Angiogenesis in The Ovary – The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow – An Overview

In the ovary, the development of new capillaries from pre‐existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin‐like growth factor (IGF), angiopoietin (ANPT) and hypoxia‐inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol‐17‐beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5–7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT‐qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF‐A, FGF‐2, IGF‐1 and IGF‐2, ANPT‐2/ANPT‐1 and HIF‐1‐alpha was found during final follicle maturation and in CL during the early luteal phase (days 1–4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.     

Isolation of Peirce's disease bacteria from grapevines in Europe

Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grape, was isolated from diseased grapevines grown in Kosova, Yugoslavia. The Kosova isolate was a rod-shaped bacterium which showed a typically rippled cell wall under electron microscopy. ELISA comparisons indicated that the Kosova isolate was closely related to the U.S. PD strains and to several other strains of X. fastidiosa. When DNA extracted from diseased grapevines collected from Kosova was used as template in PCR with primer sets specific for X. fastidiosa, a band of about 730 bp diagnostic for PD bacteria was detected. DNA from the isolated Kosova bacteria and the type strain of PD yielded the same length of DNA fragment in PCR assay. The Kosova isolate was inoculated into young healthy grapevines through the roots with negative pressure applied to the shoots. Typical scald and scorch symptoms appeared on the leaves of the inoculated grapevines 40–80 days after inoculation. The same bacteria were reisolated from these inoculated diseased plants and used to reinoculate young grapevines. The reinoculated grapevines produced the same symptoms, thereby fulfilling Koch's postulates. This is the first confirmation that PD of grapes occurs in Europe.     

Regulation of Corpus Luteum Function in Cattle – an Overview

The corpus luteum (CL) is a transient reproductive gland that produces progesterone (P), required for the establishment and maintenance of pregnancy. Although the regulation of bovine luteal function has been studied for several decades, many of the regulatory mechanisms involved are incompletely understood. We are far from understanding how these complex mechanisms function in unison. The purpose of this overview is to stress important steps of regulation during the lifetime of CL. In the first part, the importance and regulation of angiogenesis and blood flow during CL formation is described. The results underline the importance of growth factors especially of vascular endothelial growth factor A (VEGF A) and basic fibroblast growth factor (FGF-2) for development and completion of a dense network of capillaries. In the second part, the regulation of function by endocrine/paracrine- and autocrine-acting regulators is discussed. There is now more evidence that besides the main endocrine hormones LH and GH local regulators as growth factors, peptides, steroids and prostaglandins are important modulators of luteal function. During early CL development until mid-luteal stage oxytocin, prostaglandins and P itself stimulate luteal cell proliferation and function supported by the luteotropic action of a number of growth factors. The still high mRNA expression, protein concentration and localization of growth factors [VEGF, FGF-1, FGF-2, insulin-like growth factors (IGFs)] in the cytoplasm of luteal cells during mid-luteal stage suggest maintenance (survival) functions for growth factors. In the absence of pregnancy regression (luteolysis) of CL occurs. Progesterone itself regulates the length of the oestrous cycle by influencing the timing of the luteolytic signal prostaglandin F2alpha (PGF2alpha) from the endometrium. The cascade of mediators afterwards is very complex and still not well-elucidated. Evidence is given for participation of blood flow, inflammatory cytokines, vasoactive peptides (angiotensin II and endothelin-1), reactive oxygen species, angiogenic growth factors (VEGFs, FGFs, IGFs) and decrease of the classical luteotropic components as LH-R, GH-R, P450(scc) and 3beta-HSD. Despite of differences in methodology and interpretations, progress has been made and will continue to be made.     

Expression of Angiopoietin (ANPT)-1, ANPT-2 and their Receptors in Dominant Follicles during Periovulatory Period in GnRH-Treated Cow

Ovarian follicular vasculature is involved in follicular development and ovulation. Angiopoietin (ANPT)-Tie system is important for vascularization of the tissue surrounding the developing follicles and corpus luteum (CL). To determine how the expression of ANPT-1, ANPT-2 and their receptors in the follicles would be associated with the ovulatory process, the present study was conducted to examine mRNA expressions of ANPT-1, ANPT-2 and their receptors during the periovulatory phase in gonadotrophin-releasing hormone (GnRH)-treated cows. The ovaries were collected by transvaginal ovariectomy (n = 5, cows/group) and the follicles (n = 5, one follicle/cow) were classified into following groups: before GnRH administration [before luteinizing hormone (LH) surge]; 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (peri-ovulation); and early CL (days 2-3). The mRNA expression was analysed by quantitative real-time PCR (rotor-gene 3000). Angiopoietin-1 expression rapidly decreased at 3-5 h and kept low level at 10 h after GnRH treatment compared with that before GnRH, and returned to the level before LH surge in the follicles >20 h after GnRH treatment. The levels of ANPT-2 mRNA decreased at 10 and 25 h after treatment compared with other periods. The ratio of ANPT-2/ANPT-1 (an index for destabilization of blood vessels) increased in the follicles at 3-5 h after GnRH treatment only. Both of Tie-1 and Tie-2 receptor expressions decreased in the follicles at 25 h after GnRH treatment. The results of the present study indicated that mRNA expressions of ANPT-1, ANPT-2 and their receptors changed in the bovine follicles during periovulatory period. These results suggest that angiopoietin-Tie system is associated with the initiation of vasculature of follicle that grows towards ovulation.     

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.     

Dexamethasone‐Induced Eosinopenia is Associated with Lower Progesterone Production in Cattle

Eosinophilic cells accumulate in the capillaries of the bovine Graafian follicle shortly before ovulation and in the early developing corpus luteum (CL). Suppressing the migration of these eosinophilic cells by dexamethasone allowed us to evaluate their possible function in the CL development. Brown Swiss cows (n = 10) were randomly subdivided into two groups (n = 5). Every group was used once as control group and once as experimental group with two oestrous cycles between each treatment. Eighteen hours (h) after oestrus synchronization, dexamethasone or saline was given. Ovulation was induced 24 h later with gonadotropin‐releasing hormone. Another injection of dexamethasone or saline was given 12 h later. Eosinophilic cells in the blood were counted daily until day 7 after the first dexamethasone injection. The collection of ovaries took place at days 1, 2 and 5. Gene expression, protein concentration and location of angiogenic factors, chemokines, insulin‐like growth factor 1 (IGF1) and eosinophilic cells were studied. No eosinophilic cells were found in the CL of the treatment group. Blood progesterone decreased significantly in the dexamethasone group from day 8 to 17. The protein concentration of FGF2 increased significantly in CL tissue at day 2 and VEGFA decreased. Local IGF1 gene expression in the CL was not regulated. We assume from our data that the migration of eosinophilic cells into the early CL is not an essential, but an important stimulus for angiogenesis during early CL development in cattle.     

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary

The aim of this study was to characterize expression patterns of hypoxia‐inducible factor‐1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol‐17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1–2, 3–4, 6–7, >8). Experiment 3: Cows on days 8–12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.     

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.     

Ovarian function in ruminants

The purpose of this overview is to highlight important steps of ovarian regulation during follicle development, ovulation and the life span of corpus luteum (CL) in ruminants. The ovarian cycle is central to reproductive function. It is characterized by repeating patterns of cellular proliferation, differentiation and transformation that encompass follicular development and ovulation as well as the formation, function and regression of the CL. In the first part, the importance and regulation of final follicle growth and especially of angiogenesis and blood flow during folliculogenesis, dominant follicle development and CL formation are described. Our results underline the importance of growth factors especially of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) for development and completion of a dense network of capillaries (angiogenesis) during follicle growth and CL formation. In the second part, the regulation of CL function by endocrine/paracrine and autocrine acting regulators is discussed. There is evidence that besides the main endocrine hormones luteinizing hormone (LH) and growth hormone (GH) local regulators as growth factors, peptides, steroids and prostaglandins are important modulators of luteal function. During early CL development until midluteal stage oxytocin (OT), prostaglandins and progesterone (P) itself stimulate luteal cell proliferation and function supported by the luteotropic action of a number of growth factors. The still high mRNA expression, protein concentration and localization of VEGF, FGF and IGF family members in the cytoplasm of luteal cells during midluteal stage suggest that they play pivotal role in the maintenance (survival) of this endocrine tissue. The major function of the CL is to secrete P. Progesterone itself regulates the length of the estrous cycle via influencing the timing of the luteolytic PGF2alpha signal from the endometrium. At the end of a nonfertile cycle, the regression of CL commences, steroidogenic capacity is lost (functional luteolysis), cell death is initiated, and tissue involution as well as resorption occurs within a few days (structural luteolysis). The cascade of mediators during luteolysis is very complex and still awaits elucidation. Evidence is given for participation of blood flow, inflammatory cytokines, vasoactive peptides (angiotensin II and endothelin-1), and decrease of the classical luteotropic mediators.