that follicular vasculature and blood supply to individual follicles play a crucial role in the events leading to selection of the dominant follicle and ovulation in cattle. During follicular growth, an extensive vascular plexus develops in the theca layer (Fig. 2) surrounding the avascular basement membrane and granulosa layer [8]. Morphological studies of bovine follicles have revealed that the major histological characteristic of the selected (dominant) follicle is its increased vascularity in the theca layer as compared with unselected (atretic) follicles. Follicular vasculature is positively correlated with intrafollicular concentration of estradiol 17β in porcine and bovine ovaries [9]. The dominant follicle in the cow is normally identified when it reaches a diameter of 10 mm and is larger than the other follicles of the same wave. At this size, the dominant follicle acquires LH receptors on its granulosa cells and acquires ovulatory capacity in response to LH surge [10]. Color Doppler ultrasonography is a useful, noninvasive tool for evaluating follicular vascularity, allowing visual observations of the blood flow in a delimited area in the walls of preovulatory follicles in humans [11], cows [4], and mares [12]. In humans, blood flow determinations of individual follicles by Doppler ultrasonography provide an index of the intrafollicular environment and may be used to predict the developmental competence of oocytes [13]. In cows, we have previously demonstrated that the vascularity of the follicular wall is clearly different between preovulatory follicles and anovulatory, atretic follicles [4]. The results of two recent in vivo studies suggest that Doppler ultrasonography can distinguish differences in follicular vascularity between the dominant follicle and subordinate follicles [14] or to predict follicular viability after selection in mares [15] and cattle [4, 16] These observations include the time of follicle selection and establishment of follicular dominance. Color Doppler Ultrasonography An example of Doppler images and end points is shown in Fig. 3. After measuring follicular diameters, the flow mode was activated for blood flow mapping. The color Doppler generates signals so that blood flow is detectable within the follicle wall. The vascularity and blood flow within the follicle wall are quantified using color images. The degree of turbulence is indicated by a coded color signal; the brightness of the color is directly proportional to the velocity of flow within the blood vessel. Areas of color represent regions with a flow velocity higher than 1 cm/s. Pulsed Doppler mode was used to record the Doppler power spectrum during 3 cardiac cycles by placing the Doppler gate (1 mm width) across the area that displayed the most intensive color signal in order to determine blood flow velocities and vascular indices. The blood flow velocities (peak systolic velocity, PSV; time-averaged maximum velocity, TAMXV) and vascular indices were then determined. The same image was used to calculate the sectional area, and the colored area was quantified to express the follicle vascularity using the NIH Image program (ImageJ 1.31p) [15]. The ovaries of the cows were scanned by real-time color Doppler ultrasound to determine the blood flow velocities and vascular indices of the follicle walls of the 3 largest follicles of a wave beginning when the follicles were 5.0 mm (wave emergence) and continuing until the largest follicle was 12.0 mm or until ovulation. Ovulation Ovulation is a complex process by which a preovulatory follicle ruptures and releases a fertilizable oocyte into the oviductal lumen. This process occurs as a result of a dynamic interaction between the LH surge and local factors including steroids, PGs, and peptides in a time-dependent manner. The LH surge triggers structural and biochemical changes that lead to rupture of the Graafian follicle, resulting in expulsion of the oocyte and subsequent development of a corpus luteum. The collagen fibers contained in the tunica albuginea, theca layer and basal membrane contribute to the tensile strength of the follicular wall. Degradation of these collagen layers is

4 42 ACOSTA and OKUDA Fig. 4. Changes in the Doppler-derived time-averaged maximum velocity (TAMXV) of the follicle wall during the periovulatory period. The TAMXV was measured in the base of the follicle using color Doppler ultrasonography. The data is shown as the mean ± SEM for each time period (n=5 cows). Values with different superscripts are significantly different (P<0.05). accompanied by increased vascular dilatation and permeability, which are necessary for follicular rupture [17, 18]. The blood flow at the apex of the ovulatory follicle decreases while it increases at the base of the follicle [11], which facilitates follicular rupture. Several studies have reported that luteinizing hormone induces an increase in the ovarian blood flow in rats [19], rabbits [20], sheep [21], and cows [4]. However, the mechanisms of LH-induced hyperemia remain unknown. FSH and estradiol stimulate follicular growth and vascular hypertrophy. The increase in blood flow into the theca layer of the dominant follicle probably results in increased supplies of gonadotropins, other systemic biochemicals, and hormonal factors necessary for complete maturation of the follicle. The Doppler studies provided visual evidence for the time-related changes in blood flow and vascularity of the follicle wall around the time of ovulation [4]. The images of the preovulatory follicle revealed that the blood flow area and velocity (time averaged-maximum velocity; TAMXV) were temporally correlated with an increase in the plasma concentrations of estradiol and the LH-surge (Fig. 4). These findings indicate that a functional relationship exists between blood flow in the follicular wall and the plasma levels of estradiol and LH in the preovulatory follicle around the time of ovulation. We have also demonstrated that following GnRH injection, both follicular blood flow and the plasma concentrations of LH and estradiol increase simultaneously [4]. This phenomenon may induce an acute change in the metabolic function of follicular cells, resulting in increased production of steroids and vasoactive substances. Estrogen has been shown to cause a rapid dilation of blood vessels by activating endothelial nitric oxide synthase (enos). In endothelial cells, estradiol caused acute (5 min) activation of enos that was fully inhibited by concomitant treatment with estrogen receptor ERα antagonists, suggesting that the short-term effects of estrogens are mediated by ERα [22]. This local vasodilatory substance acts not only in the regulation of blood flow in the ovary but also induces apoptosis in ovarian cells [23]. Thus, the increase in blood flow to the preovulatory follicle may increase the supply of gonadotropins and facilitate follicular rupture. Local increases in the concentrations of ovarian Ang-II and PG at the time of ovulation have been observed in cows at the time of ovulation [24]. This indicates that Ang-II and PG are indeed closely related to follicular rupture, and therefore it is very likely that these vasoactive substances have major roles in the ovulatory process controlling follicular blood flow. In vitro studies have shown that LH stimulates follicular ET-1 production and that Ang- II and ET-1 may act in a local cascade-like reaction to accelerate PG production during the preovulatory period. Interestingly, Ang-II directly inhibits the release of ANP from the theca layer of the bovine mature follicle in vitro [25]. This implies that the Ang-II in the theca extra-cellular fluid precisely modulates local ANP and ET production in bovine follicles [26]. Thus, ET-1 and ANP may play a permissive or modulatory role in local ovarian Ang-II production. The complex structural, secretory, and functional changes that take place in the ovary around the time of ovulation are closely associated with a local change in blood flow within the wall of the preovulatory follicle. Conclusion Maintenance of follicular vasculature and

5 ROLE OF VASCULATURE IN FOLLICLE DEVELOPMENT AND OVULATION 43 appropriate blood supply to follicles are essential for establishment of follicular dominance. Consequently, only a dominant follicle with high vascularity has a chance to reach final maturation and acquire ovulatory capacity. Acknowledgments The authors thank Dr. Kiyoshi Okuda (Okayama University, Japan), Dr. O. J. Ginther (University of Wisconsin, Madison, WI, USA) and Dr. Akio Miyamoto (Obihiro University of Agriculture and Veterinary Medicine, Japan) for constructive guidance during the experiments and their criticism for improvement of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (C) no and (B) no from the Japan Society for the Promotion of Science. References 1. Butler W, Smith R. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J Dairy Sci 1989; 72: Fortune JE. Ovarian follicular growth and development in mammals. Biol Reprod 1994; 50: Gougeon A. Intragonadal regulation of human follicular genesis: facts and hypotheses. Annales Endocrinol 1994; 55: Acosta TJ, Hayashi KG, Ohtani M, Miyamoto A. Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows. Reproduction 2003; 125: Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol Reprod 1988; 39: Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. J Reprod Fertil 1988; 83: Ginther OJ, Beg MA, Donadeu FX, Bergfelt DR. Mechanism of follicle deviation in monovular farm species. Anim Reprod Sci 2003; 78: O Shea JD, Hay MF, Cran DG. Ultrastructural changes in the theca interna during follicular atresia in sheep. J Reprod Fertil 1978; 54: Mattioli M, Barboni B, Turriani M, Galeati G, Zannoni A, Castellani G, Berardinelli P, Scapolo PA. Follicle activation involves vascular endothelial growth factor production and increased blood vessel extension. Biol Reprod 2001; 65: Sartori R, Fricke PM, Ferreira JC, Ginther OJ, Wiltbank MC. Follicular deviation and acquisition of ovulatory capacity in bovine follicles. Biol Reprod 2001; 65: Brannstrom M, Zackrisson U, Hagstrom HG, Josefsson B, Hellberg P, Granberg S, Collins WP, Bourne T. Preovulatory changes of blood flow in different regions of the human follicle. Fertil Steril 1998; 69: Gastal EL, Gastal MO, Ginther OJ. Relationships of changes in B-mode echotexture and colour-doppler signals in the wall of the preovulatory follicle to changes in systemic oestradiol concentrations and the effects of human chorionic gonadotrophin in mares. Reproduction 2006; 131: Coulam CB, Googman C, and Rinehart LS. Colour Doppler indices for follicular blood flow as predictors of pregnancy after in-vitro fertilization and embryo transfer. Human Reproduction 1999; 14: Acosta TJ, Gastal EL, Gastal MO, Beg MA, Ginther OJ. Differential blood changes between the future dominant and subordinate follicles precede diameter changes during follicle selection in mares. Biol Reprod 2004; 71: Acosta TJ, Beg MA, Ginther OJ. Aberrant blood flow area and plasma gonadotropin concentrations during the development of dominant-sized transitional anovulatory follicles in mares. Biol Reprod 2004; 71: Acosta TJ, Hayashi KG, Matsui M, Miyamoto A. Changes in follicular vascularity during the first follicular wave in lactating cows. J Reprod Dev 2005; 51: Moor RM, Hay MF, Seamark RF. The sheep ovary; regulation of steroidogenic, haemodynamic and structural changes in the largest follicle and adjacent tissue before ovulation. J Reprod Fertil 1975; 45; Murdoch WJ, Peterson TA, Van Rick EA, Vincent DL, Inskeep EK. Interactive roles of progesterone, prostaglandins, and collagenase in ovulatory mechanism of the ewe. Biol Reprod 1986; 35: Varga B, Horvath E, Folly G, Stark E. Study of the luteinizing hormone-induced increase of ovarian blood flow during the estrous cycle in the rat. Biol Reprod 1985; 32: Janson PO. Effects of luteinizing hormone on blood flow in the follicular rabbit ovary, as measured radioactive microspheres. Acta Endocrinology 1975;

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