Effects of a commercial canine gonadotropin releasing hormone vaccine on estrus suppression and estrous behavior in mares.

We investigated the effect of immunization against gonadotropin releasing hormone (GnRH) using a commercial canine GnRH vaccine on estrus suppression and unwanted estrous behavior in mares. In experiment 1, mares were immunized (n = 6) twice with vaccine (5 mL) given intramuscularly 4 weeks apart or received a control diluent (n = 5). Transrectal ultrasonographic examination of the reproductive tracts was performed three days a week for 40 weeks after initial vaccination. Blood samples were collected weekly for GnRH antibody titer and progesterone concentration determination. In experiment 2, privately-owned mares (n = 12) were immunized twice with vaccine (1 mL) given intramuscularly 4 weeks apart. Blood samples were collected prior to each vaccination as well as 12 and 20 weeks after initial treatment, and transrectal ultrasonographic examinations of the reproductive tracts were performed 12 weeks after the first vaccination. Vaccinated mares in experiment 1 responded with a GnRH antibody titer, progesterone concentrations significantly lower than controls, and cessation of ovarian activity. Vaccinated mares in experiment 2 also responded with a GnRH antibody titer, progesterone concentrations that remained basal for the duration of the study, and cessation of ovarian activity. Owners of vaccinated mares in experiment 2 reported that the number of unwanted estrous behaviors present before vaccination significantly decreased following vaccination. In conclusion, GnRH immunization using a canine GnRH vaccine is an effective method for suppressing estrus and unwanted estrous behavior.

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.

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.

Insulin-like growth factors-1 and -2, but not hypoxia, synergize with gonadotropin hormone to promote vascular endothelial growth factor-A secretion by monkey granulosa cells from preovulatory follicles.

The midcycle gonadotropin surge promotes vascular endothelial growth factor-A (VEGF-A) production by granulosa cells in the ovulatory follicle, but it is unclear whether primary regulators of VEGF secretion in other tissues, including hypoxia and growth factors, are also important in the ovary. To address these issues, granulosa cells were collected from rhesus monkeys during controlled ovarian stimulation either before (i.e., nonluteinized granulosa cells, NLGCs) or 27 hours after (i.e., luteinized granulosa cells, LGCs) administration of an ovulatory bolus of hCG, and cultured in fibronectin-coated wells containing a chemically defined media. When NLGCs were transferred to various O2 environments (20%, 5%, or 0% O2) or media containing 100 mM CoCl2, LH (100 ng/ml)-stimulated progesterone (P4) levels were markedly (P < 0.05) suppressed by 0% O2 or CoCl2. VEGF concentrations also declined (P < 0.05) in control, CoCl2, and CoCl2 + LH groups in 0% O2, although CoCl2 modestly increased (75% above control; P < 0.05) VEGF levels in 20% and 5% O2. When NLGCs were cultured in the presence of recombinant human insulin-like growth factor (IGF)-1, IGF-2, or insulin, there was a dose-dependent increase (P < 0.05) in VEGF levels on Day 1 of culture. Whereas optimal doses of IGF-1 or IGF-2 (50 ng/ml), hCG (100 ng/ml), and IGF plus hCG stimulated VEGF levels on Day 1, only the combination of IGF-1 or IGF-2 plus hCG increased VEGF above controls and sustained levels through Day 3 of culture. The synergistic effects of IGF and hCG were also evident in P4 levels, and were not due to changes in DNA content between treatment groups. LGCs produced much higher levels of P4 and VEGF, but the responses to different O2 concentrations and insulin-related factors were qualitatively similar to those of NLGCs. These results suggest that hypoxia is not a primary regulator of VEGF production in primate granulosa cells. However, IGFs may act in concert with the gonadotropin surge to promote VEGF secretion in the ovulatory, luteinizing follicle.

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.

Changes in expression of vascular endothelial growth factor and angiopoietin-1 and -2 in the macaque corpus luteum during the menstrual cycle.

To determine the temporal expression of vascular growth factors during the lifespan of the primate corpus luteum, experiments were designed to detect mRNA for vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and Ang-2 and to localize protein expression for VEGF in macaque luteal tissue during the menstrual cycle. Corpora lutea (n = 3-5/stage) were collected during the early (3-5 days post-luteinizing hormone surge), mid- (6-8 days), mid-late (10-12 days), and late (14-16 days) luteal phase and at menstruation (17-18 days). Reverse transcription-polymerase chain reaction products equated to cDNA for VEGF, Ang-1 and Ang-2 in all corpora lutea. VEGF mRNA levels increased (P: < 0.05) from early to mid-luteal phase and declined in the late luteal phase and at menstruation. Immunostaining for VEGF was detected in the cytoplasm of steroidogenic luteal cells, with the most intense staining in the early luteal phase. Ang-1 and Ang-2 mRNA expression was low in the early to mid-luteal phase but increased (P: < 0.05) at late luteal phase before declining at menstruation. These data suggest transcriptional control of VEGF, Ang-1 and Ang-2, as well as post-transcriptional control of VEGF, in macaque corpus luteum. Dynamic expression of angiogenic/angiostatic factors appears critical for development, maintenance and regression of the luteal microvasculature during the menstrual cycle.

Angiogenesis in ovarian follicular and luteal development.

Angiogenesis is the process of new capillary formation from previously existing mature vessels. The adult ovary exhibits dramatic growth and regression of capillary networks on a cyclic basis. Ovarian follicles and the corpus luteum contain and produce endothelial cell-specific factors, which may act alone or in concert to regulate the process of angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation, as well as by metabolic cellular signals such as intracellular oxygen content and ageing. Aberrant production of these angiogenic factors may be the cause of vascular dysfunction and the development of ovarian disorders. Recent technological advances for monitoring blood flow and measuring angiogenic factors could assist in accurately diagnosing ovarian disorders. Further elucidation of specific physiological role(s) of factors involved in angiogenesis of the pre-ovulatory follicle and developing corpus luteum may be useful in addressing issues of infertility in women.

Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval.

The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates.

Impact of chronic treatment of ewes with estradiol-17beta or progesterone on oxytocin receptor gene transcription and ovarian oxytocin secretion.

Three experiments were conducted, the objectives of which were to 1) examine the effects of exogenous estradiol (E2) and progesterone (P4) on uterine concentrations of oxytocin receptors (OTR) and OTR mRNA, as well as the effect of exogenous P4 on progesterone receptors (PR) during the late luteal phase of the cycle, and 2) ascertain whether chronic E2 treatment of ewes during the cycle would alter prostaglandin F2alpha (PGF2alpha)-induced secretion of luteal oxytocin (OT). In experiment 1, 15 ewes were assigned to a control (n = 5; 2 ml corn oil [CO] s.c. on Days 4-14 of the estrous cycle) and two treatment groups (n = 5 each) receiving either 250 microg E2 s.c. (Days 4-14) or 10 mg P4 s.c. (CO on Days 4-10 and P4 on Days 11-14). Endometria and corpora lutea were removed on Day 15 of the cycle. Mean luteal weights were greater in treated than in control ewes (p < 0.05). Endometrial concentrations of OTR and OTR mRNA were significantly greater in control than in E2- or P4-treated ewes. In experiment 2, five ewes each were treated s.c. with CO or 10 mg P4 on Days 11-14 of the cycle; endometria were then removed on Day 15 for PR assay. Endometrial concentration of PR did not differ between groups. Experiment 3 consisted of 20 ewes assigned to four groups in a 2 x 2 factorial arrangement. Treatment consisted of two dosages of E2 (0 or 250 microg/day) in 2 ml CO and two dosages of PGF2alpha analogue (0 or 125 microg Estrumate). All ewes were injected s.c. with E2 or CO for 11 days as described for experiment 1. On Day 15, all ewes received an i.v. injection of PGF2alpha or saline (Time 0); then jugular blood was collected at frequent intervals for analysis of serum concentrations of OT. PGF2alpha induced a release of OT in control and E2-treated ewes (p < 0.05) compared to the value in saline-treated ewes. Collectively, these data suggest that in cycling ewes, exposure of the uterus to increased concentrations of E2 or P4 causes down-regulation of OTR as a consequence of suppression of the OTR gene. Chronic E2 treatment of ewes during the cycle does not act directly on the ovary to alter the stores of luteal OT.

Down-regulation of oxytocin receptors and secretion of prostaglandin F2alpha after chronic treatment of ewes with estradiol-17beta.

Experiments were conducted to investigate the mechanisms through which chronic treatment with estradiol-17beta (E2) prolongs the estrous cycle in the ewe. To determine whether treatment with estradiol maintained the corpus luteum (CL), mature ewes were assigned randomly to two groups receiving s.c. injections of either 500 microg E2 in corn oil or vehicle alone for 20 days beginning on Day 4 of the cycle (n = 5 per group). Laparotomy of E2-treated ewes on Day 24 revealed the presence of CL previously marked with India ink on Day 7 of the cycle. Treatment increased the mean interestrous interval for 4 of 5 animals compared with that of controls (p < 0.001). Therefore, to determine whether the increase in the interestrous interval was due to an effect on the function and/or concentration of uterine oxytocin receptors (OTr), ewes (n = 5 per group) were injected with 500 microg E2 or vehicle as described above from Days 4 to 14 of the cycle. On Day 15, a jugular blood sample was collected for progesterone (P4) analysis, after which ewes were given an i.v. injection of oxytocin (OT; 20 IU USP) or saline, and blood samples were collected at frequent intervals for 60 min to determine plasma concentrations of 13,14-dihydro-15-ketoprostaglandin F2alpha (PGFM). Laparotomies were performed after blood collection to remove uterine endometrium for OTr analysis. Mean serum concentrations of P4 were greater in treated than in control ewes on Day 15 (p < 0.05). Treatment of ewes with E2 prolonged luteal function and resulted in reduced uterine concentration of OTr compared with that of controls (p = 0.002). Treatment with E2 reduced OT-induced plasma concentrations of PGFM compared with controls (p < 0.01). Collectively, these data suggest that chronic treatment of ewes with estradiol during the cycle can prolong the interestrous interval by reducing uterine concentration of OTr and hence OT-induced secretion of prostaglandin.