Progesterone increases plasma volume independent of estradiol.

Adequate plasma volume (PV) and extracellular fluid (ECF) volume are essential for blood pressure and fluid regulation. We tested the hypotheses that combined progesterone (P(4))-estrogen (E(2)) administration would increase ECF volume with proportional increases in PV, but that P(4) would have little independent effect on either PV or ECF volume. We further hypothesized that this P(4)-E(2)-induced fluid expansion would be a function of renin-angiotensin-aldosterone system stimulation. We suppressed P(4) and E(2) with a gonadotropin-releasing hormone (GnRH) antagonist in eight women (25 +/- 2 yr) for 16 days; P(4) (200 mg/day) was added for days 5-16 (P(4)) and 17beta-estradiol (2 x 0.1 mg/day patches) for days 13-16 (P(4)-E(2)). On days 2 (GnRH antagonist), 9 (P(4)), and 16 (P(4)-E(2)), we estimated ECF and PV. To determine the rate of protein and thus water movement across the ECF, we also measured transcapillary escape rate of albumin. In P(4), P([P(4)]) increased from 2.5 +/- 1.3 to 12.0 +/- 2.8 ng/ml (P < 0.05) with no change in P([E(2)]) (21.5 +/- 9.4 to 8.6 +/- 2.0 pg/ml). In P(4)-E(2), plasma concentration of P(4) remained elevated (11.3 +/- 2.7 ng/ml) and plasma concentration of E(2) increased to 254.1 +/- 52.7 pg/ml (P < 0.05). PV increased during P(4) (46.6 +/- 2.5 ml/kg) and P(4)-E(2) (48.4 +/- 3.9 ml/kg) compared with GnRH antagonist (43.3 +/- 3.2 ml/kg; P < 0.05), as did ECF (206 +/- 19, 244 +/- 25, and 239 +/- 27 ml/kg for GnRH antagonist, P(4), and P(4)-E(2), respectively; P < 0.05). Transcapillary escape rate of albumin was lowest during P(4)-E(2) (5.8 +/- 1.3, 3.5 +/- 1.7, and 2.2 +/- 0.4%/h for GnRH antagonist, P(4), and P(4)-E(2), respectively; P < 0.05). Serum aldosterone increased during P(4) and P(4)-E(2) compared with GnRH antagonist (79 +/- 17, 127 +/- 13, and 171 +/- 25 pg/ml for GnRH antagonist, P(4), and P(4)-E(2), respectively; P < 0.05), but plasma renin activity and plasma concentration of ANG II were only increased by P(4)-E(2). This study is the first to isolate P(4) effects on ECF; however, the mechanisms for the ECF and PV expansion have not been clearly defined.     

Estrogen and progesterone effects on transcapillary fluid dynamics

The purpose of this study was to determine estrogen (E(2)) and progesterone (P(4)) effects on atrial natriuretic peptide (ANP) control of plasma volume (PV) and transcapillary fluid dynamics. To this end, we suppressed reproductive function in 12 women (age 21-35 yr) using a gonadotropin releasing-hormone (GnRH) analog (leuprolide acetate) for 5 wk. During the 5th week, the women either received 4 days of E(2) administration (17beta-estradiol, transdermal patch, 0.1 mg/day) or 4 days of E(2) with P(4) administration (vaginal gel, 90 mg P(4) twice per day). At the end of the 4th and 5th week of GnRH analog and hormone administration, we determined PV (Evans blue dye) and changes in PV and forearm capillary filtration coefficient (CFC) during a 120-min infusion of ANP (5 ng x kg body wt(-1) x min(-1)). Preinfusion PV was estimated from Evans blue dye measurement taken over the last 30 min of infusion based on changes in hematocrit. E(2) treatment did not affect preinfusion PV relative to GnRH analog alone (45.3 +/- 3.1 vs. 45.4 +/- 3.1 ml/kg). During ANP infusion CFC was greater during E(2) treatment compared with GnRH analog alone (6.5 +/- 1.4 vs. 4.9 +/- 1.4 microl. 100 g(-1) x min(-1) mmHg(-1), P < 0.05). The %PV loss during ANP infusion was similar for E(2) and GnRH analog-alone treatments (-0.8 +/- 0.2 and -1.0 +/- 0.2 ml/kg, respectively), indicating the change in CFC had little systemic effect on ANP-related changes in PV. Estimated baseline PV was reduced by E(2)-P(4) treatment. During ANP infusion CFC was approximately 30% lower during E(2)-P(4) (6.0 +/- 0.5 vs. 4.3 +/- 4.3 microl. 100 g(-1) x min(-1) mm Hg(-1), P < 0.05), and the PV loss during ANP infusion was attenuated (-0.9 +/- 0.2 and -0.2 +/- 0.2 ml/kg for GnRH analog-alone and E(2)-P(4) treatments, respectively). Thus the E(2)-P(4) treatment lowered CFC and reduced PV loss during ANP infusion.     

Effects of a gonadotrophin-releasing hormone antagonist on gonadotrophin secretion and gonadal development in neonatal pigs

Male (N = 8) and female (N = 8) pigs were assigned to receive saline or a potent GnRH antagonist ([Ac-D2Nal1,D4-Cl-Phe2,D-Trp3,D-Arg6, D-Ala10]- GnRH*HOAc; 1 mg/kg body weight) at 14 days of age. The GnRH antagonist caused LH to decline (P less than 0.01) from 1.7 ng/ml at 0 h to less than 0.5 ng/ml during 4-32 h in males and females. Concentrations of FSH in gilts declined slowly from 75 +/- 8 to 56 +/- 5 ng/ml (P less than 0.05) at 32 h. In males FSH was low (5.7 +/- 0.5 ng/ml) at 0 h and did not change significantly. To observe the effect of long-term treatment with GnRH antagonist, 10 male and 10 female pigs, 3 days of age, were treated with saline or 1 mg GnRH antagonist per kg body weight every 36 h for 21 days. Concentrations of LH were reduced (P less than 0.01) to 0.2-0.4 ng/ml throughout the experimental period in male and female piglets treated with GnRH antagonist. Plasma FSH increased in control females, but remained suppressed (P less than 0.001) in females treated with GnRH antagonist. Treatment with the GnRH antagonist suppressed FSH levels in males on Days 8 and 16 (P less than 0.05), but not on Day 24. Treatment of females with the GnRH antagonist did not influence (P greater than 0.10) oestradiol-17 beta concentrations. Administration of GnRH antagonist to males suppressed testosterone and oestradiol-17 beta values (P less than 0.01) and reduced testicular weight (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.     

Estrogen effects on osmotic regulation of AVP and fluid balance

To determine estrogen effects on osmotic regulation of arginine vasopressin (AVP) and body fluids, we suppressed endogenous estrogen and progesterone using the gonadotropin-releasing hormone (GnRH) analog leuprolide acetate (GnRHa). Subjects were assigned to one of two groups: 1) GnRHa alone, then GnRHa + estrogen (E, n = 9, 25 +/- 1 yr); 2) GnRHa alone, then GnRHa + estrogen with progesterone (E/P, n = 6, 26 +/- 3). During GnRHa alone and with hormone treatment, we compared AVP and body fluid regulatory responses to 3% NaCl infusion (HSI, 120 min, 0.1 ml. min(-1). kg body wt(-1)), drinking (30 min, 15 ml/kg body wt), and recovery (60 min of seated rest). Plasma [E(2)] increased from 23.9 to 275.3 pg/ml with hormone treatments. Plasma [P(4)] increased from 0.6 to 5.7 ng/ml during E/P and was unchanged (0.4 to 0.6 ng/ml) during E. Compared with GnRHa alone, E reduced osmotic AVP release threshold (275 +/- 4 to 271 +/- 4 mosmol/kg, P < 0.05), and E/P reduced the AVP increase in response during HSI (6.0 +/- 1.3 to 4.2 +/- 0.6 pg/ml at the end of HSI), but free water clearance was unaffected in either group. Relative to GnRHa, pre-HSI plasma renin activity (PRA) was greater during E (0.8 +/- 0.1 vs. 1.2 +/- 0.2 ng ANG I. ml(-1). h(-1)) but not after HSI or recovery. PRA was greater than GnRHa during E/P at baseline (1.1 +/- 0.2 vs. 2.5 +/- 0.6) and after HSI (0.6 +/- 0.1 vs. 1.1 +/- 1.1) and recovery (0.5 +/- 0.1 vs. 1.3 +/- 0.2 ng ANG I. ml(-1). h(-1)). Baseline fractional excretion of sodium was unaffected by E or E/P but was attenuated by the end of recovery for both E (3.3 +/- 0.6 vs. 2.4 +/- 0.4%) and E/P (2.8 +/- 0.4 vs 1.7 +/- 0.4%, GnRHa alone and with hormone treatment, respectively). Fluid retention increased with both hormone treatments. Renal sensitivity to AVP may be lower during E due to intrarenal effects on water and sodium excretion. E/P increased sodium retention and renin-angiotensin-aldosterone stimulation.     

Restoration of endocrine and ovarian function after stopping GnRH antagonist treatment in goats

We have tested if the high number of unfertilized ova and degenerated embryos found in superovulated goats previously treated with GnRH antagonist can be related to a prolongation of gonadotrophin down-regulation and/or alterations in follicular function during the period of administration of the superovulatory treatment, around 4 days after the end of the antagonist treatment. A total of 15 does were treated with intravaginal progestagen sponges and daily injections of 0.5mg of the GnRH antagonist Antarelix for 6 days, while 5 does acted as controls receiving saline. During the antagonist treatment, the mean plasma LH concentration was lower in treated than control goats (0.5 +/- 0.2 versus 0.7 +/- 0.5 ng/ml, P < 0.0005 ); however, the FSH levels remained unaffected (0.8 +/- 0.4 versus 0.8 +/- 0.5 ng/ml). In this period, treated does also showed an increase in the number of small follicles with 2-3 mm in size ( 10.7 +/- 0.7 versus 8.4 +/- 0.6, P < 0.05), and a decrease in both the number of follicles > or =4 mm in size ( 5.0 +/- 0.3 versus 6.8 +/- 0.5, P < 0.005) and the secretion of inhibin A (120.9 +/- 10.7 versus 151.6 +/- 12.6 pg/ml, P < 0.05). After cessation of the antagonist treatment, there was an increase in LH levels in treated goats from the day after the last Antarelix injection (Day 1), so that LH levels were the same as controls on Day 3 (0.6 +/- 0.1 versus 0.6 +/- 0.2 ng/ml). However, there were even greater numbers of small follicles than during the period of antagonist injections (15.4 +/- 0.6 in treated versus 8.9 +/- 0.7 in control, P < 0.0005 ). Moreover, the number of > or =4 mm follicles and the secretion of inhibin A remained lower in treated goats (3.9 +/- 0.3 follicles and 84.4 +/- 7.0 pg/ml versus 5.4 +/- 0.5 follicles, P < 0.05 and 128.9 +/- 14.2 pg/ml, P < 0.05 ). These results indicate that pituitary secretion of gonadotrophins is restored shortly after the end of antagonist treatment, but activity of ovarian follicles is affected.     

A simple technique for collecting blood of testicular origin: application to in vivo studies on testicular steroidogenesis in rats and Macaca fascicularis

A technique for rapidly collecting blood of testicular origin is described, one which can provide sufficient plasma amounts to investigate some steps of testicular steroid biogenesis in vivo in 2 species. In adult male rats, testosterone (T), androstenedione (4A) and 5-androstenediol (5AD) were determined in pampiniform plexus testicular venous blood (PPTV) and peripheral (PV) blood samples before and 2 h after human Chorionic Gonadotropin (hCG). PPTV concentration of 5AD was 0.83 +/- 0.1 ng/ml (mean +/- SEM) with a PPTV/PV ratio of 7.0 +/- 1.0, comparable to a PPTV/PV ratio for 4A of 5.8 +/- 1.8. After hCG, PPTV concentration of 5AD significantly increased to 1.28 +/- 0.15 ng/ml (P less than 0.05). Those data are in favor of a participation of 5-ene pathway to testicular biogenesis of T associated to a 4-ene pathway which is predominant. In adult male Macaca fascicularis, spermatic vein (SV) concentrations of 5AD and 4A were comparable (3.0 +/- 1.2 vs 4.3 +/- 1.0 ng/ml) as well as SV/PV ratios under basal conditions (3.5 +/- 0.9 vs 5.1 +/- 0.1), as well as 48 h after hCG, confirming in vivo that both 5-ene and 4-ene pathways are involved in testicular T biogenesis. Testicular production of estradiol (E2), estrone (E1) and their sulfates E2S and E1S showed a SV/PV ratio significantly higher than 1 (3.4 +/- 0.6; 2.4 +/- 0.1; 1.7 +/- 0.2 and 1.6 +/- 0.2, respectively).     

Failure of naloxone to stimulate luteinizing hormone secretion during pregnancy and steroid treatment of ovariectomized beef cows

The response of serum luteinizing hormone (LH) to naloxone, an opiate antagonist, and gonadotropin-releasing hormone (GnRH) was measured in cows in late pregnancy to assess opioid inhibition of LH. Blood samples were collected at 15-min intervals for 7 h. In a Latin Square arrangement, each cow (n = 6) received naloxone (0, 0.5, and 1.0 mg/kg BW, i.v.; 2 cows each) at Hour 2 on 3 consecutive days (9 +/- 2 days prepartum). GnRH (7 ng/kg body weight, i.v.) was administered at Hour 5 to all cows on each day. Mean serum LH concentrations (x +/- SE) before naloxone injection were similar (0.4 +/- 0.1 ng/ml), with no serum LH pulses observed during the experiment. Mean serum LH concentrations post-naloxone were similar (0.4 +/- 0.1 ng/ml) to concentrations pre-naloxone. Mean serum LH concentrations increased (p less than 0.05) following GnRH administration (7 ng/kg) and did not differ among cows receiving different dosages of naloxone (0 mg/kg, 1.44 +/- 0.20; 0.5 mg/kg, 1.0 +/- 0.1; 1.0 mg/kg, 0.9 +/- 0.1 ng/ml). In Experiment 2, LH response to naloxone and GnRH was measured in 12 ovariectomized cows on Day 19 of estrogen and progesterone treatment (5 micrograms/kg BW estrogen: 0.2 mg/kg BW progesterone) and on Days 7 and 14 after steroid treatment. On Day 19, naloxone failed to increase serum LH concentrations (Pre: 0.4 +/- 0.1; Post: 0.4 +/- 0.1 ng/ml) after 0, 0.5, or 1.0 mg/kg BW.(ABSTRACT TRUNCATED AT 250 WORDS)     

Total body water and ECFV measured using bioelectrical impedance analysis and indicator dilution in horses.

The purposes of this study were 1) to determine the compartmentation of body water in horses by using indicator dilution techniques and 2) to simultaneously measure bioelectrical impedance to current flow at impulse current frequencies of 5 and 200 kHz to formulate predictive equations that could be used to estimate total body water (TBW), extracellular fluid volume (ECFV), and intracellular fluid volume (ICFV). Eight horses and ponies weighing from 214 to 636 kg had catheters placed into the left and right jugular veins. Deuterium oxide, sodium thiocyanate, and Evans blue were infused for the measurement of TBW, ECFV, and plasma volume (PV), respectively. Bioelectrical impedance was measured by using a tetrapolar electrode configuration, with electrode pairs secured above the knee and hock. Measured TBW, ECFV, and PV were 0.677 +/- 0.022, 0.253 +/- 0.006, and 0.040 +/- 0.002 l/kg body mass, respectively. Strong linear correlations were determined among measured variables that allowed for the prediction of TBW, ECFV, ICFV, and PV from measures of horse length or height and impedance. It is concluded that bioelectrical impedance analysis (BIA) can be used to improve the predictive accuracy of noninvasive estimates of ECFV and PV in euhydrated horses at rest.     

Response of red cell and plasma volume to prolonged training in humans

To clarify the role of progressive heavy training on vascular volumes and hematologic status, seven untrained males [maximal O2 uptake (VO2max) = 45.1 +/- 1.1 (SE) ml.kg-1.min-1] cycled 2 h/day at an estimated 62% of VO2max. Training was conducted five to six times per week for approximately 8 wk. During this time, VO2max increased (P less than 0.05) by 17.2%. Plasma volume (PV) measured by 125I increased (P less than 0.05) from 3,068 +/- 104 ml at 0 wk to 3,490 +/- 126 ml at 4 wk and then plateaued during the remaining four wk (3,362 +/- 113 ml). Red cell (RBC) mass (RCM) measured by 51Cr-labeled RBC did not change during the initial 4 wk of training (2,247 +/- 66 vs. 2,309 +/- 128 ml). As well, no apparent change occurred in RCM during the final 4 wk of training when RCM was estimated using PV and hematocrit (Hct). Collectively, PV plus RCM, expressed as total blood volume (TBV), increased (P less than 0.05) by 10% at 4 wk and then stabilized for the final 4 wk. During the initial phase of training, reductions (P less than 0.05) were also noted in Hct (4.6%), hemoglobin (Hb, 4.0%), and RBC count (6.3%). In contrast, an increase in mean cell volume (MCV, 1.7%) and mean cell Hb (2.3%) was observed (P less than 0.05). From 4 to 8 wk, no further changes (P greater than 0.05) in Hb, RBC, and MCV were found, whereas both mean cell Hb and Hct returned to pretraining levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian steroid production in rats treated with gonadotropin-releasing hormone during early pregnancy

In the pregnant rat, luteinizing hormone (LH) stimulates the ovarian production of testosterone (T) which is aromatized to estradiol (E2). E2 promotes progesterone (P) synthesis by the ovary. To determine if the administration of gonadotropin-releasing hormone (GnRH) disrupts pregnancy by suppressing ovarian steroid production, rats were treated on days 7-12 of pregnancy with 25, 50 or 100 micrograms/day of GnRH or 0.2, 1 or 5 micrograms/day of a GnRH agonist (GnRH-Ag). The higher two doses of GnRH or GnRH-Ag within 24 h suppressed peripheral levels of plasma P and terminated pregnancy within 48 h. By day 12, P levels in the ovarian vein in rats treated with GnRH or GnRH-Ag in respective doses were 2098 +/- 261, 732 +/- 437, 110 +/- 15, and 2575 +/- 463, 49 +/- 9, 43 +/- 8 compared to 1833 +/- 433 ng/ml in controls. Daily treatment of P (4 mg) and E2 (0.5 microgram) simultaneously with GnRH-Ag at its maximum dose reversed the abortifacient effect of GnRH-Ag and maintained pregnancy. Peripheral levels of Plasma LH in all groups were higher than controls on days 10 and 12. Ovarian vein levels of T on days 10 or 12 of pregnancy were either not significantly different from controls (at 2703 +/- 607 or 3249 +/- 690 pg/ml, respectively) or increased dramatically to 9547 +/- 1769 on day 10 and to 5985 +/- 1426 pg/ml on day 12 in rats treated with 0.2 microgram of GnRH-Ag. Similarly, ovarian vein levels of E2 on days 10 or 12 were either not significantly different from controls (at 2022 +/- 227 or 2793 +/- 184 pg/ml, respectively) or increased dramatically to 2980 +/- 58 pg/ml on day 10 in rats treated with 25 micrograms of GnRH or to 3296 +/- 241 on day 10 and to 3420 +/- 325 pg/ml on day 12 in rats treated with 0.2 microgram of GnRH-Ag. These results indicate that the abortifacient effect of GnRH administration in rats is not due to its effect on the uterus, but to its suppressive effects on ovarian P secretion. There was no evidence to show that a GnRH-induced fall in ovarian secretion of either T or E2 were involved in this process.     

Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone

The effect of treatment with a GnRH agonist, hCG or progesterone (P(4)) on corpus luteum function and embryonic mortality was investigated in buffaloes inseminated during mid-winter. Italian Mediterranean buffaloes (n=309) were synchronized using the Ovsynch with timed-AI program and mated by AI at 16 h (Day 0) and 40 h after the second injection of GnRH. On Day 5, buffaloes were randomly assigned to four groups: Control (no treatment, n=69), GnRH agonist (buserelin acetate, 12.6 microg, n=73), hCG (1500 IU, n=75) and P(4) (PRID without E(2) for 10 days, n=77). Progesterone (pg/ml) was determined in milk whey on Days 5, 10, 15 and 20 and pregnancy diagnosis was undertaken on Day 26 by ultrasound and Day 40 by rectal palpation. Treatment with buserelin and hCG increased (p<0.05) P(4) on Day 15 compared with controls (456+/-27, 451+/-24 and 346+/-28 pg/ml, respectively). Buffaloes treated with a PRID had intermediate P(4) concentrations (380+/-23 pg/ml). Embryonic mortality between Days 26 and 40 (22.9%) and pregnancies at Day 40 (48.9%) did not differ between treatments. A higher (p<0.01) P(4) concentration was found on Day 20 in pregnant animals compared with non-pregnant and embryonic mortality buffaloes, which did not differ. In summary, buserelin and hCG increased P(4) concentrations on Day 15 but this was not associated with a reduced incidence of embryonic mortality in buffaloes during mid-winter.     

Enalapril attenuates endothelin-1-induced hypertension via increased kinin survival

Recent studies have shown that angiotensin-converting enzyme (ACE) inhibitors attenuate endothelin-1 (ET-1)-induced hypertension, but the mechanisms for this effect have not been clarified. Initial experiments were conducted to contrast the effect of the ACE inhibitor enalapril, the combined ACE-neutral endopeptidase inhibitor omapatrilat, and the angiotensin II receptor antagonist candesartan on the hypertensive and renal response to ET-1 in anesthetized Sprague-Dawley rats. Acute intravenous infusion of ET-1 (10 pmol x kg(-1) x min(-1)) for 60 min significantly increased mean arterial pressure (MAP) from 125 +/- 8 to 145 +/- 8 mmHg (P < 0.05) and significantly decreased glomerular filtration rate (GFR) from 0.31 +/- 0.09 to 0.13 +/- 0.05 ml x min(-1) x 100 g kidney wt(-1). Pretreatment with enalapril (10 mg/kg iv) before ET-1 infusion inhibited the increase in MAP (121 +/- 4 vs. 126 +/- 4 mmHg) before and during ET-1 infusion, respectively (P < 0.05) without blocking the effect of ET-1 on GFR. In contrast, neither omapatrilat (30 mg/kg) nor candesartan (10 mg/kg) had any effect on ET-1-induced increases in MAP or decreases in GFR. To determine whether the effect of enalapril was due to the decrease in angiotensin II or increase in kinin formation, rats were given REF-000359 (1 mg/kg iv), a selective B(2) receptor antagonist, with or without enalapril before ET-1 infusion. REF-000359 completely blocked the effect of enalapril on ET-1 infusion (MAP was 117 +/- 5 vs. 135 +/- 5 mmHg before and during ET-1 infusion, respectively, P < 0.05). REF-000359 alone had no effect on the response to ET-1 infusion (MAP was 117 +/- 4 vs. 144 +/- 4 mmHg before and during ET-1 infusion, respectively, P < 0.05). REF-000359 with or without enalapril had no significant effect on the ability of ET-1 infusion to decrease GFR. These findings support the hypothesis that decreased catabolism of bradykinin and its subsequent vasodilator activity oppose the actions of ET-1 to increase MAP.     

Prevention of endothelin-1-induced increases in blood pressure: role of endogenous CGRP

To determine the role of endothelin-1 (ET-1) and its receptors in the regulation of calcitonin gene-related peptide (CGRP) release, male Wistar rats were divided into six groups and subjected to the following treatments for 1 wk with or without ABT-627 (an ET(A) receptor antagonist, 5 mg.kg(-1).day(-1) in drinking water) or A-192621 (an ET(B)-receptor antagonist, 30 mg.kg(-1).day(-1) by oral gavage): control (Con), ET-1 (5 ng.kg(-1).min(-1) iv), Con + ABT-627, Con + A-192621, ET-1 + ABT-627, and ET-1 + A-192621. Baseline mean arterial pressure (MAP, mmHg) was higher (P < 0.05) in Con + A-192621 (122 +/- 4) and ET-1 + A-192621 (119 +/- 4) groups compared with Con (104 +/- 6), ET1 (106 +/- 3), Con + ABT-627 (104 +/- 3), and ET1 + ABT-627 (100 +/- 3) groups. Intravenous administration of CGRP(8-37) (a CGRP receptor antagonist, 1 mg/kg) increased MAP (P < 0.05) in ET-1 (13 +/- 1), Con + A-192621 (12 +/- 1), and ET-1 + A-192621 (15 +/- 3) groups compared with Con (4 +/- 1), Con-ABT-627 (4 +/- 1), and ET-1 + ABT-627 (5 +/- 1) groups. Plasma CGRP levels (in pg/ml) were increased (P < 0.05) in ET-1 (57.5 +/- 6.1), Con + A-192621 (53.9 +/- 3.4), and ET-1 + A-192621 (60.4 +/- 3.0) groups compared with Con (40.4 +/- 1.6), Con + ABT-627 (40.0 +/- 2.9), and ET-1 + ABT-627 (42.6 +/- 1.9) groups. Plasma ET-1 levels (in pg/ml) were higher (P < 0.05) in ET-1 (2.8 +/- 0.2), ET-1 + ABT-627 (3.2 +/- 0.4), Con + A-192621 (3.3 +/- 0.4), and ET-1 + A-192621 (4.6 +/- 0.3) groups compared with Con (1.1 +/- 0.2) and Con-ABT-627 (1.3 +/- 0.2) groups. Therefore, our data show that ET-1 infusion leads to increased CGRP release via activation of the ET(A) receptor, which plays a compensatory role in preventing ET-1-induced elevation in blood pressure.     

Exercise stroke volume relative to plasma-volume expansion

The effects of plasma-volume (PV) expansion on stroke volume (SV) (CO2 rebreathing) during submaximal exercise were determined. Intravenous infusion of 403 +/- 21 ml of a 6% dextran solution before exercise in the upright position increased SV 11% (i.e., 130 +/- 6 to 144 +/- 5 ml; P less than 0.05) in untrained males (n = 7). Further PV expansion (i.e., 706 +/- 43 ml) did not result in a further increase in SV (i.e., 145 +/- 4 ml). SV was somewhat higher during supine compared with upright exercise when blood volume (BV) was normal (i.e., 138 +/- 8 vs. 130 +/- 6 ml; P = 0.08). PV expansion also increased SV during exercise in the supine position (i.e., 138 +/- 8 to 150 +/- 8 ml; P less than 0.05). In contrast to these observations in untrained men, PV expansion of endurance-trained men (n = 10), who were naturally PV expanded, did not increase SV during exercise in the upright or supine positions. When BV in the untrained men was increased to match that of the endurance-trained subjects, SV was observed to be 15% higher (165 +/- 7 vs. 144 +/- 5 ml; P less than 0.05), whereas mean blood pressure and total peripheral resistance were significantly lower (P less than 0.05) in the trained compared with untrained subjects during upright exercise at a similar heart rate. The present findings indicate that exercise SV in untrained men is preload dependent and that increases in exercise SV occur in response to the first 400 ml of PV expansion. It appears that approximately one-half of the difference in SV normally observed between untrained and highly endurance-trained men during upright exercise is due to a suboptimal BV in the untrained men.     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Plasma volume expansion in humans after a single intense exercise protocol.

We used intense intermittent exercise to produce a 10% expansion of plasma volume (PV) within 24 h and tested the hypothesis that PV expansion is associated with an increase in plasma albumin content. The protocol consisted of eight 4-min bouts of exercise at 85% maximal O2 uptake with 5-min recovery periods between bouts. PV, plasma concentrations of albumin and total protein (TP), and plasma osmolality were measured before and during exercise and at 1, 2, and 24 h of recovery from exercise. During exercise, PV decreased by 15%, while plasma TP and albumin content remained at control levels. At 1 h of recovery, plasma albumin content was elevated by 0.17 +/- 0.04 g/kg body wt, accounting for the entire increase in plasma TP content. PV returned to control level at 1 h of recovery without fluid intake by the subjects, despite a 820 +/- 120-g reduction in body weight. At 2 h of recovery, plasma TP content remained significantly elevated, and plasma TP and albumin concentration were significantly elevated. At 24 h of recovery, PV was expanded by 4.5 +/- 0.7 ml/kg body wt (10 +/- 1%), estimated from hematocrit and hemoglobin changes, and by 3.8 +/- 1.3 ml/kg body wt (8 +/- 3%), measured by Evans blue dye dilution. Plasma albumin content was increased by 0.19 +/- 0.05 g/kg body wt at 24 h of recovery. If 1 g of albumin holds 18 ml of water, this increase in plasma albumin content can account for a 3.4-ml/kg body wt expansion of the PV. No significant changes in plasma osmolality occurred during recovery, but total plasma osmotic content increased in proportion to PV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal growth and reproductive performance in ewes administered GnRH agonist on day 12 post-mating

Reproductive performance and fetal growth was determined in GnRH (4 microg synthetic GnRH agonist, Receptal) administered (i.m.) to ewes on day 12 post-mating (n = 103) compared to control ewes (n = 97) during the breeding season. Plasma progesterone and LH concentrations were analyzed. A total of 13 ewes was slaughtered on day 45 of pregnancy (six from control, seven from GnRH treated groups). GnRH administration on day 12 post-mating increased plasma progesterone concentration (4.39+/-0.25 ng/ml) compared to control group (3.43+/-0.15 ng/ml) on days 13-15 post-mating (P < 0.01). GnRH administration also increased plasma LH concentration between 1 and 4 h after GnRH administration (P < 0.01). Pregnancy rate was higher in GnRH treated group (84%) than control (66%) group (P < 0.05). The ewes in GnRH administered group had more twins (P < 0.05) than those in control group. The ovarian weights (P < 0.05) and the number of corpora lutea (CL) (P < 0.01) were greater in ewes slaughtered on day 45 of pregnancy in GnRH treated group than those in control group. GnRH administration on day 12 post-mating did not have any effect on products of conception at day 45 of pregnancy except on crown-rump length (CRL) of fetuses and cotyledon weight. CRL of fetuses and cotyledon weight in GnRH treated group was higher than those in control group (P < 0.05). In conclusion GnRH administration improved reproductive performance of ewes when administered on day 12 post-mating probably through its beneficial effect on embryo survival by enhancing luteal function, but not through stimulating fetal growth.     

Influence of exogenous GnRH on sexual behavior and frozen/thawed semen viability in stallions during the non-breeding season

Twelve fertile stallions were divided into two groups, either receiving gonadotropin-releasing hormone (GnRH) (n = 6) or Placebo (n = 6). Based on the history of frozen/thawed semen characteristics three stallions within each group were assigned as being "good freezers" [GnRH (+); Placebo (+)] and three stallions were assigned as being "poor freezers" [GnRH (-); Placebo (-)]. The study was performed as a "blinded" investigation and stallions were treated twice daily by an intramuscular injection of 1 ml GnRH (Buserelin), 50 microg) or Placebo. The experiment was divided into three time periods. Period A (pre-treatment) was performed between 16 November and 20 December; Period B (treatment) was performed during 6 weeks between 21 December and 31 January; and Period C (post-treatment) was performed between 1 February and 12 February. Semen was collected every Monday, Wednesday, Friday, and analysed for motion characteristics by the use of a computerized semen analyser, and sperm morphology immediately after collection. The spermatozoa were cryopreserved, stored in liquid nitrogen, and evaluated for motility (computer assisted semen analysis), membrane integrity (carboxyfluoresceine diacetate (CFDA) combined with propidium-iodide (PI), CFDA/PI), viability and sperm morphology (Eosine-Nigrosine, EN), and osmotic reactivity (hypo-osmotic swelling test, HOS) following thawing in a water bath. The viability of spermatozoa was expressed as the difference between pre-freeze and post-thaw values. A libido score of 1-4, the number of mounts on the phantom before ejaculation, and ejaculation latency were used to evaluate the stallions sexual behavior. Effect of treatment was analysed by comparing time intervals within groups as well as comparing groups within time intervals using SAS statistics software. GnRH treatment decreased the number of mounts before ejaculation (GnRH (total): 2.5 +/- 1.14 versus 1.8 +/- 1.06, P < 0.05), and shortened ejaculation latency. Cessation of treatment increased ejaculation latency in the GnRH group (4.7 +/- 4.98 min versus 7.2+/-7.88min, P<0.05). With the exception of libido score all parameters of sexual behavior were superior in the GnRH (+) group compared to the Placebo (-) group during the treatment period (P < 0.05). GnRH administration increased progressive motility (GnRH (+): 30.7 +/- 10.74% versus 38.4 +/- 15.1%, P < 0.05; GnRH (total): 24.9 +/- 11.80% versus 31.9 +/- 14.68%, P < 0.05), membrane intact spermatozoa CFDA/PI (GnRH (-): 16.8 +/- 7.17% versus 26.2 +/- 7.02%, P < 0.05; GnRH (total): 23.1 +/- 12.33% versus 29.5 +/- 10.77%, P < 0.05) and HOS positive spermatozoa (GnRH (+): 33.2 +/- 11.29% versus 42.2 +/- 10.36%, P < 0.05; GnRH (total): 32.9 +/- 10.23% versus 40.1 +/- 10.30%, P < 0.05) of frozen/thawed spermatozoa. Following cessation of treatment, the viability of frozen/thawed spermatozoa decreased. GnRH treated stallions had lower losses of live stained spermatozoa (EN) compared to the Placebo group (GnRH (total): 17.6 +/- 4.77 versus Placebo (total): 27.2 +/- 5.44, P < 0.05). This was particularly observed in the "poor freezer" group (GnRH (-): 16.6 +/- 4.35 versus Placebo (-): 31.3 +/- 5.87; P < 0.05). In conclusion, exogenous GnRH was shown to improve sexual behavior and increase the quality of frozen/thawed spermatozoa in fertile stallions during the non-breeding season. Nevertheless, it seems that, although significance was achieved relative to improvement to post-thaw sperm quality, that the "real" change in sperm quality seems negligible in fertile stallions. The mechanism of GnRH effect was not determined but this study may support the possibility of a direct gonadal or epididymal effect of exogenous GnRH in the stallion.     

Naloxone evokes large-amplitude GnRH pulses in luteal-phase ewes

Ewes were sampled during the mid-late luteal phase of the oestrous cycle. Hypophysial portal and jugular venous blood samples were collected at 5-10 min intervals for a minimum of 3 h, before i.v. infusions of saline (12 ml/h; N = 6) or naloxone (40 mg/h; N = 6) for 2 h. During the 2-h saline infusion 2/6 sheep exhibited a GnRH/LH pulse; 3/6 saline infused ewes did not show a pulse during the 6-8-h portal blood sampling period. In contrast, large amplitude GnRH/LH pulses were observed during naloxone treatment in 5/6 ewes. The mean (+/- s.e.m.) amplitude of the LH secretory episodes during the naloxone infusion (1.07 +/- 0.11 ng/ml) was significantly (P less than 0.05) greater than that before the infusion in the same sheep (0.54 +/- 0.15 ng/ml). Naloxone significantly (P less than 0.005) increased the mean GnRH pulse amplitude in the 5/6 responding ewes from a pre-infusion value of 0.99 +/- 0.22 pg/min to 4.39 +/- 1.10 pg/min during infusion. This episodic GnRH secretory rate during naloxone treatment was also significantly (P less than 0.05) greater than in the saline-infused sheep (1.53 +/- 0.28 pg/min). Plasma FSH and prolactin concentrations did not change in response to the opiate antagonist. Perturbation of the endogenous opioid peptide system in the ewe by naloxone therefore increases the secretion of hypothalamic GnRH into the hypophysial portal vasculature. The response is characterized by a large-amplitude GnRH pulse which, in turn, causes a large-amplitude pulse of LH to be released by the pituitary gland.