Because of rapid growth followed by spontaneous regression, the ovarian corpus luteum (CL) is an excellent model to study angiogenesis in vivo. To evaluate the expression of vascular endothelial growth factor (VEGF) protein during luteal development, ovaries were collected from FSH-stimulated ewes throughout the estrous cycle. VEGF was immunolocalized in tissue sections by using an affinity-purified antibody. VEGF protein localized exclusively to the thecal layer of preovulatory follicles, while the granulosa was devoid of staining. Associated with the periovulatory period was intense expression of VEGF by thecal cells at the basement membrane and subsequent invasion of the granulosa layers by these VEGF-positive cells immediately after ovulation. The early CL showed staining for VEGF in thecal-derived compartments, and strong staining for VEGF was also seen in cells within the granulosa-derived parenchymal lobules. Dual immunohistochemical localization of VEGF and smooth muscle cell alpha-actin indicated that the VEGF-positive cells were capillary pericytes or vascular smooth muscle cells. In another experiment, we quantified proliferation of endothelial cells and pericytes throughout luteal development. Pericytes represented a large proportion of the proliferating cells during the early luteal phase and then decreased dramatically. Perivascular cells, therefore, may play a critical role in angiogenesis that occurs during transformation of the follicle into the highly vascular CL of the sheep. As angiogenesis occurs only at the level of capillaries, and pericytes are integral members of these microvessels, regulation of pericytes may provide a novel mechanism for regulating luteal growth and tissue growth in general.

Nitric oxide (NO) has emerged as a novel regulator of several ovarian events, such as ovulation, steroidogenesis, and apoptotic cell death. The NO synthases (NOS) are a family of enzymes that catalyze the oxidation of L-arginine to NO and L-citrulline. The purpose of the present study was to localize NOS isoforms in the rat ovary and to examine their hormonal regulation. We conducted immunohistochemistry and Western blot analysis using isoform-specific antibodies against brain NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Immature rats were superovulated by injecting PMSG (10 I.U. s.c.) followed by an injection of human CG (hCG; 10 I.U. s.c.) 48 h later. Ovaries were obtained from control rats (no PMSG), 24 h and 48 h after PMSG treatment and 2 h, 8 h, 12 h, 20 h or 6 days and 10 days after hCG injection (n = 3-5 rats/group). Rat ovaries were clearly devoid of brain NOS staining at any of the time points studied. In control ovaries, eNOS was detected in the theca cell layer, ovarian stroma, and on the surface of oocytes. During follicular development, eNOS staining was still expressed in the theca cell layer and was also present in mural granulosa cells. After ovulation, homogenous eNOS staining was observed within cells of the corpus luteum (CL). Western blots of ovarian homogenates demonstrated that during PMSG-induced follicle growth, eNOS levels increased by 2.5-fold relative to control rats (P < 0.05). eNOS levels were further increased 12 h and 20 h after hCG injection (5-fold and 7-fold, respectively, relative to control; P < 0.05). The greatest amount of eNOS was observed in ovaries 10 days after hCG injection (15-fold relative to control; P < 0.05). We also detected expression of iNOS in the ovary, but the pattern and cell-specific staining differed from that observed for eNOS. In immature ovaries and during follicular development, iNOS staining was found within the theca cell layer and stroma. After ovulation, iNOS staining was present only in the external layers of the developing CL, but in the degenerating CL (10 days post-hCG), strong staining in nonparenchymal cells was observed within the entire CL. Western blots showed no changes in levels of ovarian iNOS protein during follicular development, but a significant increase (6-fold relative to control; P < 0.05) was observed after an ovulatory dose of hCG. The highest level of iNOS was observed in ovaries 10 days after hCG injection (10-fold relative to control; P < 0.05). Our data demonstrate that ovarian eNOS and iNOS show distinct cell-specific expression patterns and are differentially regulated during follicular and luteal development.

Evidence supports the involvement of nitric oxide (NO) in ovulation, steroidogenesis, and atresia-related apoptosis. This study was designed to investigate the role of endothelial nitric oxide synthase (eNOS)-derived NO in ovulation, oocyte meiotic maturation, and ovarian steroidogenesis using wild-type (WT) mice and mice in which the gene for eNOS had been deleted (eNOS knock-out). We observed that mature eNOS knock-out females have significantly fewer pups born in each litter and a higher mortality rate of pups than those born to heterozygote or WT females (P < 0.05). To determine the influence of eNOS deficiency on ovarian function, immature WT and eNOS knock-out mice were superovulated by injecting PMSG (5 IU) followed by an injection of hCG (5 IU, i.p.) 48 h later. To determine whether murine oocytes expressed eNOS before (0 and 8 h post-hCG) and after ovulation (16 h post-hCG). we performed immunofluorescent staining. Positive specific staining for eNOS was observed on the surface of ovarian and ovulated oocytes obtained from WT mice, but not on oocytes from eNOS knock-out mice. To determine the role of eNOS-derived NO in ovulation, ovulated oocytes were counted 16 h post-hCG. eNOS knock-out females showed a significant reduction (by 63%; P < 0.0001) in ovulatory efficiency compared with WT females. The reduction in the ovulation rate in eNOS-deficient mice compared with that in WT mice was also associated with a higher concentration of estradiol (P < 0.01) without significant changes in the plasma progesterone level. eNOS deficiency impaired not only ovulation, but also oocyte meiotic maturation. Ovulated oocytes were classified as being in one of the following stages of meiosis: metaphase I, metaphase II, or showing atypical (degenerative) morphology. We observed that fewer oocytes from eNOS knock-out mice had entered metaphase II of meiosis, and a greater percentage remained in metaphase I or were atypical (P < 0.002) relative to those in WT mice. Furthermore, many oocytes that showed either a delay in meiotic maturation or abnormal morphology were undergoing cell death. Our results support a role for NO in the ovulatory process. The ovarian defects observed in the eNOS knock-out mice suggest that eNOS-derived NO is a modulator of oocyte meiotic maturation.

This study was conducted to determine the rates of growth and cellular proliferation of ovine corpora lutea (CL) throughout the estrous cycle. To determine the cellular labeling index (LI), ewes received an iv injection of bromodeoxyuridine (BrdU) 1 h before death on days 2, 4, 8, 12, or 15 (day 0 = estrus; n = 6-12 ewes/day). At death, CL were weighed, and samples of each were fixed in Carnoy's solution or frozen until analyzed for DNA, protein, and progesterone contents. Nuclear incorporation of BrdU was determined in paraffin-embedded tissue sections by using a primary antibody against BrdU and a fluorescent (fluorescein isothiocyanate-labeled) secondary antibody, and sections were counterstained with propidium iodide (a nuclear stain). The labeling index (BrdU-labeled nuclei as a proportion of propidium iodide-labeled nuclei) of each CL was determined by using dual channel interactive laser cytometry and image analysis. Moreover, BrdU and 3 beta-hydroxysteroid dehydrogenase (a marker for steroidogenic cells) or BrdU and factor VIII (a marker for endothelial cells) were immunolocalized in tissue sections by using double immunohistochemical or dual immunofluorescent staining, respectively. Results demonstrated that cellular proliferation was greatest (LI, 34.1 +/- 2.1%) on day 2 and decreased (P < 0.01) through day 15 (LI, 0.7 +/- 0.1%) of the estrous cycle. The results of the immunohistochemical studies provide evidence that both parenchymal (steroidogenic) and nonparenchymal (e.g. endothelial, fibroblastic) luteal cells proliferated throughout the ovine estrous cycle. Conversely, from days 2-12 of the estrous cycle, fresh weight and DNA content of CL increased linearly (P < 0.01; 8- and 10-fold, respectively), then decreased (P < 0.02) from days 12-15. Ratios of protein/DNA on days 2, 4, and 8 were similar and were greater (P < 0.02) than those on days 12 and 15, which also were similar. These data demonstrate that growth of the ovine CL is extremely rapid, linear from days 2-12, and primarily due to hyperplasia. In addition, the high rate of cellular proliferation is associated primarily with nonsteroidogenic cells, a large proportion of which appear to be endothelial cells. Data such as these will enable us to determine the factors that are important in regulating luteal growth and development in normal and pathological conditions.

Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.