Cortisol and testosterone levels on a weekend and a work day in three mountain villages in the Selska Valley of northwest Slovenia.

BACKGROUND: In contemporary western populations, a week commonly involves 5 days of paid work (work days) and two non-working days (weekend). Work days are usually perceived as being more stressful than non-work days and this hypothesis has been tested in several studies, most of which selected subjects with jobs that are perceived to have high stress. AIM: The study measured salivary cortisol and testosterone on a work day and a weekend in a community-based sample of people going about their everyday lives and tested the hypothesis that hormone levels will be higher on a work day. SUBJECTS AND METHODS: Slovenian alpine villagers (30 females and 25 males) were sampled without reference to their occupation. Each individual was measured on two occasions, a day on a weekend and a work day as they went about their usual activities in the afternoon. RESULTS: Cortisol (mean = 3.32 ng ml(-1), range 0.4-27.9) and testosterone (mean = 121 pg ml(-1), range 17-424) values were similar to other populations. Neither the age of subjects nor the time in the afternoon of sample collection were associated with hormone concentrations. On each day of collection, cortisol and testosterone values were correlated for each sex, with the estimate of the correlation coefficient ranging from 0.57 to 0.88. For females, testosterone values were higher on the weekend than the work day (102 pg ml(-1) and 60 pg ml(-1), respectively) but not for males (mean across both days 134 pg ml(-1)). Independent of this effect, the presence of a spouse or other adult in the house was significantly associated with lower testosterone levels in both sexes. Husband and wife testosterone values are correlated on the weekend (r = 0.67, p = 0.02) but not on the work day. Mean cortisol values for the weekend and work day were not different and there was no correlation between levels on these two days. CONCLUSIONS: These results, although based upon a small sample size, reveal potential relationships between testosterone, work-rest activities, and the presence-absence of a social partner that warrant further investigation.

AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

The expression of apoptosis related genes in the first trimester human placenta using a short term in vitro model.

Using in vitro model for studying the induction and inhibition of spontaneous apoptosis in human first trimester placental villi, mediated by the free radical scavenger SOD, we have examined the expression of bcl-xL, bax, Caspase-3 and PARP (Poly ADP-ribosyl). An increase in apoptosis was associated with activation of PARP and an increase and activation of Caspase-3. There was no significant change in bcl-x or bax. Therefore bcl-x and bax do not appear to have a significant role in apoptosis in the first trimester in vitro. Cleavage of Caspase-3 rather than transcriptional regulation appears to be the main determinant of Caspase-3 activity in first trimester placental villi.

Patterns of apoptosis in the corpora lutea of the rat during the oestrous cycle, pregnancy and in vitro culture.

Apoptosis is associated with regression of corpora lutea (CL) in a number of species. The present work compared apoptotic rates, assessed by low molecular weight DNA labelling, in rat CL of the oestrous cycle, pregnancy, the immediate post-partum period, and in vitro culture. Apoptosis was significantly related to cycle phase with peak activity at oestrus. This pattern was similar in CL formed from the most recent ovulation and from the two previous generations. Apoptosis was evident during pregnancy, albeit at low rates. It declined on the day of parturition and increased on the following day. Apoptosis was greatly increased in cultured CL to reach 20-fold higher levels than those achieved during the oestrous cycle or pregnancy. Collectively, these results suggest that apoptosis has lesser significance in rat CL functional regression than in other species with more precipitate structural changes. However, rat CL clearly retain the potential for high rates of apoptosis and so present a useful model for examining molecular mechanisms of apoptotic cell death.

Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea.

Apoptosis is a morphologically defined type of cell death initiated by various stimuli that results in the activation of caspases (cysteine-containing aspartate-specific proteases). In the present study, it was determined that caspases are present during, and play a role in, corpus luteum (CL) apoptosis in vitro. Pseudopregnancy was induced in rabbits with 100 IU human chorionic gonadotrophin. On Day 11 of pseudopregnancy, CL were isolated and cultured for 0, 2, 4, 6, and 8 h in the absence of trophic support to induce spontaneous apoptosis. Total RNA was extracted and analysed for caspase-I expression by Northern blot analysis. The results demonstrated caspase-I expression from 4 h. In the second part of the study, CL were incubated without trophic support for 4 h with increasing concentrations of three general caspase inhibitors, sodium aurothiomalate (SAM), iodoacetic acid (IAA) and N-tosyl-L-phenylalanylchloromethylketone (TPCK), and two specific caspase inhibitors, N-acetyl (Ac)-Tyr-Val-Ala-Asp (YVAD)-chloromethylketone (CMK) (Ac-YVAD-CMK) and Ac-Asp-Glu-Val-Asp (DEVD)-aldehyde (CHO) (Ac-DEVD-CHO). At completion, DNA was isolated and integrity assessed. Treatment of CL with SAM, IAA or Ac-DEVD-CHO effectively suppressed apoptotic DNA fragmentation. The final component of the study was to examine caspase-3 protein expression. Western blot analysis revealed a significant increase in caspase-3 expression over the experimental time-course. The results of the present study clearly demonstrate a time-dependent link between the caspases, specifically caspase-3 and spontaneous apoptosis in the rabbit CL.

Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids.

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11 beta-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.

Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression.

We have recently reported that members of the bcl-2 gene family are expressed and estradiol regulated in rabbit luteal cells during corpus luteum (CL) regression, and that estradiol and hCG are effective inhibitors of apoptosis in the rabbit CL in vivo and in vitro. As Bcl-2 and related proteins are known to regulate levels of reactive oxygen species or their intermediates in cells as one possible mechanism to control apoptosis, the present studies were designed to examine if oxidative stress plays a role in luteal cell apoptosis during CL regression in the rabbit. In the first set of experiments, healthy CL obtained from day 11 pseudopregnant rabbits were incubated in serum-free medium for 2 h in the absence or presence of superoxide dismutase (SOD; 1.5-150 U/ml), ascorbic acid (1-100 mM), N-acetyl-L-cysteine (25 and 50 mM), or catalase (10-1000 U/ml). Cells within CL incubated in medium alone exhibited extensive apoptosis (examined by analysis of extracted DNA using 3'-end labeling), and this onset of apoptosis was blocked in a dose-dependent fashion by treatment with SOD, ascorbic acid, N-acetyl-L-cysteine, or catalase. In the second set of experiments, expression of bax and bcl-x in CL after in vitro treatment without and with 100 U/ml SOD was examined. Although SOD treatment did not alter the levels of bcl-x messenger RNA (mRNA) over the 2-h incubation period, this antioxidant enzyme significantly reduced the levels of bax mRNA in incubated CL. In the final set of experiments, we observed that expression of mitochondrial- or manganese-containing SOD was significantly increased by treatment of isolated CL with 1 microg/ml hCG in vitro, whereas bax mRNA levels were significantly reduced under the same culture conditions. Collectively, these data indicate that the gonadotropin-mediated inhibition of apoptosis in rabbit luteal cells involves enhanced expression of the oxidative stress response gene, manganese-containing SOD, whose protein product may then function to protect luteal cells directly from the damaging effect of reactive oxygen species and/or indirectly by acutely down-regulating expression of Bax, a prooxidant member of the Bcl-2 protein family.

Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat.

We have demonstrated that continuous administration of a GnRH-agonist (GnRH-Ag) in the rat during early pregnancy suppressed plasma progesterone levels within 8 h after the commencement of treatment. The purpose of the present study is to evaluate the hypothesis that in vivo GnRH-Ag treatment induces apoptotic cell death during early pregnancy. Rats were treated individually on day 8 of pregnancy with 5 microg/day of GnRH-Ag via an osmotic minipump. Sham-controlled rats received no treatment. Rats were killed at 4, 8 or 24 h after the treatment. At autopsy, blood samples were obtained and ovaries were removed. The corpora lutea (CL) from one ovary were removed for DNA laddering and RNA studies. As early as 8 h after the commencement of treatment, GnRH-Ag suppressed serum progesterone levels as expected, and increased the degree of DNA degradation in the CL that was time-dependent. At 24 h after the treatment, GnRH-Ag increased the Bax, a cell death gene, expression in the CL. Collectively, these data suggest that GnRH-Ag treatment induces apoptosis during early pregnancy in the rat.

Induction of myometrial 11beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid and protein expression late in rat pregnancy.

Myometrial function in pregnancy is regulated by a range of hormonal stimuli, including glucocorticoids, particularly in the period leading up to parturition. Glucocorticoid hormone action is dependent not only on expression of glucocorticoid receptor (GR) within target cells, but also on local expression of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD). Therefore, this study examined changes in myometrial 11betaHSD bioactivity and expression (messenger RNA and protein) of the 11betaHSD-1 isoform and whether 11betaHSD-1 and GR are colocalized to myometrial cells. Myometrial 11-oxoreductase activity (conversion of [3H]11-dehydrocorticosterone to [3H]corticosterone) was only just detectable (<6%) at the postestrus stage of the cycle and on days 5 and 10 of pregnancy, but then increased markedly by day 16 (45 +/- 2%). This activity increased further to maximal levels on day 22 of pregnancy (55 +/- 3%) and remained high on day 23 (term; 34 +/- 3%) before decreasing by 24 h postpartum (9 +/- 2%). High 11beta-dehydrogenase activity was evident before (87 +/- 1%) and during the first half (day 5,91 +/- 1%; day 10, 88 +/- 2%) of pregnancy, was lower on days 16 (55 +/- 2%), 22 (39 +/- 3%), and 23 (58 +/- 1%), then returned to prepregnancy levels 24 h postpartum (86 +/- 1%). The marked induction of 11-oxoreductase activity late in pregnancy was strongly and positively correlated with both 11betaHSD-1 messenger RNA expression (by Northern analysis) and protein (by Western analysis; r = 0.96 and 0.98, respectively; P < 0.001). Moreover, 11betaHSD-1 and GR immunoreactivity were colocalized to the smooth muscle cells of the myometrium and the uterine epithelium late in pregnancy. Collectively, these data demonstrate that a marked induction of 11betaHSD-1 expression occurs in the rat myometrium near term, and this is associated with increased 11-oxoreductase bioactivity. As GR is coexpressed in the myometrium, we suggest that the induction 11betaHSD-1 serves to enhance local glucocorticoid actions and thus facilitate parturition.