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For singleton, twin, and triplet pregnancies, uteri were collected on day 140 of pregnancy. For each ewe (n = 18), placentomes were fixed by arterial perfusion supplying the fetal (cotyledon) and maternal placenta (caruncle). Tissue sections were stained for determination of vascularity by image analysis. Further, protein expression for factor VIII, vascular endothelial growth factor (VEGF) and its receptor, VEGFR1, as well as basic fibroblast growth factor (FGF2) and its receptor, FGFR, in tissue sections was determined by immunohistochemistry and image analyses. Cotyledonary and caruncular samples were analyzed for expression of mRNA for Vegf and its two receptors, Vegfr1 and Vegfr2, as well as Fgf2 and Fgfr. Fetal number did not affect placental capillary density or factor VIII expression, whereas increased fetal number reduced total cotyledon and caruncle capillary volume. While expression of Vegf, Vegfr1, Vegfr2, and Fgfr mRNA in cotyledonary but not caruncular tissue was greater in twin pregnancies compared to singleton and triplet pregnancies, protein expression of VEGF in the placentome decreased with increasing numbers of fetuses, VEGFR1 did not change, and FGFR was greater in twin versus singleton and triplet pregnancies. Fetal number did not affect the expression of Fgf2 mRNA in placental tissues, whereas FGF2 protein expression was less in triplet compared to singleton and twin pregnancies. Reduced fetal and placental weights in twins and/or triplet pregnancies are associated with an overall decrease in total placental vascularity, VEGF and FGF2 and/or FGFR protein expression, but not in angiogenic factor mRNA expression or VEGFR1 protein expression in sheep.

We have previously established an ovariectomized (OVX) ewe model to study how steroid removal and replacement affects uterine blood vessel and tissue growth. Using this model, endometrial expression of mRNA for 14 angiogenic factors (7 genes and their respective receptors) in caruncular (CAR) and intercaruncular (ICAR) endometrium were evaluated by quantitative real time RT-PCR at 0 (control), 2, 4, 8, 16, or 24 h after treating OVX ewes with an estradiol-17beta (E2) implant. In CAR and ICAR, compared to 0 h, the mRNA expression of vascular endothelial growth factor (VEGF), VEGF receptor (R)1, soluble guanylate cyclase (GUCY1B3; the R for nitric oxide [NO]), hypoxia inducible factor (HIF)1alpha, and placental growth factor (PlGF) increased by 4 h after E2-treatment, but basic fibroblast growth factor (FGF2), endothelial NO synthase (NOS3), angiopoietin (ANGPT)1, ANGPT2, ANGPT receptor Tie2 by 2 h after E2. Expression of mRNA for FGFR2 IIIc was increased at 2 h by E2-treatment in ICAR, but not in CAR. By contrast, expression of neuropilin (NP)1 mRNA was increased at 2 h in CAR, but not ICAR. The mRNA expression of VEGF, FGF2, HIF1 alpha, and PlGF was positively correlated with mRNA expression of NOS3, VEGFR1, and Tie2 suggesting some E2-stimulated interactions between these factors in promoting blood vessel growth. Thus, several major angiogenic factors and their receptors are increased within hours after E2-treatment, which indicates that E2 plays a role in regulation of angiogenesis in the uterus. By using the OVX ewe model, we may begin to understand the molecular basis of E2 effects on angiogenesis in the endometrium and, eventually, how angiogenesis is regulated in normal versus pathological conditions.

The objective of current study was to evaluate the expression of Cx37 in ovarian follicles and in corpora lutea (CL) during the estrous cycle in sheep. Ovine Cx37 was cloned and characterized to design speciesspecific probe and primers. In Exp. 1, ovaries were collected on d 13, 14, 15, and 16 of the estrous cycle, or from FSH-induced ewes at 0, 2, 4, 8, 12, 24, and 48 h after hCG treatment on d 15 of the estrous cycle. In Exps. 2 and 3, CL were collected on d 5, 10, and 15 of the estrous cycle, or at 0, 4, 8, 12, and 24 h after prostaglandin F2alpha (PGF2alpha)-induced luteal regression on d 10 of the estrous cycle, respectively. Ovarian tissues (e.g., granulosa cells, theca cells, ovarian follicles, and /or CL) were used for Cx37 immunostaining followed by image analysis or for determination of Cx37 mRNA expression by real-time RT-PCR. We demonstrated that (1) Cx37 protein was expressed in granulosa and cumulus oocyte complex compartments, ovarian blood vessels, and on the luteal cell borders, (2) expression of Cx37 mRNA was greater in granulosa than in theca cells of preovulatory follicles, (3) Cx37 mRNA expression in granulosa but not theca cells was affected by hCG treatment, (4) Cx37 protein and mRNA expression were dependent on the stage of luteal development, and (5) Cx37 expression changed during PGF2alpha- induced luteal regression. Thus, Cx37 may play a role in follicular development and ovulation as well as in luteal tissue growth, differentiation, and regression.

We have demonstrated that vascular endothelial growth factor (VEGF) is expressed in capillary pericytes of the developing corpus luteum (CL) and others have shown that basic fibroblast growth factor (FGF2) and angiopoietins (ANGPT) are present in the CL. VEGF and FGF2 target endothelial cells to initiate angiogenesis and stimulate nitric oxide (NO) production. Conversely, NO may increase VEGF expression by vascular smooth muscle cells and pericytes. To investigate the relationship between these angiogenic factors and NO in the CL, microvascular pericytes and endothelial cells were isolated from CL collected from superovulated ewes (n = 5) on d 9 of the estrous cycle. Pericytes were identified by their morphology in culture and by immunofluorescent staining for smooth muscle cell actin. Pericytes were incubated with or without varying doses of the NO-donor DETA-NO for 8 h. Then, total cellular RNA was extracted from the cells and evaluated for expression of mRNA for VEGF, FGF2, ANGPT1, ANGPT2, and NO receptor, guanylate cyclase 1, soluble beta3 (GUCY1B3), using real-time quantitative RT-PCR. NO caused a dose-dependent increase in VEGF (p < 0.001), FGF2 (p < 0.001), ANGPT2 (p < 0.06), and GUCY1B3 (p < 0.03) mRNA expression. Expression of mRNA for ANGPT1 in luteal pericytes was not affected by the NO treatment. These data provide further evidence of the role of the luteal pericyte and NO in angiogenic factor expression, and of the potential interactions of pericytes with endothelial cells via NO production.

Throughout each estrous cycle, the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are involved in regulation of folliculogenesis. We have shown that LH or FSH affect cellular interactions mediated by gap junctions in bovine granulosa and thecal cells in vitro. To evaluate further the hypothesis that gonadotropins influence gap junctional intercellular communication (GJIC) and expression of gap junctional proteins known as connexins (Cx), throughout antral follicle development, granulosa and thecal cells from large (>10 mm; n = 13), medium (5-10 mm; n = 20), and small (<5 mm; n = 27) follicles were cultured (n = 4 cultures per size) with or without LH, FSH, or LH + FSH for 24 h. GJIC was evaluated (n = 125-150 cells/treatment group) by using the fluorescent recovery after photobleaching technique and laser cytometry. Additionally, Cx43, Cx32, and Cx26 were detected in cultured cells by immunocytochemistry and Cx43 by Western immunoblot analysis. Finally, progesterone production by cultured cells was evaluated by radioimmunoassay. Across all follicles and treatments, GJIC was greater (p < 0.01) for granulosa than thecal cells (4.9 +/- 0.05 vs 3.8 +/- 0.04%/min). For granulosa cells of large and medium follicles, LH and/or FSH did not affect GJIC. For granulosa cells of small follicles, FSH increased (p < 0.05), but LH or LH + FSH had no effect on GJIC. For thecal cells of large follicles, LH increased (p < 0.01) GJIC, whereas FSH or LH + FSH had no effects. For thecal cells of medium and small follicles, LH and/or FSH did not affect GJIC. These results demonstrate that FSH influenced GJIC of granulosa cells from small, but not from medium or large, follicles, and LH influenced GJIC of thecal cells from large, but not from medium or small, follicles. Cx43 was present as punctate staining between granulosa or thecal cells from all cultures, indicating assembled gap junctions. LH + FSH increased (p < 0.05) expression of Cx43 only by thecal cells from large follicles. Cx32 was detected in the perinuclear cytoplasm of cultured granulosa or thecal cells, and in the cytoskeleton of a few cells per culture dish in all sizes of follicles. Cx26 was present in a regular pattern throughout the cytoplasm of granulosa or thecal cells in all sizes of follicles. For granulosa cells from large follicles, progesterone production was stimulated (p < 0.05) with LH or FSH alone but was unaffected by LH + FSH. For granulosa cells from medium and small follicles, progesterone production was unaffected by LH and/or FSH. For thecal cells from all sizes of follicles, LH, FSH, and LH + FSH stimulated (p < 0.05) production of progesterone. These data indicate that LH and FSH influence gap junction function and expression, which likely contributes to the development and maintenance of ovarian follicles.

The ovarian corpus luteum plays a critical role in reproduction because it is the primary source of circulating progesterone. After ovulation, as the corpus luteum forms from the wall of the ruptured follicle, it grows and vascularizes extremely rapidly. In fact, the rates of tissue growth and angiogenesis in the corpus luteum rival those of even the fastest growing tumors. Thus, the corpus luteum provides an outstanding model for studying the factors that regulate the angiogenic process, which is critical for normal tissue growth, development, and function. In agreement with data from other tissues, vascular endothelial growth factors (VEGF) seem to be a major angiogenic factor responsible for vascularization of the developing corpus luteum. Recent data suggest that luteal expression of VEGF occurs primarily in specific perivascular cells, including arteriolar smooth muscle and capillary pericytes, and is regulated primarily by oxygen levels. In addition, soon after ovulation, pericytes derived from the thecal compartment appear to be the first vascular cells to invade the developing luteal parenchyma. The granulosa-derived cells produce a factor that stimulates pericyte migration. Moreover, nitric oxide (NO), which is a potent vasodilator and can stimulate VEGF production and angiogenesis, is expressed in endothelial cells of luteal arterioles and capillaries, often in association with expression of VEGF by luteal perivascular cells. Thus, we have proposed a model for the initial process of luteal vascularization in which hypoxia plays a major role. In this model, which we believe will apply to other tissues as well, a paracrine loop exists between the vascular endothelial cells, which produce NO, and the peri-endothelial cells (vascular smooth muscle and pericytes), which produce VEGF, to ensure coordinate regulation of luteal vasodilation and angiogenesis.

Detection of connexin (Cx) proteins has been used as an indicator of the presence of structural and functional gap junctions in tissues. To examine the role of gap junctions during follicular growth and atresia, the presence of three major connexins, Cx43, Cx32, and Cx26, was evaluated in bovine ovaries by using immunohistochemistry and Western immunoblot analysis. Cx43 was not present in primordial follicles, but was present in granulosa cells of primary/secondary and antral follicles. Cx43 also was present on the borders between granulosa cells and the oocyte. Expression of Cx43 increased in healthy developing antral follicles, but decreased during follicular atresia. Cx32 was not present in healthy follicles but was present in granulosa cells of atretic antral, and especially small antral follicles. Cx26 was present in the oocyte of primordial and primary/secondary follicles, and in the granulosa and/or thecal cell layers of healthy antral follicles. The percentage of healthy antral follicles that expressed Cx26 also increased during follicular development, but decreased during atresia. Cx32 and Cx26 also were detected in ovarian blood vessels and in stromal tissues adjacent to the tunica albuginea in some ovaries. The pattern of expression of these Cx indicates that gap junctional proteins may be involved in the control of follicular growth and atresia in cows.

Wound healing involves the interactions of many cell types, and is controlled in part by growth factors. Intercellular communication mediated by gap junctions is considered to play an important role in the coordination of cellular metabolism duringthe growth and development of tissues and organs. Basic fibroblast growth factor (bFGF), known to be important in wound healing, has been found to increase Cx43 expression and intercellular communication in endothelial cells and cardiac fibroblasts. It has been proposed that an increased coupling is necessary for the coordination of these cells in wound healing and angiogenesis, and that one of the actions of bFGF is to modulate intercellular communication. The aim of our study was to evaluate the effects of bFGF on gap junctional intercellular communication (GJIC) in vitro, and the presence of gap junctional proteins connexin (Cx) 26, Cx32, and Cx43 in fibroblasts of diabetic and nondiabetic individuals. Fibroblast cell lines (n = 10) were cultured for 3 d in serum-free media with or without bFGF (3 ng/mL). Cells were evaluated for the rate of GJIC by using laser cytometry, and for the presence of Cx26, Cx32, and Cx43 by immunohistochemical and Western analyses. All cell types communicated via contact-dependent mechanisms. The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (4.1 +/- 0.01 vs 3.3 +/- 0.01%/min). bFGF increased (p < 0.01) the rate of GJIC for diabetic (4.9 +/- 0.01 vs 4.1 +/- 0.01%) and nondiabetic (4.1 +/- 0.01 vs 3.3 +/- 0.01%) fibroblasts. Immunohistochemistry identified Cx26 in the cytoplasm, Cx32 was not detected, and Cx43 was present on the cellular borders in all cultures. Image analysis of immunofluorescent staining demonstrated that bFGF increased (p < 0.05) Cx43 expression in diabetic and nondiabetic fibroblasts. Western immunoblot analysis revealed bands at 43-46 kD that were similar in volume for diabetic and nondiabetic fibroblasts. Thus, gap junctions involving Cx43 and GJIC among fibroblasts appear to be targets for bFGF. Fibroblasts of diabetic individuals appear to have an increased rate of cell-cell coupling, correlating with a decreased rate of proliferation.

Ovarian follicles from days 13, 14, 15, and 16 and corpora lutea (CL) from days 2, 4, 8, 12, and 15 of the estrous cycle were evaluated for the presence of connexins by immunohistochemistry. In addition, CL from days 5, 10, and 15 of the estrous cycle were used for immunofluorescent detection of Cx43 followed by image analysis, and for Western immunoblot. In all tissues, staining for all connexins appeared punctate, indicating the presence of assembled gap junctions. Cx26 was present in the ovarian surface epithelium, stroma, and blood vessels within the stroma and hilus, and in the CL. In healthy antral follicles, Cx26 was present only in the theca layer, whereas Cx43 was present in granulosa and theca layers. In the majority of atretic follicles, connexins were not detected, but in 13% of the atretic follicles, Cx43 was present in the theca layer. Cx32 was detected in the blood vessels of ovarian stroma and in the CL, and Cx43 was detected in the CL. Localization and/or expression of connexins depended on stage of luteal development. Western analysis demonstrated that expression of Cx32 in luteal tissues was similar across the estrous cycle. The area of positive staining for Cx43 and expression of Cx43 in luteal tissues decreased (p < 0.05) as the estrous cycle progressed. The pattern of expression of connexins indicates that gap junctional proteins may be important in the regulation of folliculogenesis and follicular atresia, as well as growth, differentiation, and regression of the CL.

Cellular interactions mediated by contact-dependent pathways may be important to maintain luteal function. The objective of the present experiment was to evaluate the role of LH and prostaglandin F(2α) (PGF) in regulation of contact-dependent, gap junctional intercellular communication (GJIC) of ovine luteal cells from several stages of luteal development. Corpora lutea (CL) obtained from superovulated ewes on days 5 (n=7), 10 (n=8), and 15 (n=9) after estrus were dispersed with collagenase and cell types were separated by elutriation. Cells were plated as a mixed population (nonelutriated), or as small or large luteal cell fractions, and incubated in serum-free media containing no hormone, LH (100 ng/mL), PGF (100 ng/mL), LH+PGF, or dibutyryl cAMP (dbcAMP; 2 mM) for 18-24 h. Media were collected for evaluation of progesterone (P4) concentrations and replaced with media containing fluorescent dye. Then the rate of GJIC was evaluated by using the fluorescence recovery after photobleaching technique and laser cytometry. The rate of GJIC was determined for selected cells: small luteal cells in contact only with small luteal (S-S) cells; large luteal cells in contact only with small luteal (L-S) cells; and large luteal cells in contact only with large luteal (L-L) cells. LH increased (p<0.01) GJIC for S-S on d 5 and 10 and for L-S cells across the estrous cycle, but did not affect GJIC for L-L cells. PGF increased (p<0.05) GJIC for L-L cells on d 10 and 15, and decreased (p<0.05) GJIC for S-S cells from d 5 and 10 of the estrous cycle. LH+PGF increased (p<0.05) GJIC for S-S cells on d 5 and 10, and for L-S and L-L cells on d 10 and 15 of the estrous cycle. In addition, PGF diminished (p<0.05) LH-stimulatory effects on GJIC for S-S cells from d 5 and 10, and for L-S cells from d 5 of the estrous cycle. Dibutyryl cAMP stimulated (p<0.05) GJIC between all evaluated cell types across the estrous cycle. LH and dbcAMP stimulated (p<0.05) P4 secretion by mixed and small luteal cell fractions, PGF alone did not affect basal P4 secretion, but LH+PGF stimulated (p<0.05) P4 production by small luteal cells across the estrous cycle. PGF diminished (p<0.05) LH-stimulatory effects on P4 production in mixed populations of luteal cells across the estrous cycle.These data demonstrate that both luteal cell types communicate with each other, and the rate of communication was affected by LH, PGF, and dbcAMP. Modulation of gap junctional contact-dependent intercellular communication may be an important mechanism by which regulatory signals are transduced during luteal growth, differentiation, and regression in sheep.