Timothy M. Hazzard

Regulation and action of angiogenic factors in the primate ovary.

The ephemerality of the maturing follicle and subsequent corpus luteum as they perform their gametogenic and/or endocrine functions during the ovarian cycle is associated with remarkable changes in local vasculature. Studies on the angiogenic and angiolytic process in the ovary, rare in healthy adult tissues, complement recent efforts to understand vasculogenesis in embryonic tissues and to control angiogenesis in pathologic states such as cancer. Several reports indicate that the newly discovered vascular-specific angiogenic factors are expressed in the ovary, notably members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) families plus their receptors (VEGF-Rs, neuropilins, Tie). Unlike in many other tissues, gonadotropic hormones (particularly luteinizing hormone, [LH]) are major stimulators of angiogenesis and VEGF/Ang expression in the ovary. However, local factors such as insulin-like growth factors or oxygen tension likely modulate the angiogenic processes. Recent studies employing systemic or local administration of anti-angiogenic drugs (TNP-470 or fumagillin) or specific VEGF antagonists (VEGF antibody or soluble VEGFR-1) demonstrate a vital role for normal angiogenesis and VEGF action in follicle development, ovulation, or corpus luteum function. Further studies discerning the various angiogenic factors and their roles in controlling the growth, maturation, function, and regression of the vasculature in ovarian compartments during the menstrual cycle could yield novel strategies for manipulating fertility or for alleviating infertility.

Injection of Soluble Vascular Endothelial Growth Factor Receptor 1 into the Preovulatory Follicle Disrupts Ovulation and Subsequent Luteal Function in Rhesus Monkeys

Abstract Remarkable changes in vascular permeability and neovascularization occur within the ovulatory, luteinizing follicle. To evaluate the importance of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in periovulatory events, sequential experiments were designed in which vehicle (PBS/0.1% BSA; controls, n = 13) or a low dose (1.5 μg; n = 4) or a high dose (7.5 μg; n = 4) of a VEGF antagonist, soluble VEGF receptor 1 (sVEGFR1) chimera, was injected directly into the preovulatory follicle of rhesus monkeys the day before (Day −1) or the day of (Day 0) the midcycle LH surge during spontaneous menstrual cycles. After vehicle injection, animals typically exhibited patterns and levels of serum progesterone (P4) that were comparable to those of untreated animals in our colony. Following low-dose sVEGFR1 injection, serum P4 levels were diminished in two of four animals from the early to midluteal phase, but were similar to vehicle controls thereafter. In contrast, high-dose sVEGFR1 injection decreased serum P4 levels throughout the luteal phase compared with levels in controls (P < 0.05), but it did not cause premature menstruation. Control follicles displayed indices of rupture (protruding stigmata) and luteinization. However, sVEGFR1-injected follicles exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization, but not necessarily timely ovulation. Histological evaluation of serial sections from ovaries removed on Day 3 after treatment revealed that all (n = 3) vehicle-injected follicles ovulated, whereas half (n = 3 of 6) the sVEGFR1-injected follicles failed to ovulate and still contained an oocyte in the antrum. No appreciable differences were apparent between treatment groups in numbers of cells in luteal tissue (Day 3 or 6 after treatment) that stained positive for immunochemical or histochemical markers of proliferative (Ki67), endothelial (platelet endothelial cell adhesion molecule 1), and steroidogenic (3β-hydroxysteroid dehydrogenase) cells. However, there was a dose-dependent increase (P < 0.05) in extracellular space in the corpus luteum by midluteal phase in sVEGFR1-treated animals. The data suggest that acute exposure to a VEGF antagonist can impair ovulation, and the subsequent development and functional capacity of the primate corpus luteum. The results are consistent with a critical role for VEGF in normal ovarian function during the periovulatory interval in primates.

Chitosan Dressing Provides Hemostasis in Swine Femoral Arterial Injury Model

Objective. Chitosan dressings have been shown to be effective in improving survival of severe parenchymal injuries in an animal model andin treating prehospital combat casualties. Our goal was to test the efficacy of chitosan acetate dressings in providing durable hemostasis in a high-flow arterial wound model. Methods. A proximal arterial injury was created with 2.7-mm vascular punches in both femoral arteries of fourteen anesthetized swine. By using a crossover design, 48-ply gauze (48PG) or a chitosan dressing (HC) was applied with pressure to the injury for 3 minutes andthen released. If hemostasis was not maintained for 30 minutes, a second identical attempt was made by using the same dressing type. If hemostasis was still not achieved, the dressing was considered an acute failure andthe alternate dressing type was applied. If failure of hemostasis occurred between 30 and240 minutes after application, the dressing was considered a chronic failure andthe artery was ligated. Results. All 25/25 (100%) of the HC tests and3/14 (21%) of the 48PG maintained hemostasis for 30 minutes. At 240 minutes, 21/25 (84%) of the HC tests and1/14 (7%) of the 48PG maintained hemostasis. Statistical analysis by Fischers exact test shows a significant (p < 0.001) difference in hemostatic efficacy between the 48PG andHC groups in this model, both at 30 minutes andat 240 minutes. Conclusion. Chitosan acetate hemorrhage control dressings provided superior hemostasis to 48 ply gauze in high inguinal femoral arterial injuries. Chitosan-based dressings may provide prehospital treatment options for hemostasis in patients with severe hemorrhagic arterial injuries.

Injection of antiangiogenic agents into the macaque preovulatory follicle: disruption of corpus luteum development and function.

Ovulation and conversion of the follicle into the corpus luteum involve remarkable changes in vascular permeability and neovascularization of the luteinizing granulosa layer. To evaluate the importance of these vascular events in follicle rupture and luteal development, sequential experiments were designed in which vehicle or angiogenic inhibitors (TNP-470 or angiostatin) were injected directly into the preovulatory follicle of rhesus monkeys during spontaneous mentstrual cycles. After control injections, 13 of 14 animals exhibited serum levels of progesterone (P) during the subsequent luteal phase that were comparable to untreated animals in our colony. Following low-dose (400 pg/mL) TNP-470, serum P levels increased normally until d 8 of the luteal phase, but then declined prematurely by d 9 (p < 0.05 compared to controls) and remained below controls until menses. Following high-dose (2 µg/mL) TNP-470, serum P levels were diminished in the early luteal phase (d 3–5; p<0.05 compared to controls), but reached typical levels at mid luteal phase, only to decline prematurely by d 9 (p<0.05) and remain low until menses. Control ovaries displayed indices of follicle rupture (protruding stigmata) and luteinization. TNP-470-treated ovaries exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization; however, a well-formed stigmata was not observed. A “trapped” oocyte was not observed in serial sections of developing corpora lutea from control or TNP-470-treated animals. However, the early corpus luteum of TNP-470-injected ovaries contained pockets of excessive numbers of blood cells that were absent in controls. Angiostatin did not alter serum P levels or ovarian morphology compared to controls. These data suggest that acute exposure to the antiangiogenic agent TNP-470 impairs the development and functional capacity of the primate corpus luteum in a dose-dependent manner. The results are consistent with a critical role for angiogenesis in cyclic ovarian function in primates.

Gene expression in pre-term, pre-labour and parturient canine placenta.

Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), vascular endothelial growth factor (VEGF)-A, VEGF-A receptor (Flt-1) and KiSS-1 receptor (KiSS-1R) all play a role in trophoblast invasion in a number of mammalian species. However, mRNA expression of factors regulating trophoblast invasion has not been studied in dogs. Abnormal expression of these factors at the end of canine gestation may contribute to placental retention and/or subinvolution of placental sites. Therefore, we sought to determine the relative mRNA expression of these factors in canine chorioallantois tissue at 61 ± 1 day past the LH surge (pre-term; n = 4), following elective c-section at 64 ± 1 day past the LH surge prior to first-stage labour (pre-labour; n = 4) and following natural delivery (parturient; n = 3). Total RNA was isolated, real-time RT-PCR was performed, and relative expression was calculated using the relative quantitation (2-ΔΔCT) method. MMP-9 mRNA expression was significantly higher in pre-term samples compared to pre-labour and parturient samples. The results showed no significant difference between MMP-2, TIMP-2, VEGF-A and Flt-1 mRNA expression among the three groups. KiSS-1R mRNA was not expressed in any tissues studied. Gene expression of MMP-9 may be related to the onset of labour, whereas MMP-2, VEGF-A, Flt-1, TIMP-2 and KiSS-1R mRNA do not appear to play a role at the end of gestation in the dog.

Cellular Characteristics of Cultured Canine Trophoblasts

CONTENTS Shallow trophoblast invasion is detrimental in human pregnancies, but represents normal endotheliochorial placentation in dogs. Factors regulating shallow trophoblast invasion into the canine decidua are not well described, but it is known that matrix metalloproteinases (MMPs) play a crucial role in trophoblast invasion in many species. Following the methods previously described for isolating human trophoblasts, canine trophoblasts were isolated using collagenase and trypsin digestions with Percoll density gradient centrifugation. In addition, placental pieces were cryopreserved prior to primary culture following methods previously described for human tissue. Expression of cytokeratin-7, MMP2 and MMP9 was confirmed using fluorescent immunocytochemistry. Cellular morphology was similar to that reported for trophoblasts. More than 97% of the cells cultured expressed cytokeratin-7. More cultured canine trophoblasts expressed MMP9 (54.7 ± 3.4%) compared with MMP2 (40.3 ± 1.8%) (p = 0.02). Although both MMPs were immunolocalized to the cytoplasm, MMP2 was found in large, coalescing granules, whereas MMP9 was more diffusely expressed throughout the cell. Cryogenic freezing of placental tissue prior to primary cell culture had no effect on cell proliferation (p = 0.37). This research has established a baseline for future studies investigating the canine placenta as a model for disorders of shallow trophoblast invasion in humans.

Effect of different dosages of PG-600 on ovulation and pregnancy rates in ewes during the breeding season

Abstract This study compared the reproductive effects of different dosages of PG-600 (Intervet/Merck Animal Health, Madison, NJ) during the breeding season of ewes. PG-600 is a single-dose injectable product labeled for estrous induction in swine, containing equine chorionic gonadotropin (80 IU/mL) and human chorionic gonadotropin (40 IU/mL). PG-600 is routinely used off-label for out-of-season estrous induction in sheep. However, at the most common dose administered to ewes (5 mL), PG-600 is likely to overstimulate the ovaries, resulting in reduced pregnancy rates. Following estrous synchronization with intravaginal progesterone and cloprostenol, Polypay ewes were treated with 5 mL PG-600 (T1; n = 8), 1.5 mL PG-600 (T2; n = 8), or 5 mL saline (C; n = 8) and then mated to rams. Jugular vein samples were collected prior to the PG-600 injection (0 hr) and at 2, 4, 8, 12, and 24 hr after injection. Serum estradiol-17β was determined by chemiluminescence and among groups using repeated measures analysis of covariance. Ovulation and pregnancy rates were determined by transrectal ultrasonography and compared by one-way ANOVA and chi-square, respectively. Estradiol-17β concentrations were greater in T1 compared to T2 and C (P < 0.001). Ovulation rate was greater (P < 0.001) but pregnancy rate was lower (P < 0.001) in the T1 compared to C and T2. These data confirm that a 5 mL dose of PG-600 administered to ewes during the breeding season overstimulates the ovaries, which may then reduce fertilization or embryo survival. Future research will focus on the effects of different dosages of PG-600 on pregnancy rate of ewes during the nonbreeding season.

Intraovarian Control of Ovulation: Lessons from Steroid Ablation/Replacement in Monkeys

An important action of estrogen secreted by the maturing follicle is to trigger the release of surge levels of the gonadotropic hormones, follicle stimulating hormone (FSH), and luteinizing hormone (LH) from the pituitary. The gonadotropin surge initiates a cascade of events within the follicle that promotes oocyte maturation, ovulation, and conversion of the follicle wall into a new endocrine structure, the corpus luteum. The latter process, termed luteinization, involves major changes in the structure—function of the tissue, including development of the molecular and cellular components required to produce large quantities of the steroid hormone progesterone. As the periovulatory interval proceeds, the luteinizing follicle switches from primarily an estrogen-secreting to a progesterone-secreting tissue. In some series, the corpus luteum is devoid of estrogen synthetic capacity, whereas in others (e. g., Old World macaques and women) significant production of estrogen (and its bioactive precursor, androgen) remains. Thus, the periovulatory follicle experiences remarkable changes in the steroid milieu as androgen and estrogen initially rise and then decline, and appreciable levels of progestins are achieved well before follicle rupture (1, 2).