Cardiac ischemia model for +Gz using miniature swine and baboons.

BACKGROUND: Military aircrew with minimal coronary artery disease (MCAD) may be restricted from flying high-performance aircraft due to possible ischemia during high +Gz. An animal model is presented to provide ischemia data for a more informed decision. METHODS: There were 18 swine that were placed on a high cholesterol/high fat diet for up to 57 wk. Five control swine were maintained on a standard swine diet. Also, nine male baboons had a constrictor placed around the left anterior descending coronary artery. Two baboons were sham-operated controls. The unanesthetized swine and baboons were infused with Tc-99m at the end of +Gz exposure and scanned for myocardial perfusion. RESULTS: Five swine died unexpectedly before +Gz exposure with moderate-to-severe CAD. Dysrhythmias during +Gz were seen equally in both the control and experimental swine and in the baboons before and after stenosis, with or without propranolol. During +Gz, ECG ST-T wave changes suggesting ischemia were observed in the cholesterol swine but not the control swine, and in the baboons before and after stenosis, with or without propranolol. There was a positive relationship between a normal/abnormal ECG and a normal/abnormal myocardial perfusion scan in the swine and a weak relationship in the baboon before stenosis, but somewhat better after stenosis. Coronary histopathology showed normal vessels from the control swine and stenoses ranging from 0-95% from the cholesterol swine. Baboon stenosis averaged 37.6 +/- 15.0%. CONCLUSIONS: In the swine and the baboon extended high levels of +Gz, were associated with evidence of myocardial ischemia.

The phosphodiesterase 3 inhibitor ORG 9935 inhibits oocyte maturation in the naturally selected dominant follicle in rhesus macaques.

BACKGROUND: The study was conducted to determine whether the phosphodiesterase (PDE) 3 inhibitor ORG 9935 prevents the resumption of meiosis in primate oocytes during natural menstrual cycles. STUDY DESIGN: Regularly cycling adult female macaques (n=8) were followed during the follicular phase and then started on a 2-day treatment regimen of human recombinant gonadotropins to control the timing of ovulation. Monkeys received no further treatment (controls) or ORG 9935. Oocytes were recovered by laparoscopic follicle aspiration 27 h after an ovulatory stimulus, cultured in vitro in the absence of inhibitor and inseminated. The primary outcome was the meiotic stage of the oocyte. RESULTS: In six ORG 9935 cycles, five of the recovered oocytes were germinal vesicle (GV)-intact, and one exhibited GV breakdown (GVBD). In contrast, all three oocytes that recovered during control cycles were GVBD (p<.05). None of the ORG 9935-treated oocytes underwent fertilization compared with 2/3 (67%) from controls. CONCLUSIONS: These results demonstrate that ORG 9935 blocks resumption of meiosis in the naturally selected dominant follicle in primates and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women.

Evaluation of the vervet (Clorocebus aethiops) as a model for the assisted reproductive technologies.

The vervet monkey was evaluated as a primate model for use in assisted reproductive technologies (ARTs). Eight adult female vervets were hormonally monitored for their potential use as egg donors and those six females displaying regular menstrual cycles were subjected to controlled ovarian stimulation with recombinant human gonadotropins. Three animals failed to respond while laparoscopic follicular aspiration was performed on the other three females at 27-30 h post-human chorionic gonadotropin administration. A total of 62, 40, and 18 oocytes was recovered from these three animals of which 30, 20, and 4, respectively, matured to the metaphase II stage and were subsequently inseminated using intracytoplasmic sperm injection. An average of 40+/-15% (SEM) of the inseminated oocytes were fertilized based on pronucleus formation and timely cleavage. One embryo from each of the two stimulated females developed into expanded blastocysts. Two adult male vervets were assessed as sperm donors. Neither adjusted well to the restraint and collection procedure required for penile electroejaculation. Samples collected via rectal electroejaculation were very low in sperm motility and concentration; however, cauda epididymal aspirations from one male yielded an adequate concentration of motile sperm. These results emphasize the need to establish species-specific ovarian stimulation protocols and semen collection techniques if vervets are to be considered for basic and applied (ARTs) research on primate gametes or embryos.

Assessment of menstruation in the vervet (Cercopithecus aethiops).

Vervet monkeys (Chlorocebus aethiops) are Old World nonhumans that display attenuated menstruation that requires detection by vaginal swab. The physiology underlying attenuated menstruation in this species has not been previously studied. To fill this gap, we evaluated endometrial cell proliferation, steroid receptor localization and expression of menstruation-associated matrix metalloproteinase (MMP) enzymes in vervets during natural and artificial menstrual cycles. The artificial cycles were induced by sequentially treating ovariectomized animals with estradiol (E(2)) and progesterone (P). Because menstrual flow is exceptionally light in this species, menses was detected by vaginal swab. We found that both natural and artificially cycled animals menstruated 3-5 days after the decline of P at the end of the cycle. As in other primates, P withdrawal at the end of artificial cycles triggered endometrial expression of MMPs, including MMP-1, 2, 3, 7, 10, 11, 13 and 26 transcripts. In both the natural and artificial menstrual cycle, menstrual sloughing was restricted to the upper one-fourth of the endometrium, and MMP-1 and 2 were strongly expressed by the stroma of the sloughing zone. MMP-7 was localized in the endometrial glands during late menses. As in macaques, epithelial cell proliferation was localized to the functionalis zone during the estrogen-dominated proliferative phase and to the basalis zone glands during the P-dominated secretory phase. Regulation of estrogen and progestin (or estradiol and progesterone) receptors was similar to that reported for macaques. Because strong similarities exist between the endometrium of vervets, macaques and women, we conclude that vervets can provide a useful animal model for studies on hormone regulation of menstruation.

Characteristics and regulation of the ovarian cycle in vervet monkeys (Chlorocebus aethiops).

This study was designed to evaluate the timecourse of ovarian and pituitary endocrine events throughout the menstrual cycle in the vervet monkey, and whether circulating luteinizing hormone (LH) or the uterus regulates the functional lifespan of the vervet corpus luteum. Daily saphenous blood samples were collected from adult females (1) during spontaneous menstrual cycles (n = 7), and (2) during cycles in which a gonadotropin-releasing hormone antagonist (acyline) was administered for 3 days at midluteal phase (n = 3), and (3) for 30 days following recovery from hysterectomy (n = 4). Estradiol (E) and progesterone (P) levels were assayed using electrochemoluminescent assays. Gonadotropin levels were measured by radioimmunoassay using reagents developed for the assay of follicle-stimulating hormone and LH in macaques. Spontaneous cycles exhibited a midcycle E rise (476+/-49 pg/ml), engendering an LH surge, 12+/-1 days after onset of menses, followed by a luteal phase with peak P levels of 4.7+/-0.9 ng/ml. Histologic evaluation of the ovaries at late follicular phase or early luteal phase revealed the presence of a single, large Graafian follicle or developing corpus luteum, respectively. Acyline treatment caused a significant (P<0.05) decline in P levels (2.9+/-0.5 vs 0.5+/-0.3 ng/ml, 0 vs 48 h post-treatment) and premature menstruation compared with untreated controls (P<0.05). Hysterectomy had no apparent effect on the monthly pattern or levels of circulating E or P. Thus, the characteristics and regulation of the ovarian cycle in vervets appear similar to those in women and macaques, with cyclicity dependent on pituitary gonadotropin hormones and independent of a uterine luteolytic factor.

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 menstrual 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 microg/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.

Understanding the basis of CD4(+) T-cell depletion in macaques infected by a simian-human immunodeficiency virus.

The efficacy of candidate AIDS vaccines to mediate protection against viral infection and pathogenesis is evaluated, at a preclinical stage, in animal models. One model that is favored because the infecting virus is closely related to HIV-1 and because of the rapidity of pathogenic outcomes is the infection of Old World monkeys by simian-human immunodeficiency virus (SHIV) chimerae. We investigated the basis for the depletion of CD4(+) T lymphocytes in a SHIV-macaque model. Molecularly cloned SHIVs, SHIV-89.6 and SHIV-KB9, differ in the ability to cause CD4(+) T-cell loss at a given level of virus replication in monkeys. The envelope glycoproteins of the pathogenic SHIV-KB9 mediate membrane-fusion in cultured T lymphocytes more efficiently than the envelope glycoproteins of the non-pathogenic SHIV-89.6. The minimal envelope glycoprotein region that specifies this increase in membrane-fusing capacity was sufficient to convert SHIV-89.6 into a virus that causes profound CD4(+) T-cell depletion in monkeys. Conversely, two single amino acid changes that decrease the membrane-fusing ability of the SHIV-KB9 envelope glycoproteins also attenuated the CD4(+) T-cell destruction that accompanied a given level of virus replication in SHIV-infected monkeys. Thus, the ability of the HIV-1 envelope glycoproteins to fuse membranes, which has been implicated in the induction of viral cytopathic effects in vitro, contributes to the capacity of the pathogenic SHIV to deplete CD4(+) T lymphocytes in vivo.