Ovariectomized Guinea Pigs Research Articles

Quantitative analysis of underivatized 17 β-estradiol using a high-throughput LC–MS/MS assay – Application to support a pharmacokinetic study in ovariectomized guinea pigs

Difference in female sex hormone, β-estradiol (E2), levels can contribute to sex differences in biological processes that underlie target tissue functions (QT interval), vulnerability to diseases (hepatitis or HIV), and response toward therapies. Accurate quantification of plasma E2 level is thus an important aspect in both basic science research examining hormone-regulated physiological mechanisms and in clinical settings to support patient care associated with altered E2 levels. Due to lack of a high-throughput high-sensitivity analytical method, we developed and validated a LC–MS/MS assay for accurate low-level quantification of E2 and demonstrated its application to a guinea pig pharmacokinetic study in which guinea pigs were treated with 10 or 40 μg/kg E2 subcutaneously and blood samples collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post-dosing. E2 was extracted using 90 μL ovariectomized guinea pig plasma by liquid-liquid extraction. The method was robust, sensitive with linear range from 3.9 to 1000 pg/mL, and the assay met acceptance criteria for validation parameters listed in the current FDA Guidance on Bioanalytical Method Validation. Compared to the 10 μg/kg dose, more than dose proportional increase in maximum E2 plasma concentration (Cmax) and AUC0-∞ and correspondingly longer half-life were observed after 40 μg/kg dose. This assay is a significant improvement over existing E2 quantification methods in bioanalytical field, with high precision and accuracy, low sample and injection volumes, no derivatization, and short assay run time of 3 min. This assay is amenable in high-throughput settings requiring low-level E2 quantitation in basic science research and clinical settings.

Effect of ovariectomy on intracellular Ca2+ regulation in guinea pig cardiomyocytes.

This study addressed the hypothesis that long-term deficiency of ovarian hormones after ovariectomy (OVx) alters cellular Ca2+-handling mechanisms in the heart, resulting in the formation of a proarrhythmic substrate. It also tested whether estrogen supplementation to OVx animals reverses any alterations to cardiac Ca2+ handling and rescues proarrhythmic behavior. OVx or sham operations were performed on female guinea pigs using appropriate anesthetic and analgesic regimes. Pellets containing 17β-estradiol (1 mg, 60-day release) were placed subcutaneously in selected OVx animals (OVx + E). Cardiac myocytes were enzymatically isolated, and electrophysiological measurements were conducted with a switch-clamp system. In fluo-4-loaded cells, Ca2+ transients were 20% larger, and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials, creating a larger "window" current. SR Ca2+ stores were 22% greater in the OVx group, and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations, and a greater percentage of these cells showed delayed afterdepolarizations after exposure to isoprenaline compared with sham myocytes. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx + E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pigs lead to changes in myocyte Ca2+-handling mechanisms that are considered proarrhythmogenic. 17β-Estradiol replacement prevented these adverse effects.NEW & NOTEWORTHY Ovariectomized guinea pig cardiomyocytes have higher frequencies of Ca2+ waves, and isoprenaline-challenged cells display more early afterdepolarizations, delayed afterdepolarizations, and extra beats compared with sham myocytes. These alterations to Ca2+ regulation were not observed in myocytes from ovariectomized guinea pigs supplemented with 17β-estradiol, suggesting that ovarian hormone deficiency modifies cardiac Ca2+ regulation, potentially creating proarrhythmic substrates.

Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

Use of long-acting progestin only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+ medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+ progesterone (P4) were analyzed by quantitative Real-time (q)-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA) treated women and ovariectomized guinea pigs (GPs) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR-mediated transcription. The resultant PR and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding.

Estrogen Alters Remodeling of the Vaginal Wall after Surgical Injury in Guinea Pigs1

Loss of pelvic organ support (i.e., pelvic organ prolapse) is common in menopausal women. Surgical reconstruction of pelvic organ prolapse is plagued with high failure rates. The objective of this study was to determine the effects of estrogen on biomechanical properties, lysyl oxidase (LOX), collagen content, and histomorphology of the vagina with or without surgical injury. Nulliparous ovariectomized guinea pigs were treated systemically with either 50 μg/kg/day estradiol (E2,) or vehicle. After 2 wk, vaginal surgery was performed, and animals were treated with either beta-aminopropionitrile (BAPN, an irreversible LOX inhibitor), or vehicle to determine the role of LOX in recovery of the vaginal wall from injury with or without E2. Estradiol resulted in (i) significant growth, increased smooth muscle, and increased thickness of the vagina, (ii) increased distensibility without compromise of maximal force at failure, and (iii) increased total and cross-linked collagen. In the absence of E2, BAPN resulted in decreased collagen and vaginal wall strength in the area of the injury. In contrast, in E2-treated animals, increased distensibility, maximal forces, and total collagen were maintained despite BAPN. Interestingly, LOX mRNA was induced dramatically (9.5-fold) in the injured vagina with or without E2 at 4 days. By 21 days, however, LOX levels declined to near baseline in E2-deprived animals. LOX mRNA levels remained strikingly elevated (12-fold) at 21 days in the estrogenized vagina. The results suggest that prolonged E2 induced increases in LOX, and collagen cross-links may act to sustain a matrix environment that optimizes long-term surgical wound healing in the vagina.

Effects of puerarin on cholinergic enzymes in the brain of ovariectomized guinea pigs

Estrogen has beneficial effects on neurodegenerative disorders and cognitive function of postmenopausal women. Puerarin, isolated from Pueraria lobota, has been classified as a phytoestrogen, which can be highly effective against cerebrovascular diseases. In this study, the effects of puerarin on neural cholinergic system in the brain of ovariectomized guinea pigs were studied. The puerarin at the doses used (15 mg/kg body weight (bw)/day and 30 mg/kg bw/day) for 10 days had the estrogenic activity indicated by the attenuation of the reduction of uterine weight induced by ovariectomy. In brain, puerarin treatment increased choline acetyltransferase (ChAT) activity and expression in hippocampus, and increased ChAT immnuopositive signals in septal diagonal region. Puerarin treatment could suppress the increase of acetylcholinesterase expression and activity to the levels of the intact group, although they were not significantly different from those of the ovariectomized animals. Moreover, puerarin decreased the β-amyloid immunopositive staining in hippocampus. In brief, the present study suggests that puerarin prevents the dysfunction of the neuronal cholinergic system and ameliorates the increase of β-amyloid caused by estrogen deficiency.

17β-estradiol enhances cholecystokinin-induced stimulation of colonic motility in ovariectomized guinea pigs

17β-estradiol enhances cholecystokinin-induced stimulation of colonic motility in ovariectomized guinea pigs

Estrogen Regulation of Genes Important for K+ Channel Signaling in the Arcuate Nucleus

Estrogen affects the electrophysiological properties of a number of hypothalamic neurons by modulating K(+) channels via rapid membrane actions and/or changes in gene expression. The interaction between these pathways (membrane vs. transcription) ultimately determines the effects of estrogen on hypothalamic functions. Using suppression subtractive hybridization, we produced a cDNA library of estrogen-regulated, brain-specific guinea pig genes, which included subunits from three prominent K+ channels (KCNQ5, Kir2.4, Kv4.1, and Kvbeta(1)) and signaling molecules that impact channel function including phosphatidylinositol 3-kinase (PI3K), protein kinase Cepsilon (PKCepsilon), cAMP-dependent protein kinase (PKA), A-kinase anchor protein (AKAP), phospholipase C (PLC), and calmodulin. Based on these findings, we dissected the arcuate nucleus from ovariectomized guinea pigs treated with estradiol benzoate (EB) or vehicle and analyzed mRNA expression using quantitative real-time PCR. We found that EB significantly increased the expression of KCNQ5 and Kv4.1 and decreased expression of KCNQ3 and AKAP in the rostral arcuate. In the caudal arcuate, EB increased KCNQ5, Kir2.4, Kv4.1, calmodulin, PKCepsilon, PLCbeta(4), and PI3Kp55gamma expression and decreased Kvbeta(1). The effects of estrogen could be mediated by estrogen receptor-alpha, which we found to be highly expressed in the guinea pig arcuate nucleus and, in particular, proopiomelanocortin neurons. In addition, single-cell RT-PCR analysis revealed that about 50% of proopiomelanocortin and neuropeptide Y neurons expressed KCNQ5, about 40% expressed Kir2.4, and about 60% expressed Kv4.1. Therefore, it is evident that the diverse effects of estrogen on arcuate neurons are mediated in part by regulation of K(+) channel expression, which has the potential to affect profoundly neuronal excitability and homeostatic functions, especially when coupled with the rapid effects of estrogen on K(+) channel function.

The effects of the novel selective progesterone receptor modulator (SPRM) asoprisnil on the morphology of the reproductive tract of cycling and ovariectomized guinea pigs

Asoprisnil, its active metabolite J912, and J956 are novel SPRMs that exhibit partial (mixed) agonist/antagonist activities in animal and humans. SPRMs inhibit endometrial proliferation and induce amenorrhea in non-human primates and humans. To evaluate the effects of asoprisnil, J912, and J956 on weights and morphology of the uterus and vagina in intact and ovariectomized (OVX) guinea pigs. The effects of SPRMs were compared with those of promegestone (R5020, pure progestin), progesterone (P) and the antiprogestins mifepristone (RU486) and onapristone (ONA). Intact guinea pigs (n=5/group) were treated subcutaneously (s.c.) with either the SPRMs or the reference compounds RU486, ONA, R5020, P or vehicle from cycle day 8–14 (dose/animal/day: 1.0mg for promegestone and 10.0mg for the other compounds). OVX guinea pigs (n=7/group) were primed s.c. with estradiol (E2) (0.03μg/day for 5 days) and then treated with the SPRMs and the reference compounds for 14 days in the presence of E2 (same dose). Autopsy was performed 24 hours later. Histological evaluation was based on serial HE-stained paraffin sections (6–10μm). SPRMs, progesterone and R5020, but not the antiprogestins, significantly (p<0.05) reduced the uterine and vaginal weights in intact animals. Proliferation and cornification of the vaginal epithelium prevailed after ONA and RU486. The employed E2 dose, when administered alone, led to minor increase of uterine and vaginal weights. ONA enhanced E2 effects on the uterus and vagina. Vaginal and endometrial histology revealed a massive epithelial proliferation, consistent with an “unopposed” estrogenic effect. The endometrium of animals exposed to ONA+E2 was highly hyperplastic with cystic changes. In contrast, the SPRMs and R5020 restored secretory appearance of the endometrium and induced mucification of the vaginal epithelium. Asoprisnil and the structurally related SPRMs exhibited P agonist effects on endometrial and vaginal morphology in both intact and OVX guinea pigs. In contrast, the antiprogestins induced a condition of “unopposed” estrogenic effects in the vagina and uterus. Asoprisnil has the potential to control hormone-dependent gynaecological disorders without inducing “unopposed” estrogen effects.

Modulation of uterine prostaglandin secretion by the selective progesterone receptor modulator (SPRM) asoprisnil, progestins, and antiprogestins in cycling and ovariectomized guinea pigs

In cycling guinea pigs, the uterine PGF2α controls luteolysis. Hysterectomy prolongs the life span of the corpus luteum (CL), resulting in long-term elevation in serum progesterone (P) levels. To study the effects of asoprisnil, J912, and J956 (SPRMs with partial agonist/antagonist activities), promegestone (R5020) and progesterone (P) (pure PR agonists), and mifepristone (RU486) and onapristone (ONA) (PR antagonists) on uterine PGF2α production and COX I and COX II mRNA expression in intact and ovariectomized (OVX) guinea pigs.

Guinea pigs as models to study the hypocholesterolemic effects of drugs.

Guinea pigs are useful models to investigate the mechanisms of the hypocholesterolemic effects of drugs. Like humans, guinea pigs are one of the few species that carry the majority of cholesterol in LDL. This animal model has also been shown to develop atherosclerosis when challenged with hypercholesterolemic diets. In addition, plasma lipid profiles in males, females and ovariectomized guinea pigs, a model for menopause, follow similar patterns to those observed in humans. In this report, drugs aimed at lowering plasma cholesterol and triglycerides in hyperlipidemic individuals are reviewed. Studies analyzing the hypolipidemic effect of HMG-CoA reductase inhibitors, acyl CoA cholesterol acyltransferase inhibitors, fibrates, bile acid resins, apical sodium bile acid transporter inhibitors, and others show that guinea pigs and humans have comparable responses to drug therapy. In addition, results from the limited clinical reports addressing specific effects of drugs on LDL catabolism or VLDL synthesis are in agreement with observations in guinea pigs. From the review of these studies, it is apparent that the guinea pig is a useful animal model to further explore the mechanisms of action of lipid lowering drugs including effects on specific receptors and regulatory enzymes involved in cholesterol metabolism and on early atherosclerosis development. ACAT, acyl-CoA:cholesterol acyltransferase; ASBT, apical sodium co-dependent bile acid transporter; ApoB, apolipoprotein B; CHD, coronary heart disease; CYP7, cholesterol 7alpha-hydroxylase; HDL, high density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; FCR, free catabolic rate; LDL, low density lipoprotein; PPAR, peroxisome proliferators-activated receptor; TC, total cholesterol; TG, triglycerides; VLDL, very low density lipoprotein.

Comparative distribution of estrogen receptor α and β immunoreactivities in the forebrain and the midbrain of the female guinea pig

Estrogen plays an important role in regulating gonadotropin secretion and reproductive behavior. The estrogen receptor α (ERα) was believed to be the only receptor which mediated the actions of the hormone until the identification of a novel ER called ERβ. In the present study, the map of ERα immunoreactive (IR) neurons was compared with the distribution pattern of ERβ-IR neurons in the forebrain and midbrain of ovariectomized guinea pigs using immunocytochemistry. The immunoreactivities appeared to be mainly nuclear in their subcellular distribution. Both ERα- and ERβ-like immunoreactivities were highly expressed in the bed nucleus of the stria terminalis and the ventrolateral hypothalamic nucleus but were found to be differentially expressed in discrete subregions of the amygdaloid complex. A large number of intensely labeled ERα cells were observed throughout the rostrocaudal extent of the preoptic region, whereas only a few ERβ-IR neurons were found in the periventricular preoptic nucleus bordering the third ventricle or scattered in the medial preoptic area. In contrast, only ERα-immunoreactivity was seen in the septum, and in the magnocellular supraoptic, paraventricular, arcuate, and premammillary nuclei. In the midbrain, neurons containing ERα were observed throughout the rostrocaudal extent of the gray matter, whereas ERβ was only detected within the dorsal raphe nucleus. These observations provide evidence of a distinct neuroanatomical pattern for the two subtypes of the ER which may have different roles in regulating behavior and the neuroendocrine mechanisms of reproduction. Species similarities and differences in the distributions of ERα and ERβ immunoreactivities are discussed.

A New Estrogenic Pathway

Although estrogens have classically been believed to act through nuclear receptors that function as transcription factors, it has become increasingly apparent that, as with other steroid hormones, rapid effects of estrogens can be initiated at the plasma membrane. The receptors and pathways through which these peripheral effects are mediated, however, remain unclear. Qiu et al. investigated modulation of γ-aminobutyric acid (GABA)-mediated activation of heterotrimeric guanosine triphosphate-binding protein-coupled inwardly rectifying K + (GIRK) channels by 17β-estradiol (E 2 ) in hypothalamic neurons. They identified a signaling pathway that involved activation of Gα q after E 2 binding to a transmembrane estrogen receptor. The authors used whole-cell recordings of GABA-, proopiomelanocortin-, and dopamine-expressing neurons in hypothalamic slices from ovariectomized guinea pigs to investigate the effects of E 2 and various selective estrogen receptor modulators (SERMs) on GIRK activation by the GABA B receptor agonist baclofen. E 2 inhibited baclofen-mediated GIRK activation, as did raloxifene, STX (a SERM without activity at nuclear estrogen receptors), and membrane-impermeant E 2 bound to bovine serum albumen, whereas tamoxifen did not. Pharmacological analysis indicated that this effect of E 2 involved protein kinase C δ acting upstream of protein kinase A, but not mitogen-activated protein kinase pathways. The authors used a peptide inhibitor to implicate Gα q in this signaling pathway. These data extend the mechanisms whereby estrogens are known to act within the central nervous system; moreover, the identification of STX as selective for this pathway may facilitate the development of highly specific approaches to estrogen therapy. J. Qiu, M. A. Bosch, S. C. Tobias, D. K. Grandy, T. S. Scanlan, O. K. Rønnekleiv, M. J. Kelly, Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C. J. Neurosci 23 , 9529-9540 (2003). [Abstract] [Full Text]

Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy

Asoprisnil is a novel selective steroid receptor modulator that shows unique pharmacodynamic effects in animal models and humans. Asoprisnil, its major metabolite J912, and structurally related compounds represent a new class of progesterone receptor (PR) ligands that exhibit partial agonist and antagonist activities in vivo. Asoprisnil demonstrates a high degree of receptor and tissue selectivity, with high-binding affinity for PR, moderate affinity for glucocorticoid receptor (GR), low affinity for androgen receptor (AR), and no binding affinity for estrogen or mineralocorticoid receptors. In the rabbit endometrium, both asoprisnil and J912 induce partial agonist and antagonist effects. Asoprisnil induces mucification of the guinea pig vagina and has pronounced anti-uterotrophic effects in normal and ovariectomized guinea pigs. Unlike antiprogestins, asoprisnil shows only marginal labor-inducing activity during mid-pregnancy and is completely ineffective in inducing preterm parturition in the guinea pig. Asoprisnil exhibits only marginal antiglucocorticoid activity in transactivation in vitro assays and animal models. In male rats, asoprisnil showed weak androgenic and anti-androgenic properties. In toxicological studies in female cynomolgus monkeys, asoprisnil treatment abolished menstrual cyclicity and endometrial atrophy. Early clinical studies of asoprisnil in normal volunteers demonstrated a dose-dependent suppression of menstruation irrespective of the effects on ovulation, with no change in basal estrogen concentrations and no antiglucocorticoid effects. Unlike progestins, asoprisnil does not induce breakthrough bleeding. With favorable safety and tolerability profiles thus far, asoprisnil appears promising as a novel treatment of gynecological disorders, such as uterine fibroids and endometriosis.

Grape Polyphenols Decrease Plasma Triglycerides and Cholesterol Accumulation in the Aorta of Ovariectomized Guinea Pigs

Female ovariectomized guinea pigs, a model for menopausal women, were fed either a control diet or a diet containing 10 g/100 g of a lyophilized grape preparation for 12 wk. The macronutrient composition of the grape preparation was: simple carbohydrates, 90 g/100 g; protein, 4 g/100 g; and dietary fiber, 6 g/100 g. Control and grape diets had the same composition except for the percentage of macronutrients provided by the grape preparation. Polyphenols were present in the grape preparation at 0.58 g/100 g and included flavans, anthocyanins, quercetin, myricetin, kaempferol and resveratrol. Dietary cholesterol was 0.33 g/100 g to raise plasma cholesterol concentrations and ensure the development of atherosclerosis. Plasma LDL cholesterol concentrations did not differ between groups, whereas plasma triglycerides and VLDL cholesterol were 39 and 50% lower, respectively in guinea pigs fed the grape diet compared with controls (P < 0.05). Significant modifications in LDL particles included 58 and 30% lower triglycerides and phospholipids, respectively (P < 0.0001). Hepatic acyl CoA:cholesteryl acyltransferase activity was 27% lower (P < 0.05) in the grape diet-fed group compared with controls. In addition, concentrations of cholesterol in the aorta were 33% lower (P < 0.05) in guinea pigs fed the grape diet. These results suggest that grape intake in ovariectomized guinea pigs alters hepatic cholesterol metabolism, which may affect VLDL secretion rates and result in less accumulation of cholesterol in the aorta.

Gender and hormonal status affect the regulation of hepatic cholesterol 7α-hydroxylase activity and mRNA abundance by dietary soluble fiber in the guinea pig

Dietary soluble fiber (SF) consistently lowers plasma LDL cholesterol (LDL-C) concentrations, however, secondary mechanisms governing this reduction are not completely defined. Moreover, these mechanisms appear to differ with gender. Male, female and ovariectomized (to mimic menopause) guinea pigs were used to assess effects of gender, hormonal status and SF on activity and expression of hepatic cholesterol 7α-hydroxylase (CYP7). Diets were identical except for fiber source (control 10% cellulose, SF 5% psyllium/5% pectin). SF intake resulted in 44% lower plasma total cholesterol, 51% lower plasma LDL-C and 22% lower plasma triacylglycerol (TAG) concentrations. However, ovariectomized guinea pigs fed either the control or SF diets, had the highest plasma LDL-C and TAG levels ( P<0.01). SF altered hepatic cholesterol metabolism by effectively reducing hepatic free cholesterol, TAG and microsomal free cholesterol, while activity of CYP7, the rate-limiting enzyme of cholesterol catabolism, was up-regulated. Hepatic CYP7 mRNA abundance paralleled the increase in enzyme activity. Ovariectomized guinea pigs had lowest activity and expression of hepatic CYP7 even after intervention with SF. These results suggest that induction of hepatic CYP7 activity may account, in large part, for the hypocholesterolemic effect of SF. Gender and hormonal status influence metabolic responses to dietary SF with estrogen deprivation leading to the most detrimental lipid profile.

Dietary fat saturation and gender/hormonal status modulate plasma lipids and lipoprotein composition

Male, female and ovariectomized (to mimic menopause) guinea pigs were fed a saturated (SFA) or a polyunsaturated (PUFA) fat diet for 4 weeks to determine the effects of dietary fat saturation on lipoprotein levels and composition and to assess whether gender and hormonal status modulate the cholesterolemic response. Both diets contained 15g/100 g fat and 0.04 g/100 g cholesterol and were identical in composition except for the type of fat. The SFA diet contained 50% saturated fat (25% lauric + myristic fatty acids), 25% PUFA and 25% monounsaturated fatty acids while the PUFA diet had 50% PUFA (linoleic acid), 25% monounsaturated and 25% SFA fatty acids. Plasma LDL cholesterol (LDL-C) was an average 21% lower in guinea pigs fed PUFA compared to those fed SFA (P < 0.05). In addition, ovariectomized guinea pigs, both in the SFA and PUFA groups, had 20–33% higher LDL-C than either males or females (P < 0.01). VLDL cholesterol was 70% higher in the PUFA-fed animals (P < 0.0001). A gender effect was observed in plasma HDL cholesterol (HDL-C) with females and ovariectomized guinea pigs having 30–42% higher HDL-C than males (P < 0.01). LDL susceptibility to oxidation was not affected by dietary fat saturation or gender. In contrast, VLDL and LDL composition were significantly influenced by diet and gender. VLDL particles were larger in size in guinea pigs fed the SFA diets (P < 0.01) while LDL particles were larger in female guinea pigs (P < 0.001). Hepatic lipids were influenced by the interaction between diet and group. Hepatic cholesterol (P < 0.01) and TAG concentrations (P < 0.0001) were highest in female guinea pigs fed the PUFA diet. Since the liver is the major site of lipoprotein synthesis and catabolism, these results suggest that not only diet but also gender may play a major role in determining the composition and size of lipoproteins.

Soluble fiber and soybean protein reduce atherosclerotic lesions in guinea pigs. Sex and hormonal status determine lesion extension.

These studies were undertaken to assess guinea pigs as potential models for early atherosclerosis development. For that purpose, male, female, and ovariectomized (to mimic menopause) guinea pigs were fed a control or a TEST diet for 12 wk. Differences between diets were the type of protein (60% casein/40% soybean vs. 100% soybean) and the type of fiber (12.5% cellulose vs. 2.5% cellulose/5% pectin/5% psyllium) for control and TEST diets, respectively. Diet had no effect on plasma cholesterol or triacylglycerol (TAG) concentrations; however, there were significant effects related to sex/hormonal status. Ovariectomized guinea pigs had higher plasma cholesterol and TAG concentrations than males or females (P < 0.01). In contrast to effects on plasma lipids, hepatic cholesterol and TAG were 50% lower in the TEST groups (P < 0.01) compared to controls. Low density lipoproteins (LDL) from guinea pigs fed the TEST diet had a lower number of cholesteryl ester (CE) molecules and a smaller diameter than LDL from controls. Atherosclerotic lesions were modulated by both diet (P < 0.0001) and sex (P < 0.0001). Guinea pigs fed the TEST diet had 25% less lesion extension whereas males had 20% larger occlusion of the arteries compared to both female and ovariectomized guinea pigs. Significant positive correlations were found between LDL CE and atherosclerotic lesions (r = 0.495, P < 0.05) and LDL size and fatty streak area (r = 0.56, P < 0.01). In addition, females fed the TEST diet had the lowest plasma and hepatic cholesterol concentrations, the smallest LDL particles, and the least atherosclerosis involvement compared to the other groups. These data indicate that dietary factors and sex/hormonal status play a role in determining plasma lipids and atherosclerosis in guinea pigs.

Pregnancy and estradiol decrease GTPase activity in the guinea pig uterine artery.

The mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner are poorly understood. We propose that it is consequential to estrogen-mediated alterations in G protein-mediated signal transduction. Aortas and uterine (UAs) and mesenteric arteries (MAs) were obtained from late-pregnant, nonpregnant, or ovariectomized guinea pigs chronically treated with 17beta-estradiol. High-affinity GTPase activity was assayed enzymatically. The cGMP generated in response to the endothelium-dependent agonist ACh was measured in UAs incubated with or without cholera toxin (CTX, which inhibits G(s)alpha). Pregnancy significantly decreased UA but not aorta or MA GTPase activity. 17beta-Estradiol decreased UA GTPase activity compared with untreated ovariectomized animals. ACh increased cGMP in pregnant but not nonpregnant UAs. Pretreatment of nonpregnant UAs with CTX increased ACh-induced cGMP levels similar to pregnancy. Thus pregnancy and estradiol decrease the GTPase activity of a CTX-sensitive G protein in UAs, increasing receptor-dependent cGMP release. This alteration in receptor-mediated G protein coupling in UAs may contribute to the characteristic cardiovascular adaptation to pregnancy.

Gender and Hormonal Status Affect the Hypolipidemic Mechanisms of Dietary Soluble Fiber in Guinea Pigs

The objective of this study was to assess the effects of gender on the secondary mechanisms by which dietary soluble fiber lowers plasma LDL cholesterol. For that purpose, male, female and ovariectomized (to mimic menopause) guinea pigs (8-10 per group) were allocated to two dietary treatments. Diets were identical in composition except for the fiber source: the control diet contained 10 g/100 of cellulose and 2.5 g/100 g of guar gum, while the soluble fiber (SF) diet contained 5 g/100 of psyllium, 5 g/100 of pectin and 2.5 g/100 g of guar gum. SF intake resulted in 44% lower plasma LDL cholesterol, 64% lower apo B and 22% lower plasma triacylglycerol (TAG) concentrations (P < 0.01) compared to guinea pigs fed the control diet. However, ovariectomized guinea pigs had higher plasma cholesterol, apo B and TAG concentrations (P < 0.01) compared to males and females, even those fed SF. Plasma HDL-cholesterol was higher in females than in males (P < 0.05). LDL size, as measured by LDL composition and fast protein liquid chromatography, was larger in females than males. Guinea pigs fed SF had smaller LDL than controls. LDL susceptibility to oxidation was 80% lower in male and females fed the SF diet (P < 0.001) than in controls, while there was no effect of diet in ovariectomized guinea pigs. Hepatic free cholesterol and TAG were lower, and activities of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7alpha-hydroxylase were higher in guinea pigs fed SF (P < 0.05) than in controls. These results indicate that gender plays an important role in the metabolic responses to dietary soluble fiber and that estrogen deprivation leads to a detrimental lipoprotein profile.

Quantitative studies of progesterone receptor and nitric oxide synthase colocalization with somatostatin, or neurotensin, or substance P in neurons of the guinea pig ventrolateral hypothalamic nucleus: an immunocytochemical triple-label analysis.

Previous double-label studies have demonstrated that progesterone receptors (PR) may coexist with nitric oxide synthase (NOS) and also with neuroactive peptides such as somatostatin (SOM), neurotensin (NT) and substance P (SP) in neurons of the ventrolateral hypothalamic nucleus (VL) of the guinea pig. In the present study, triple-label immunofluorescence histochemistry was used to determine whether neurons co-expressing both PR and NOS also contain one neuropeptide (SOM, NT or SP) and to quantify these colocalization relationships. Ovariectomized guinea pigs were primed with estradiol to induce PR immunoreactivity and treated with colchicine to visualize immunoreactive (IR) peptidergic neurons. Using three primary antibodies raised in different species and labeled with three different markers, it is possible to recognize which type(s) of immunoreactivity each cell contains on the same sections. We observed that PR, NOS and SOM co-occurred extensively, whereas PR, NOS and NT or PR, NOS and SP were colocalized infrequently in neurons of the VL. Combining our various quantitative observations, we found that SOM immunoreactivity appeared in 33% of the PR/NOS-IR neurons in the caudal aspect of the nucleus and that nearly all neurons containing SOM and NOS contained PR. The occasional localization between PR, NOS and SP seen in the rostral subdivision of the VL represented a very low proportion of the PR/NOS-IR neurons but 48% of NOS/SP-IR neurons. The relatively high proportion of PR/NOS-IR cells expressing SOM in the VL suggests that these neurons are part of a neural circuitry involved in a variety of steroid-dependent functions.