Expression of Vascular Endothelial Growth Factor Receptors in Bovine Cystic Follicles

Cystic follicles have excess fluid derived from blood flow in the theca interna of the follicle; therefore, the vasculature network is related to cystic follicle formation. Vascular endothelial growth factor (VEGF) is a potent stimulator of blood vessel permeability and angiogenesis. The aim of this study was to examine the expression of VEGF receptors proteins and mRNA in cystic follicles to elucidate the VEGF system in cystic follicles. The expression of protein for VEGF receptors; fms‐like‐tyrosine kinase‐1 (Flt‐1) and foetal liver kinase‐1 (Flk‐1) was detected by the immunohistochemical method. The mRNA expression of Flt‐1 and Flk‐1 in cystic follicles was determined by RT‐PCR. Concentration of oestradiol‐17β and progesterone in the follicular fluid of cystic follicles was determined using ELISA. Flt‐1‐ and Flk‐1 proteins were localized in granulosa and theca interna cells and endothelial cells of theca layers. The intensity of Flt‐1 and Flk‐1 immunoreaction was similar among cystic follicles with various ratios of oestradiol‐17β/progesterone concentrations. The expression of Flt‐1 and Flk‐1 mRNA was similar, regardless of the ratio of oestradiol‐17β to progesterone in follicular fluid. These results demonstrate that cystic follicles have both VEGF receptors in the granulosa and theca interna layers, which may be responsible for the increased permeability of microvessels, causing the accumulation of follicular fluid in cystic follicles.     

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4–8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.     

Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

Comparison of cadherin and integrin localization in bovine cystic and healthy follicles

As stage progresses in the cystic follicle, granulosa cells are lost. We hypothesized that the granulosa and theca interna layers are detached in association with weakened expression of cell adhesion molecules such as cadherin (cell–cell adhesion) and integrin (cell–extracellular matrix adhesion) in cystic follicles. To elucidate this hypothesis, we immunolocalized these molecules in the granulosa and theca interna and compared them between cystic and small healthy follicles. Sections were immunostained with cadherin and integrin β₁ antibodies and their localizations were compared. Cadherin‐positive reaction was seen in the cytoplasma of all granulosa cells. No increase in the frequency of cadherin‐positive area in the granulosa layers and the intensity of cadherin immunoreaction in the theca interna was detected in cystic follicles compared with healthy ones. A dense immunoreaction product of integrin β₁ was detected in the theca interna in both cystic and healthy follicles. Intensity of integrin β₁‐immuno reaction in the granulosa layers and integrin β₁‐positive area in the theca interna was significantly lower in the cystic follicle than in the healthy follicles. These results suggest that granulosa and theca interna cells are detached while maintaining the cell–cell adhesion, resulting in the consequent loss of these layers from the cystic follicle.     

Tumor necrosis factor-alpha (TNF alpha) inhibits progesterone and estradiol-17beta production from cultured granulosa cells: presence of TNFalpha receptors in bovine granulosa and theca cells

The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.     

Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

Immunohistochemical localization of 3beta-hydroxysteroid dehydrogenase in the granulosa and theca interna layers of bovine cystic follicles

The aim of the present study was to determine whether the alteration of population of cells containing 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is responsible for the formation of cystic follicles. Paraffin sections of healthy (2 to 5 mm in diameter), atretic (2 to 5 mm) and cystic follicles (more than 25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine 3beta-HSD. The 3beta-HSD-positive cells were counted in 4 different regions of the follicles from the apical to the basal side. The frequencies of 3beta-HSD-positive granulosa cells in cystic follicles were significantly higher than those in the healthy follicles (P<0.05), although the number of 3beta-HSD-positive granulosa cells in the cystic follicle were fewer than half the cells (30 to 40%) and was much smaller than that in preovulatory follicles (Conley et al., 1995). The frequencies of 3beta-HSD-positive cells were higher in the granulosa layer and lower in the theca interna layer of the cystic follicles than the atretic follicles. These results suggest that the differentiation of granulosa cells to express 3beta-HSD might be insufficient in cystic follicles and accordingly they fail to ovulate. The differences of frequencies of 3beta-HSD-positive cells in the granulosa and theca interna layers between cystic and atretic follicles may be one of the reasons why regression is delayed in cystic follicles.     

Intraovarian localization of growth factors in induced cystic ovaries in rats

We hypothesized that the special hormonal environment present in animals with cystic ovarian disease (COD) interferes with cellular production of growth factors (GFs). The objective of the present study was to characterize the expression of insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in induced COD using immunohistochemistry. We used an experimental model based on the exposure to constant light of adult rats during 15 weeks. We quantified the expression of GFs in cystic and normal ovaries by the Immunohistochemical Stained Area (IHCSA). In animals with COD, a significant reduction in the IHCSA of IGF-I in the follicular fluid, theca and granulosa layers of cysts occurred; and an increase in the interstitial tissue with regard to the control group. We found moderate immunoreactivity of FGF-2 in granulosa and theca layers of secondary and tertiary follicles and lower expression in the granulosa and theca interna layers of cystic follicles. Immunoexpression of VEGF was found in granulosa and theca cells of secondary and tertiary follicles. This study shows changes in the ovarian expression of IGF-I, FGF-2 and VEGF in induced COD. We can propose that an alteration in the control of the follicular dynamic, through the GFs, added to other features, could be involved in the ovarian cyst pathogenesis.     

Characteristics of developing prolonged dominant follicles in cattle

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.     

Lectin histochemical pattern on the normal and cystic ovaries of sows

The lectin histochemical pattern (LHP) was characterized and compared in normal and cystic ovaries of sows. Six biotinylated lectins (PNA, SBA, WGA, RCA‐1, DBA and UEA‐1) were used on tissue sections. In the normal ovaries, the reaction to UEA‐1 and SBA was mild to moderate in mesothelial and endothelial cells. RCA‐1 staining was mild to moderate in theca interna of growing follicles, corpora luteum and mesothelium. In addition, this lectin presented strong reaction in endothelial cells, granulosa cells of atretic follicles, zona pellucida of growing follicles and plasma. DBA showed strong intensity in mesothelial and endothelial cells. There was mild to moderate reactivity to WGA in granulosa cells, corpus luteum and theca interna of follicles in development, and moderate in zona pellucida, in granulosa cells of atretic follicles and mesothelium. PNA staining was mild to moderate in oocytes and in the adventitia and media of medullary arteries. Changes in the LHP of the cystic ovaries were noted; however, there were no differences in these findings between the follicular and luteinized cysts. UEA‐1 reactivity in the cystic ovaries was moderately reduced in the mesothelial and endothelial cells, whereas there was mild reduction in the DBA staining in the granulosa cells. Reaction to RCA‐1 and WGA in the cysts also was decreased in theca interna, zona pellucida and granulosa cells of atretic follicles. Furthermore, endothelium and theca interna in the cystic ovaries presented mild reduction of marcation to SBA, whereas there was decreased reactivity to PNA in the oocytes and adventitia and media layers of the medullary arteries. The results of the current study show that cysts modify the LHP in swine ovaries. These changes of glycoconjugates in many ovarian structures could modify diverse process and may be one of the reasons for decreased fertility in sows.     

Insulin-like growth factor I in sera, ovarian follicles and follicular fluid of cows with spontaneous or induced cystic ovarian disease

The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle.     

Cell Proliferation in the Atretic Follicles of Buffalo and Cattle Ovary

The present study was carried out to describe the proliferative activity of granulosa and theca cells in healthy antral and atretic follicles of Philippine buffaloes (BU) and Holstein-Friesian (HF) cows. Paraffin sections of ovary were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). Then the follicles were classified into healthy and various stages of atretic follicles. The granulosa layer of healthy follicles had a significantly higher frequency of PCNA-positive cells than the early and advanced atretic follicles in both breeds. In the theca interna, significantly reduced populations of the PCNA-positive cells were found in both breeds as atresia progressed. Moreover, HF had significantly higher PCNA-positive cells in the theca interna of healthy, early atretic and advanced atretic follicles than BU. A reduction of PCNA-positive cells during atresia was also noted in the theca externa in both animals although differences were not significant. The results of the present work suggest that the proliferative activity of granulosa and theca cells decreases in association with follicular atresia in the BU similar to HF. Furthermore, a significantly deficient cell proliferative activity of theca interna was found in BU compared with HF.     

Relationship between apoptosis and proliferation in granulosa and theca cells of cystic follicles in sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.     

Changes in the expression of toll-like receptor mRNAs during follicular growth and in response to lipopolysaccharide in the ovarian follicles of laying hens

The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.     

Distribution of immunoreactive von Willebrand factor in the microvascular network of bovine cystic follicles

The present study was undertaken to examine whether the von Willebrand factor (vWF) expression in the follicular microvasculature in the cystic follicles differs from that in the atretic follicles. Paraffin sections of healthy, atretic and cystic follicles were immunostained with rabbit monoclonal antibody to vWF. The vWF-positive cells were counted in four different regions of a follicle from the apical to the basal side. In all types of follicles, immunoreactions for vWF were observed in the endothelial cells of capillaries as well as veins and arteries in the theca interna and externa. In the theca interna, vWF-positive areas were significantly lower in the Type A and B cystic follicles compared to advanced and late atretic follicles. In the theca externa, the vWF-positive blood vessels and vWF-positive area were significantly smaller in all types of cystic follicles than in the healthy or atretic follicles. From these results, it is suggested that in the cystic follicles the induction of vWF in the follicular microvasculature system is reduced, which may suppress the degeneration of vascular system. Continuation of stability in vasculature may be one of the factors that delays the tissue regression in the cystic follicles, and also contributes to the accumulation of follicular fluid that originates from the serum.     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Steroids and cAMP in follicles of postpartum beef cows treated with norgestomet.

To determine time of occurrence of follicular changes that may be associated with the length of the subsequent luteal phase, follicles were collected at different times before ovulation from cows expected to have corpora lutea of short (control) or normal (norgestomet-treated) life span. Beginning on d 20 to 23 postpartum (d 0 of study), 34 crossbred beef cows received either a 6-mg implant of norgestomet for 9 d or served as untreated controls. Ovaries were removed from norgestomet-treated cows on d 6 (N6; n = 9), d 8 (N8; n = 8), or the day after implant removal (N10; n = 8). Control cows were ovariectomized on d 6 (C6; n = 4) or d 10 (C10; n = 5). The largest and second largest follicles greater than 8 mm (F1 and F2, respectively) were dissected from the ovaries. Granulosal and thecal layers and follicular fluid were separated and assayed for estradiol-17 beta, progesterone, androstenedione, and testosterone. Cyclic 3'5'adenosine monophosphate (cAMP) was determined in thecal and granulosal tissue. Diameters of the F1 (14.6 +/- .4 mm) and F2 (10.6 +/- .4 mm) did not differ due to treatment. A greater proportion (P less than .05) of the F1 (20/33) than of the F2 (4/27) had estradiol:progesterone ratios of greater than 1 in follicular fluid. Contents of estradiol, androstenedione, and testosterone in theca and granulosa and follicular fluid, androstenedione in theca, and testosterone in theca and follicular fluid (all P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascularity and expression of angiogenic factors in bovine dominant follicles of the first follicular wave

To determine the relationships among vascularity, expression of angiogenic factors, and selected intrafollicular factors in dominant and nondominant follicles of the first follicular wave, ovaries were obtained on d 3 of the estrous cycle from mature cross-bred beef heifers (n = 8) after a synchronized estrus. Follicular fluid (FF) was collected from all follicles > or = 3 mm for determination of estradiol-17beta (E), progesterone (P4), vascular endothelial growth factor (VEGF), and IGFBP concentrations. The ovaries were then perfusion-fixed and used for histochemical detection of lectin BS-1 (a marker of endothelial cells and thus vascularization) binding, and immunolocalization of VEGF, endothelial nitric oxide synthase (eNOS), and proliferating cell nuclear antigen, followed by image analysis of selected follicles. Follicles were classified, based on E and P4 concentrations in FF, as dominant, estrogen-active (EA; E:P4 > or = 1) or nondominant, estrogen-inactive (EI; E:P4 <1). Concentrations of E and VEGF in FF, the area of positive staining for lectin BS-1, VEGF, and eNOS, and the labeling index (an index of the percentage of cells proliferating) in granulosa and theca layers were greater (P < 0.05) in the EA than in the EI follicles, but concentrations of P4 and IGFBP in FF were less (P < 0.05) in EA than in EI follicles. In addition, vascularity was positively correlated (P < 0.05) with VEGF and eNOS protein expression, and tended (P < 0.1) to be positively correlated with the E:P4 ratio in FF but tended (P < 0.1) to be negatively correlated with IGFBP and P4 concentrations in FF. These data highlight the importance of vascularity, angiogenic factors, and IGFBP in the health of the dominant follicle in heifers, and indicate that the FF concentrations of E, VEGF, IGFBP, and P4, and the E:P4 ratio can be used as markers of dominant follicles.     

Expression of mRNA for the angiopoietin-tie system in granulosa cells during follicular development in cows

Recent findings indicate that the changing profile of angiopoietins (ANPT) and their receptor Tie2 are closely associated with development and regression of the vascular network in the cyclic ovary. We previously reported that mRNA expression for the ANPT-Tie system in theca interna changes during bovine follicular development and atresia, and both ANPTs affect steroidogenesis in the preovulatory follicle. The aim of this study was to investigate mRNA expression for ANPT1, ANPT-2 and Tie2 in granulosa cells (GC) during follicular development in the cow. Bovine follicles were classified according to the estradiol-17beta (E(2)) concentration in follicular fluid (FF) as follows: (1) E(2)<0.5, (2) 0.5180 ng/ml FF. Semi-quantitative RT-PCR analysis revealed that the expression of ANPT-1 mRNA was not detected in most of the follicle with E(2)<5 ng/ml (diameter of 5-10 mm), but clearly detected in all follicles with E(2)>5 ng/ml (diameter of >10 mm). The mRNA expression for ANPT-2 was drastically decreased in the follicles with E(2)>5 ng/ml. Tie2 mRNA expression remained unchanged at the different stages of follicular development. The present data show that ANPT-1 becomes predominant in the follicle producing high levels of E(2), indicating the possible switch-over from ANPT-2 (antagonist) to ANPT-1 (agonist). Thus, the result suggests that the ANPT-Tie system in bovine GC may stimulate E(2) secretion rather than angiogenesis in the late stages of follicular development.