Effects of oestrus induction with progestagens or prostaglandin analogues on ovarian and pituitary function in sheep.

The aim of the current study was to determine possible differences in ovarian and pituitary features explaining lower fertility rates in sheep with oestrus induced with intravaginal progestagens or prostaglandin analogues (group FGA and PGF, n=8 in both) when compared to a control group (group C, n=8). The growth profiles and the mean individual sizes of preovulatory follicles were similar between groups; however, the number of preovulatory follicles per ewe and, consequently, the number of ovulations were higher in groups FGA and PGF (2.3±0.3 and 2.0±0.1, respectively) than in group C (1.4±0.1, P<0.05). However, plasma oestradiol concentrations were similar between groups suggesting a defective function in some preovulatory follicles of groups FGA and PGF. In group FGA, the basal LH levels during the follicular phase were lower (0.21±0.0 ng/mL, P<0.005) than in groups C (0.41±0.1 ng/mL) and PGF (0.55±0.1 ng/mL); the onset of preovulatory discharge being later (21.0±2.3h vs. 12.8±1.5 in C and 14.5±1.5 in PGF; P<0.05 for both). Finally, luteal activity was also found to be affected in group FGA; the rate of progesterone secretion per total luteal tissue was lower (range: 0.46-0.65 ng/mL/cm(2)) than in ewes treated with cloprostenol (2.1-3.3 ng/mL/cm(2)) and control sheep (2.0-3.4 ng/mL/cm(2)).

Cardiological aging in SAM model: effect of chronic treatment with growth hormone.

The purpose of this study was to investigate the effect of aging on different parameters related to inflammation, oxidative stress and apoptosis in hearts from two types of male mice models: senescence-accelerated mice (SAM-P8) and senescence-accelerated-resistant (SAM-R1), and the influence of chronic administration of Growth Hormone (GH) on old SAM-P8 mice. Forty male mice were used. Animals were divided into five experimental groups: two 10 month old untreated groups (SAM-P8/SAM-R1), two 2 month old young groups (SAM-P8/SAM-R1) and one 10 month old group (SAM-P8) treated with GH for 30 days. The expression of tumor necrosis factor-alpha, interleukin 1, interleukin 10, heme oxygenases 1 and 2, endothelial and inducible nitric oxide synthases, NFkB, Bad, Bax and Bcl-2 were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Results were submitted to a two way ANOVA statistical evaluation using the Statgraphics program. Inflammation, as well as, oxidative stress and apoptosis markers were increased in the heart of old SAM-P8 males, as compared to young controls and this situation was not observed in the old SAM-R1 mice. Exogenous GH administration reverted the effect of aging in the described parameters of old SAM-P8 mice. Our results suggest that inflammation, apoptosis and oxidative stress could play an important role in the observed cardiovascular alterations related to aging of SAM-P8 mice and that GH may play a potential protective effect on the cardiovascular system of these animals.

Hormonal regulation of pro-inflammatory and lipid peroxidation processes in liver of old ovariectomized female rats.

There is now a large body of evidence suggesting that the decline in ovarian function with menopause is associated with spontaneous increases in pro-inflammatory cytokines. On the other hand, oxidative stress has been implicated in the pathogenesis of several alterations due to menopause, and can arise through the increased production of lipid peroxides (LPO) and/or a deficiency of antioxidant defense. The aim of the present study was to investigate the effect of aging and ovariectomy on various physiological parameters related to inflammation and oxidative stress in livers obtained from old female rats and the influence of chronic exogenous administration of estrogens, phytoestrogens and growth hormone on these. Thirty-six female Wistar rats of 22 months of age were used in the present study. Twelve of them remained intact, and the other 24 had been ovariectomized at 12 months of age. Intact animals were divided into two groups and treated for 10 weeks with GH or saline, and ovariectomized animals were divided into four groups and treated for the same time with GH, estrogens, phytoestrogens or saline. A group of 2 month old intact female rats was used as young control. Protein expression of iNOS, HO-1, IL-6, TNFalpha, and IL-1beta were determined by Western blot analysis. The levels of NO( x ), LPO, TNFalpha, IL-1beta, IL-6 and IL-10 were determined in different fractions of the liver. Levels of LPO in the liver homogenates as well as iNOS protein expression and NO( x ) levels were increased in old rats as compared to young animals; this effect was more evident in ovariectomized animals. Pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 were significantly increased and anti-inflammatory IL-10 decreased during ageing and after ovariectomy. Aging also significantly increased expression of HO-1 protein and ovariectomized rats showed an additional increase. Hormonal administration to the ovariectomized groups decreased NO( x ), LPO levels and pro-inflammatory cytokines as compared with untreated rats. Significant rise in IL-10 and reductions in the iNOS, IL-6, TNFalpha and IL-1beta proteins expression were also found. Oxidative stress and inflammation induced during aging in the liver are more marked in castrated than in intact old females. Administration of the different hormonal replacement therapies was able to inhibit the induction of pro-inflammatory cytokines and iNOS, decreased the levels of oxidative stress markers and had therapeutic potential in the prevention of liver injury.

Improvement of leucocyte functions in ovariectomised aged rats after treatment with growth hormone, melatonin, oestrogens or phyto-oestrogens.

Ageing is accompanied by an impairment of the physiological activity of the nervous, endocrine and immune system, as well as in neuroendocrine-immune communication. However, age-related changes in this communication axis have been scarcely studied. In mammals, the process of ageing is associated with an important decline in the secretion of several hormones, such as growth hormone (GH), melatonin (MEL) and oestrogens (Os). Ovariectomy, a model of menopause in rats, has been found to lead to premature immunosenescence. In the present study, the effect of ovariectomy and the role of replacement therapies with GH, MEL, O and natural phyto-oestrogens (POs) have been assessed on several functions in leucocytes from the spleen and the axillary nodes of intact and ovariectomised rats. Chemotaxis, lymphoproliferative response to the mitogen concanavalin A (Con A), the release of interleukin-2 (IL-2) and the natural killer (NK) cell activity have been investigated. Age-controlled rats were used to compare immune functions in hormone treated aged rats with those in younger untreated animals. In all experimental groups, the immune impairment caused by ageing and ovariectomy was partially or completely reversed by hormone treatments. Since the immune system is a marker of health and a predictor of longevity, the results suggest that treatment with hormones could slow down the effects of the ageing process.

Ovarian follicular dynamics and plasma steroid concentrations are not significantly different in ewes given intravaginal sponges containing either 20 or 40 mg of fluorogestone acetate.

Although various progestagens are often used to induce and synchronize estrus and ovulation in ruminants, concerns regarding residues are the impetus to develop alternative approaches, including reduced doses of progestagens. Therefore, the objective was to determine whether ovarian function was affected by halving the dose of fluorogestone acetate in intravaginal sponges for synchronizing ovulation in sheep during the physiologic breeding season. Twenty Manchega ewes, 4-6-year-old, were randomly allocated to receive an intravaginal sponge containing either 20mg (P20, n=10) or 40 mg of fluorogestone acetate (P40, n=10). Cloprostenol (125 microg) was given at sponge insertion, and all sponges were removed after 6d. Ovarian follicular dynamics (monitored by daily ultrasonography) and other aspects of ovarian function did not differ significantly between the two groups. Ovulatory follicles (OF) grew at a similar growth rate (r=0.62; P<0.001), with comparable initial and maximum diameters (4.2+/-0.4 to 6.0+/-0.3mm in P20 vs. 4.6+/-0.6 to 5.7+/-0.2 mm in P40, mean+/-S.E.M.). Plasma estradiol concentrations (determined once daily) increased linearly during the 72 h interval after sponge removal (1.3+/-0.1 to 3.3+/-0.1 pg/mL for P20, P<0.005 and 1.4+/-0.1 to 3.1+/-0.2 pg/mL for P40, P<0.005). Ten days after sponge removal, ovulation rates (1.2+/-0.2 for P20 and 1.4+/-0.3 for P40), and plasma progesterone concentrations (3.8+/-0.35 ng/mL for P20 and 3.9+/-0.38 ng/mL for P40) were similar. In conclusion, reducing the dose of fluorogestone acetate from 40 to 20mg did not affect significantly ovarian follicular dynamics or other aspects of ovarian function.

Melatonin is able to prevent the liver of old castrated female rats from oxidative and pro-inflammatory damage.

The aim of this study was to investigate the effect of aging and ovariectomy on various physiological parameters related to inflammation and oxidative stress in livers obtained from old female rats, and the influence of chronic administration of melatonin on these animals. Twenty-four female Wistar rats of 22 months of age were used. Animals were divided into four experimental groups: two intact groups that were untreated or given melatonin (1 mg/kg/day), and two ovariectomized groups that also untreated and treated with melatonin (1 mg/kg/day). After 10 wk of treatment, rats were sacrificed by decapitation, and livers were collected and homogenized. A group of 2-month-old female rats was used as young controls. Protein expression of inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), IL-6, TNF-alpha and IL-1beta were determined by Western blot analysis. The levels of nitric oxide metabolites (NO(x)), lipid hydroperoxide (LPO), TNF-alpha, IL-1beta, IL-6 and IL-10 were determined. Levels of LPO in the liver homogenates as well as iNOS protein expression and NO(x) levels were increased in old rats as compared with young animals; this effect was more evident in ovariectomized animals. Pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 were significantly increased and anti-inflammatory IL-10 decreased during aging and after ovariectomy. Aging also significantly increased the expression of HO-1 protein, and ovariectomized rats showed an additional increase. Administration of melatonin, both to intact and to the ovariectomized animals significantly reduced NO(x), LPO levels and pro-inflammatory cytokines in the liver as compared with untreated rats. Significant rice in IL-10 and reductions in the iNOS, HO-1, IL-6, TNF-alpha and IL-1beta protein expression were also found in rats treated with melatonin. Oxidative stress and inflammation induced during aging in the liver are more marked in castrated than in intact females. Administration of melatonin reduces both these situations.

Preovulatory follicle development in goats following oestrous synchronization with progestagens or prostaglandins.

The study reports on differences in the dynamics of growth and functionality of preovulatory follicles in response to oestrous synchronization, either by the administration of two doses of prostaglandin or by an intravaginal progestagen sponge, in goats. The progestagen-treated group (n = 8) showed more follicles of preovulatory size (> or =5.5 mm) than the cloprostenol group (n = 8) during the follicular phase (4.5 +/- 0.6 vs 1.9 +/- 0.2, p < 0.01). The diameters of the largest follicles (LF1, LF2 and LF3) were also larger in the progestagen group (LF1, 7.8 +/- 0.3 vs 7.0 +/- 0.2 mm, p < 0.05; LF2, 6.7 +/- 0.2 vs 5.6 +/- 0.2 mm, p < 0.01; LF3, 5.5 +/- 0.3 vs 4.2 +/- 0.2 mm, p < 0.01). The study of the preovulatory follicles showed that 27.2% (3/11) of the follicles were in the static phase in the cloprostenol group, whilst 71.4% (10/14) were static in progestagen group (p < 0.05). Higher plasma oestradiol levels were recorded in the progestagen-treated goats during the 48 h prior to cloprostenol injection or progestagen withdrawal (4.2 +/- 0.4 vs 3.0 +/- 0.2 pg/ml, p < 0.05). In conclusion, goats with oestrus synchronized by progestagen showed a higher number of preovulatory-sized follicles, but a decreased oestradiol secretion when compared with does with oestrus synchronized by using prostaglandin analogues. These would support the development of alternative protocols for assisted reproduction.

Effect of chronic treatments with GH, melatonin, estrogens, and phytoestrogens on oxidative stress parameters in liver from aged female rats.

The aging theory postulates that this process may be due to the accumulation of oxidative damage in cells and molecules. The present study has investigated the effect of castration in old female rats on various parameters related to the antioxidant properties of several cellular fractions obtained from the liver, and the influence of several chronic treatments on it, both in intact and castrated animals. Sixty-one 22-month-old Wistar female rats, were used. About 21 intact animals were divided into three groups and treated for 10 weeks with GH, melatonin or saline, and 40 ovariectomized (at 12 months of age) animals were divided into five groups and treated for the same time with GH, melatonin, estrogens (Eos), phytoestrogens (Phyt) or saline. All animals were sacrificed at 24 months of age by decapitation. The activity of glutathione peroxidase (GPx) in cytosolic fraction, glutathione-S-transferase (GST) in cytosol and microsomal fractions, and the levels of nitric oxide (NO) and cytochrome C in mitochondrial and cytosol fractions of liver were determined. A decrease in GST activity was detected in cytosol and in the microsomal fraction in ovariectomized animals as compared to intact rats. The activity of GPx was also decreased in ovariectomized as compared with the intact group. NO level was increased and cytochrome C decreased in the mitochondrial fraction of the liver in ovariectomized females as compared with the intact group, respectively. No significant changes after melatonin or GH treatments were found in GPx, GST activity and NO level in mitochondrial fraction in the intact group. Administration of GH, melatonin, Eos and Phyt in the ovariectomized groups significantly increased the GPx, and GST activity in the cytosol and microsomal fraction and decreased the level of NO in the mitochondrial fraction as compared with the untreated rats. A significant increase in the level of cytochrome C in the mitochondrial fraction and a decrease in the cytosol fraction were also found with all treatments. The administration of GH, melatonin, Eos and Phyt to castrated females seem to reduce oxidative changes in the liver from old ovariectomized rats.

Effect of exogenous administration of melatonin and growth hormone on pro-antioxidant functions of the liver in aging male rats.

Aging is accompanied by changes in the morphology and physiology of organs and tissues, such as the liver. This process might be due to the accumulation of oxidative damage induced by reactive oxygen (ROS) and reactive nitrogen species (RNS). Hepatocytes are very rich in mitochondria and have a high respiratory rate, so they are exposed to large amounts of ROS and permanent oxidative stress. Twenty-four male Wistar rats of 22 months of age were divided into three groups. One group remained untreated and acted as the control group. The second was treated with growth hormone (GH) (2 mg/kg/d sc) and the third was submitted to treatment wit 1 mg/kg/d of melatonin in the drinking water. A group of 2-months-old male rats was used as young controls. After 10 wk of treatment the rats were killed by decapitation, and the liver was dissected and homogenized. Mitochondrial, cytosolic and microsomal fractions were obtained and cytochrome C, glutathione peroxidase, s-transferase and nitric oxide (NO) were measured. Aging induced a significant increase in mitochondrial nitric oxide. An increase in cytochrome C in the cytosolic fraction and a reduction in the mitochondrial fraction with age was also observed. Both GH and melatonin treatments significantly reduced the enhanced measures and increased the reduced values. A reduction in glutathione peroxidase and glutathione S-transferase was found in old control rats when compared with the group of young animals. Treatment for 2.5 months of old rats with GH and melatonin were able to increase the enzymes reaching values similar to those found in young animals. In conclusion, GH and melatonin treatment seems to have beneficial effects against age-induced damage in the liver.

Effects of breed on follicular dynamics and oestradiol secretion during the follicular phase in sheep.

Current study evaluates effects from breed and management background on follicular dynamics and endocrine output during the follicular phase of sheep oestrous cycle. Follicular phases were synchronized with three cloprostenol doses, 10 days apart, in three groups of 10 females of different non-prolific Spanish breeds (Manchega, Rubia del Molar and Negra de Colmenar). Development of all follicles reaching antral diameters >or=2 mm was assessed by daily transrectal ultrasonographies, whereas follicular function was evaluated by measurement of plasma oestradiol concentrations. All the ovulatory follicles were present at induced luteolysis in Manchega sheep, while a 93.7% were detected in Rubia del Molar and Negra de Colmenar ewes. The mean size of these ovulatory follicles was similar between breeds at 0 h, but their growth rates were higher in Manchega ewes, reaching a larger size at oestrous detection than in Negra de Colmenar and Rubia del Molar sheep (p < 0.05). Conversely, the oestradiol levels increased with time in Rubia del Molar and Negra de Colmenar (p < 0.05); whilst remained stable in Manchega females. However, the patterns of follicular turnover were similar between breeds. These results indicate that, though differences in follicular size and size distribution, patterns of follicular turnover in sheep are affected neither by the breed nor by the background of management and selection.

Effects of progestagens on follicular growth and oocyte developmental competence in FSH-treated ewes.

Previous research has reported evidence for negative effects of progestagens on follicular growth and oocyte competence. In the present study, negative effects of progestagens on follicular growth and oocyte developmental competence were assessed. During the breeding season, 20 Sarda ewes were treated with two doses of cloprostenol, 10 days apart, to assure the presence of a corpus luteum (CL). On day 5 after the second cloprostenol dose, 10 ewes were treated with a progestagen sponge while 10 females remained untreated. Starting on day 7 after the second cloprostenol dose, all the ewes were treated with 6 equal doses of 24 I.U. of FSH (Ovagen, ICP, NZ), every 12h. The number of follicles > or =2mm in diameter increased (P<0.0005) in all the ewes from 24 h before to 60 h after the first FSH dose (from 12.8+/-1.1 to 23.4+/-1.3 in treated and from 12+/-0.6 to 22+/-1.2 in untreated ewes, n.s.). There were no significant differences in follicle dynamics between groups, but concentrations of estradiol in control ewes were higher than in the progestagen group (P<0.05). Twelve hours after the last FSH dose, oocytes were collected by ovum pick-up. Recovery rates were lower for progestagen-treated ewes (71.1 versus 83%; P<0.001). After IVP procedure, cleavage rate was also lower in the progestagen group (39.1 versus 82.6%; P<0.001). Furthermore, blastocysts output revealed that oocyte developmental competence was lower in progestagen group (17.3 versus 30.4%; P=0.245), although differences were not significant. These results suggest deleterious effects from progestagen on oocyte developmental competence and set the basis for new protocols for in vitro embryo production.

Effect of isoflavone administration on age-related hepatocyte changes in old ovariectomized femal Wistar rats.

Aging seems to be due to the accumulation of oxidative damage in cells and molecules. On the other hand, menopause and ovariectomy induce deleterious effects on different organs and systems that have been shown to be counteracted by estrogens and in a not so evident form also with phytoestrogens. The present study has investigated whether the administration of a commercial soy extract that contains approximately 10% isoflavones was able to modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female Wistar rats. Eighteen 22-month-old animals that had been previously ovariectomized at 12 months of age were divided into four groups: ovariectomized control rats, estradiol-treated ovariectomized females and ovariectomized rats treated with isoflavones. Six intact female rats of 2 months of age were used as reference group. Hepatocytes were isolated and cultured, and carbon monoxide (CO) and nitric oxide (NO) release, as well as adenosyl triphosphate (ATP), cyclic guanosyl monophosphate (cGMP), phosphatidylcholine (PC) and lipid peroxide (LPO) content of cells were evaluated. Uterus was also removed and weighed. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content as compared to young animals. Age also induced an increase in LPO cell content. NO, CO and cGMP were augmented with age, and PC synthesis showed a dramatic reduction. Treatment with either estradiol or isoflavones were able to improve all the mentioned parameters altered in hepatocytes isolated from old ovariectomized rats, and the magnitude of the improvement was similar for both treatments. Ovariectomy induced a significant reduction in uterine weight, which was significantly counteracted by estradiol treatment but not by isoflavone administration. In conclusion, the administration of a soy extract containing isoflavones seems to prevent oxidative changes in hepatocytes isolated from old ovariectomized female rats, without modifying uterus weight.

Causes, characteristics and consequences of anovulatory follicles in superovulated sheep.

Efficiency of superovulatory protocols is affected by the occurrence of reproductive abnormalities, such as the presence of anovulatory follicles. The objective of current study was to assess the incidence and possible causes of anovulatory follicles in superovulated sheep, in order to characterize the endocrine functionality of these follicles in terms of estradiol production and to evaluate their relationship with development of embryos from other follicles. The number and size of all follicles present in the ovaries of 12 sheep treated with a superovulatory FSH step-down treatment was assessed by ultrasonography. On Day 3 after subsequent estrus behaviour, the number of corpora lutea and anovulatory follicles were recorded and the fluid of anovulatory follicles >or=5mm in size was aspirated and assayed for estradiol. At once, embryos were recovered to evaluate their viability. In current study, anovulatory structures averaged 34.6% of the follicles developing to preovulatory sizes. The number of anovulatory follicles was determined by the existence of follicular dominance effects, since they increased with a higher difference in size between the largest and the second largest follicle at the beginning of the superovulatory treatment (P<0.05, r(2)=0.420). Most of the anovulatory follicles showed signs of functionality failures, indicated by a low mean estradiol concentration (9.9+/-1.1 ng/ml). However, a 22.4% of them were highly estrogenic (>200 ng/ml) and their permanence beyond the ovulation was related to a drop in the embryo viability rate (P<0.005), leading to decreased final superovulatory yields.

Effects of progestagens and prostaglandin analogues on ovarian function and embryo viability in sheep.

Current study assessed differences in the response of sheep to estrus synchronization either by the administration of two doses of prostaglandin or by the insertion of an intravaginal progestagen sponge. The preovulatory follicular dynamics and estradiol secretion, the ovulatory response and progesterone secretion and the number and quality of embryos were studied in 27 ewes treated with two doses of 100 microg of cloprostenol, 10 days apart, and in 29 sheep treated with progestagen sponges for 14 days. Percentage of sheep responding to the synchronization treatments with signs of estrus behaviour was similar between both groups (81.5% versus 72.4%, respectively). The use of progestagen resulted in a higher diameter of the largest follicle (6.6+/-0.2 versus 5.9+/-0.2, P<0.05), and a lower number of small (6.7+/-0.3 versus 9.6+/-0.4, P<0.005) and total follicles (10.3+/-0.3 versus 12.9+/-0.4, P<0.005). However, mean plasma estradiol concentration during the follicular phase was higher in cloprostenol treated sheep (P<0.005). The mean ovulation rate was similar in both treatments (1.7+/-0.2 versus 1.7+/-0.3), but progesterone concentration during the early luteal phase was again higher in sheep treated with cloprostenol (P<0.05). The mean number of retrieved oocytes/embryos was very similar in both treatments (1.2+/-0.2 versus 1.4+/-0.2) and showed similar fertilization rates (70.6% versus 66.7%), but, although differences did not reach statistical significance, final viability rate was higher in cloprostenol than in progestagen treated ewes (58.9% versus 46.1%, P=0.07). Current results give new evidences supporting the negative effects of progestagens on the functionality of ovulatory follicles and support the development of new protocols for assisted reproduction including the use of prostaglandin analogues.

Body composition and vascular effects of growth hormone administration in old female rats.

Aging is associated with alterations in cardiovascular system and changes in body composition. The aim of this study was to investigate the effect of GH on body composition, vascular function and structure in old female rats. Old (20 months) and adult (4 months) female Wistar rats were used. One group of old animals was treated with GH (2 mg/kg/day) for four weeks. Periuterine fat weight, specific gravity index (SGI), dose response to Acetylcholine, Isoprenaline, Phenylephrine and Acetylcholine in the presence of L-NAME and vascular morphology in aortic rings, were studied. Old rats showed increased fat weight and decreased SGI (p<0.05) as compared to adult animals. GH reduced fat weight (p<0.05) and tended to increase SGI (NS). Old rats showed impaired vasodilatation to Acetylcholine and Isoprenaline (p<0.05), and GH improved these responses (p<0.05). Contraction response to Phenylephrine was higher in old than in adults rats (p<0.05), but GH did not show any effect. Contraction induced by Acetylcholine+L-NAME was higher in old rats than in adults, and GH tended to reduce this response, although not significantly. Aortic media area was increased in old rats, and GH reduced this parameter (p<0.05). In conclusion, GH shows beneficial effects on body composition, vascular function and morphology in old female rats.

Growth hormone and aging.

In elderly people, vascular alterations and degenerative alterations of the Central Nervous System (CNS) are two of the most common reasons for illness and death. Lipid pattern modifications and menopause in women are some of the causes for the appearance of these alterations. Vascular endothelium is in part responsible for vascular homeostasis, through the production of several vasoactive factors. Growth hormone (GH) exerts effects on the CNS and on the vascular endothelium, since GH deficient subjects exhibit endothelium-dependent alterations, which recover under substitutive GH treatment. Growth hormone has important actions on lipid metabolism that also play a role on vascular and endothelial function. Moreover, cardiac function improves when GH is associated to angiotensin II receptor blockers. Elderly people exhibit a physiological GH deficiency that could affect their vascular and cerebral functions. A study was carried out using old Wistar rats to clarify the effects of GH on the vessels under chronic "in vivo" conditions. The response to various vasoactive substances in aortic rings has been evaluated. An increase in the aortic media thickness was seen in old rats, which showed also a reduction in the vasodilator response to isoprenaline as compared to young animals. GH treatment partially restored the vasodilator response and reduced media thickness. Neuronal population was reduced in the hypocampus of old rats as compared to young ones and GH treatment was able to significantly enhance the number. Neurotransmitters were measured in several cerebral areas to establish differences between young and old GH-treated or untreated animals. Glutamine, Arginine and Aspartate were reduced in old animals whereas Citruline was increased. GH treatment restored in all cases the levels corresponding to young rats.

Circadian rhythms in airline pilots submitted to long-haul transmeridian flights.

BACKGROUND: Circadian rhythms shift out of phase after transmeridian flights. Desynchronization between body rhythms and the environment is linked to jet lag, which depends on age, flight direction, and number of time zones crossed. METHODS: To investigate this problem in airline pilots, we performed a multivariate analysis of their circadian systems during Madrid-Mexico-Madrid flights (-7 time zones, n = 12) and Madrid-Tokyo-Madrid flights (+8 time zones, n = 21). Telemetry was used to record pilots' activity, skin temperature, and heart rate, obtaining 6 d of continuous data, including 2 d before the flight, the flights themselves, 2 d at the stopover, and 1 d after the return flight. Time series were analyzed by cosinor, and the resulting parameters of the rhythms were compared by ANOVA and Tukey contrasts in every category formed by the age groups (under and over 50 yr old) and flight direction groups. Subjective time estimation of short, intermediate, and long intervals was recorded. Other psychological variables were measured, including anxiety, tiredness, and performance. RESULTS AND CONCLUSIONS: Activity/rest and heart rate rhythms appeared to be linked to a "weak oscillator." Temperature rhythms manifested a rigid response after the phase shifts of the light/dark cycle, closely related to the biological clock. Subjective time appreciation tended to be overestimated without exhibiting a clear circadian component, but attributable to fatigue and stress. Psychometric evaluation showed that desynchronization affected all the pilots. Some results showed an age-related variability with a more marked influence in younger pilots. No consistent effects regarding flight direction were found.

Circadian urinary 6-sulphatoxymelatonin, cortisol excretion and locomotor activity in airline pilots during transmeridian flights.

Airline pilots divided into two groups of age (over and under 50 years) were studied before, during and after westbound (Madrid-Mexico City-Madrid, n = 12) and eastbound (Madrid-Tokyo-Madrid, n = 21) flights. A group of 10 age-matched people staying in Madrid were submitted to the same tests and served as a control group. Changes in urinary 6-sulphatoxymelatonin (6-aMTs) and free cortisol excretion (determined in 6-hr intervals) were measured by radioimmunoassay. Using wrist actigraphy, the circadian locomotor activity rhythm (LAR) was also monitored. Maximal baseline excretion of 6-aMTs occurred between 00:00 and 12:00 hr and maximal excretion of cortisol took place between 6:00 and 12:00 hr in the control group. Analysed globally, older pilots exhibited significantly lower values of 6-aMTs than younger ones. In both flight directions, pilots maintained the pattern of excretion of 6-aMTs, corresponding to baseline. The return flight to Madrid from Mexico and Tokyo coincided with a maximum in 6-aMTs excretion. Pilots kept the cortisol pattern found in the control group, with those over 50 years of age exhibiting significantly lower cortisol values than the younger ones. A 7-hr delay in acrophase of LAR after 2 days in Mexico City was found after cosinor analysis, and similar pre-flight values were found after returning to Madrid. An 8-9-hr acrophase advance of LAR was observed after arriving in Tokyo, with acrophase on the post-return flight day still being advanced 3 4 hr as compared to pre-flight values. Decreases in the amplitude of LAR in older pilots were found at Mexico City, as well as at Tokyo stopover and on post-flight day. Data confirm the occurrence of internal desynchronization in airline crewmembers after transmeridian flights.

GH gene expression in the submaxillary gland in normal and Ames dwarf mice.

High local GH-releasing hormone (GHRH) levels are capable of inducing transdifferentiation in salivary cells to synthesize GH. However, the factors implicated in this process remain unknown. To study this subject, normal and Ames dwarf mice were implanted in the submaxillary gland with a slow release pellet releasing 21 microgram GHRH (1-29)-NH(2)/day for 2 months. Control animals received placebo pellets at the same site. After 60 days, heart blood was collected and submaxillary glands were removed. Circulating levels of GH and IGF-I were significantly decreased (P<0.05) in dwarf mice in comparison with controls, and GHRH treatment did not modify either of these two parameters. Controls carrying GHRH pellets showed a significantly higher GH content (P<0.05) in the submaxillary gland than the placebo-treated normal mice. There were no differences between the IGF-I concentrations of placebo- and GHRH-treated salivary tissue from normal mice. Analysis of GH mRNA by RT-PCR followed by Southern blot revealed that GH transcripts were present in the salivary gland samples carrying the placebo pellets in both normal and dwarf mice. The expression of GH was significantly (P<0.05) increased by the GHRH pellets in salivary tissue from normal mice, but not in submaxillary glands from dwarf mice. Pit-1 mRNA was not detected in the GHRH-treated glands of normal and dwarf mice by RT-PCR or by Southern blot. Using these highly sensitive methods, we have been able to detect the transcription of both GH and Pit-1 in pituitaries from Pit-1-deficient Ames dwarf mice. The present experiment demonstrates that salivary tissue synthesizes GH when it is exposed to the influence of GHRH. Both basal and GHRH-induced salivary GH expression appear to be independent of Pit-1.

Daily rat tibial growth in vivo following hypothalamic sex reversal with neonatal and pubertal treatments with gonadal steroids.

A striking sex-related difference in postpubertal growth and growth hormone (GH) secretory pattern in the rat has been described. Although this sexual dimorphism seems to be determined by the neonatal effects of gonadal steroids on the hypothalamus, peripubertal exposure to steroids also plays an important role. In order to study the real influence of the hypothalamic sex and/or peripubertal gonadal steroids, the growth pattern of female and male rats in response to neonatal and peripubertal sexual steroid treatments was studied using microknemometry, a technique that allows non-invasive daily measurements of rat tibial growth rate. Neonatal steroid environment in males was modified by castration on day 1, whereas in females it was changed by a single neonatal testosterone administration on day 5 followed by castration at 13 days of age. From the onset of puberty to adulthood, both female and male animals received testosterone or estrogens, respectively. Neonatal treatment alone, i.e. androgenization of female and castration of male rats, were only able to induce a partial reversal of the original sex-dependent growth pattern. Additional peripubertal treatments achieved a complete change in the sex-linked growth pattern. Consistent with the effects observed on growth, the pituitary GH concentration was significantly increased in females, and diminished in males, when they were treated both at the neonatal and peripubertal stages. However, only this latter group, whose growth was more seriously compromised, showed decreased plasma insulin-like growth factor-I (IGF-I) levels. In conclusion, a complete feminization of male tibial growth pattern or masculinization of female pattern can only be achieved by maintaining the new steroid environment from puberty to adulthood.