Multicentre study of Y chromosome microdeletions in 1,808 Chinese infertile males using multiplex and real-time polymerase chain reaction.

Azoospermia factor (AZF) genes on the long arm of the human Y chromosome are involved in spermatogenesis, and microdeletions in the AZF region have been recognised to be the second major genetic cause of spermatogenetic failure resulting in male infertility. While screening for these microdeletions can avoid unnecessary medical and surgical treatments, current methods are generally time-consuming. Therefore, we established a new method to detect and analyse microdeletions in the AZF region quickly, safely and efficiently. In total, 1,808 patients with spermatogenetic failure were recruited from three hospitals in southern China, of which 600 patients were randomly selected for screening for Y chromosome microdeletions in AZF regions employing real-time polymerase chain reaction with a TaqMan probe. In our study, of 1,808 infertile patients, 150 (8.3%) were found to bear microdeletions in the Y chromosome using multiplex PCR, while no deletions were found in the controls. Among the AZF deletions detected, two were in AZFa, three in AZFb, 35 in AZFc, three in AZFb+c and two in AZFa+b+c. Our method is fast-it permits the scanning of DNA from a patient in one and a half hours-and reliable, minimising the risk of cross-contamination and false-positive and false-negative results.

Neuroprotective effects of NEP1-40 and fasudil on Nogo-A expression in neonatal rats with hypoxic-ischemic brain damage.

The hypoxic-ischemic encephalopathy caused by peripartum asphyxia is a serious disease in newborn infants, and effective therapies need to be developed to reduce injury-related disorders. We evaluated the effects of NEP1-40 and fasudil on Nogo-A expression in neonatal hypoxic-ischemic brain damage (HIBD) rats. Seven-day-old Wistar rats were randomly divided into control, HIBD, NEP1-40, and fasudil groups. NEP1-40 and fasudil groups were injected intraperitoneally with these compounds. Rat brains at 6, 24, 72 h, and 7 days after HIBD were collected to determine histopathological damage and the expression levels of Nogo-A. Histopathological damage was reduced in NEP1-40 and fasudil groups compared with the untreated HIBD group. The expression of Nogo-A in the HIBD group was significantly higher than that in control, NEP1-40 and fasudil groups at the same times. Compared with the fasudil group, the expression levels of Nogo-A were significantly reduced in the NEP1-40 group. We conclude that NPE1-40 and fasudil have potential for neuroprotective effects in the neonatal rat HIBD model, mediated by inhibiting Nogo-A/ Rho pathways.

Overexpression of clusterin correlates with tumor progression, metastasis in gastric cancer: a study on tissue microarrays.

Clusterin (CLU) is expressed in a wide variety of human tissues and fluids. Overexpression of cytoplasmic clusterin (sCLU) has been implicated in cancer development and progression. The aim of the present study is to evaluate the association of sCLU overexpression with clinicopathological features of human gastric carcinomas (GC).We constructed a gastric cancer tissue microarray containing 173 primary gastric carcinomas and 70 paired non-neoplastic mucosa specimens. The expression of sCLU was studied by immunohistochemistry. The correlations between sCLU expression and clinicopathological features, p53 abnormality, as well as Ki67 activation were analyzed. Overexpressions of sCLU was detected in 28.5% (n=165) of primary GCs by immunohistochemical staining, but not in non-neoplastic mucosa. Clinical association study found that overexpression of sCLU was significantly correlated with lymph-node metastasis (p < 0.001), tumor invasion (p < 0.001) and TNM stage (p < 0.001). In Kaplan-Meier survival analysis, overexpression of sCLU was significantly correlated with unfavorable survival in advanced GCs (p < 0.03). Furthermore, the association of sCLU with abnormal expression of p53 was ascertained. These results suggested that overexpression of sCLU was involved in the progression of GC and it's oncogenic function might be associated with p53 abnormality. Overexpression of sCLU seems to be related with patient's shorter survival in late stage GC.

A dendritic cell-based tumour vaccine for lung cancer: full-length XAGE-1b protein-pulsed dendritic cells induce specific cytotoxic T lymphocytes in vitro.

XAGE-1b is regarded as one of the most immunogenic antigens and the most promising targets for lung adenocarcinoma immunotherapy. In this study, we sought to determine whether monocyte-derived dendritic cells (DCs) pulsed with purified full-length XAGE-1b could induce specific cytotoxic T lymphocytes (CTLs) against tumour cells from patients with non-small cell lung cancer (NSCLC) in vitro. XAGE-1b mRNA expression was examined in primary cultures of lung cancer cells and normal lung epithelial cells established from fresh tissues surgically resected from 30 patients with NSCLC using reverse transcription-polymerase chain reaction (RT-PCR). XAGE-1b mRNA expression was observed in 11 of 18 (61.1%) adenocarcinomas and one of 12 (8.3%) lung cancers of other histological types (P = 0.015). The 246-base pairs XAGE-1b gene was inserted into a recombinant expression vector. Full-length XAGE-1b was then expressed in BL21 (DE3) Escherichia coli and purified by AKTA-fast performance liquid chromatography (FPLC). DCs generated from peripheral blood mononuclear cells were pulsed with XAGE-1b by incubation with the protein at an immature stage. The XAGE-1b-pulsed DCs induced CTLs following 14 days of co-culture. Finally, an adherent target detachment (ATD) assay was performed to test the cytotoxicity of the XAGE-1b-specific CTLs against cancer cells and normal lung epithelial cells. The XAGE-1b-specific CTLs had a stronger lytic effect on autologous XAGE-1b mRNA-positive cancer cells than on autologous XAGE-1b mRNA-negative cancer cells or allogenous XAGE-1b mRNA-positive cancer cells. The CTLs had no lytic activity against normal lung epithelial cells. These results can be used to develop simple and effective cancer/testis antigen-based immunotherapies for NSCLC.

Adrenergic and serotoninergic receptors mediate the immunological activation of corticosterone secretion in male rats.

In order to elucidate the mechanism of action of immune agents on corticosterone secretion, the present study evaluated the possible involvement of some neuronal pathways (serotoninergic, noradrenergic/adrenergic) in the lipopolysaccharide (LPS)-induced corticosterone release in male rats. Serotoninergic antagonists, mianserin (5-HT2C receptor blocker) or pindolol (5HT1A receptor blocker) or noradrenergic/adrenergic antagonists, prazosin (alpha 1-adrenoceptor blocker) or propranolol (beta-adrenoceptor blocker), were intraperitoneally (i.p.) injected before (5 min) the administration of LPS. In each experiment a group of rats i.p. injected with vehicle served as controls. Animals were sacrificed by decapitation 90 min after administration of LPS and trunk blood was collected for corticosterone radioimmunoassay. Results showed that pretreatment with mianserin, but not with pindolol, significantly reduced plasma corticosterone levels following administration of LPS (p < 0.05); prazosin attenuated the plasma corticosterone response to LPS (p < 0.05), while propranolol did not induce significant change. The present study indicated that serotoninergic and noradrenergic/adrenergic pathways are involved in the immunoneuroendocrine modulation of hypothalamus-pituitary-adrenal function in rats. In particular, it is probably mediated by the activation of 5-HT2C receptors and of alpha 1-adrenoceptors, while type 1A serotonin receptors or beta-adrenoceptors do not seem to be involved in such a phenomenon.

Estrous cycle- and acute stress-related changes of rat ovarian immunoreactive corticotropin-releasing factor.

Corticotropin-releasing factor (CRF), the major regulator of the stress response within the central nervous system, is also present at peripheral sites, including the gonads, and the gene encoding its own receptor can be finely induced in selective ovarian compartments in both control and stressful conditions during the gonadal life cycle. The present study, therefore, investigated the influence of both gonadal function and estrous cycle on the immunoreactive CRF (irCRF) contents in the immature and adult rat ovary. In addition, the effect of an acute (5 min) or chronic intermittent (twice a day for 4 days) cold swimming stress on ovarian irCRF contents was evaluated. High-performance liquid chromatography (HPLC), gel-chromatography (Sephadex G-75, 45 x 1 cm) and a direct radioimmunoassay were performed to measure irCRF ovarian contents. The HPLC elution profile of irCRF in ovarian tissues of adult rats was superimposable on that of synthetic rat/human CRF and gel-chromatograms performed according to the phase of the estrous cycle revealed higher irCRF contents at proestrus. Total irCRF ovarian content was undetectable both in control and acute stressed immature rats, while adult rats showed the highest values at proestrus (p < 0.0001). The acute stress exposure induced a significant increase (p < 0.0001) of irCRF ovarian contents only at proestrus, without affecting irCRF at the other phases of the estrous cycle. Finally, no significant changes were found in ovarian irCRF after chronic intermittent stress. The proestrus-related changes of ovarian irCRF, confirming the adult ovary as an extrahypothalamic source of CRF, may constitute a neuropeptidergic signal involved in the gonadal reproductive cycle. Furthermore, the stress-related changes of ovarian irCRF indicated that the gonad may be locally sensitive to acute stressful stimuli.

Bicuculline enhances the corticosterone secretion induced by lipopolysaccharide and interleukin-1 alpha in male rats.

Lipopolysaccharide (LPS)-induced inflammatory stress activates the hypothalamus-pituitary-adrenal (HPA) function. Interleukin-I (IL-1) is one of the key factors during this event; however, the mechanisms mediating IL-1 stimulation of HPA axis are still unclear. The present study evaluated the possible involvement of gamma-aminobutyric acid (GABA) in LPS-induced activation of HPA axis in adult male rats. In addition, the possible existence of diurnal changes of LPS-induced HPA axis activity was also investigated. Bicuculline (0.8 mg/kg BW), a GABA-A receptor antagonist and GABA (1 g/kg BW) were intraperitoneally (ip) injected 15 min before LPS (2 mg/kg BW, ip) or recombinant human IL-1 alpha (microgram/rat) administration in intact rats. Control animals received an equivalent volume of 0.9% saline. Rats were sacrificed at 60 min or 90 min after LPS, or IL-1 alpha or saline injection. Plasma corticosterone levels were measured by radioimmunoassay. Results showed that pretreatment with bicuculline enhanced both LPS- and IL-1-induced corticosterone secretion; while pretreatment with GABA significantly reduced the LPS-stimulated corticosterone release (p < 0.05, vs LPS alone). The effect is dependent on the time of sampling and such effect of bicuculline or GABA was not observed when rats were stimulated in the evening. In addition, the maximal changes of plasma corticosterone following LPS administration in the evening were significantly lower than in the morning (p < 0.01). The present study provides evidence that GABA is involved, at least in part, in the neuroendocrine regulation of LPS/interleukin-1a-induced corticosterone secretion via GABA-A receptor in rats. In addition, the response of plasma corticosterone to LPS has a diurnal variation, which corresponds to a diurnal change of GABAergic modulation of the immunoneuroendocrine response.

Allopregnanolone concentration in hippocampus of prepubertal rats and female rats throughout estrous cycle.

Hippocampus plays an important role in cognition, neuroendocrine function and sexual behaviour. Changes of hippocampal neuropeptide and neurotransmitter concentrations are associated to behavioural changes occurring throughout reproductive life. The present study focused the attention on the presence of a neurosteroid, 5 alpha-pregnan-3 alpha-ol-20-one (termed allopregnanolone) in hippocampus. In particular, hippocampal allopregnanolone concentration in male and female prepubertal rats and in female rats throughout estrous cycle were evaluated. Hippocampal extracts were eluted on high pressure liquid chromatography and allopregnanolone concentration was measured by radioimmunoassay. Prepubertal male and female rats (15 days old) showed highest values which significantly decreased with advancing age (25 and 60 days) (p < 0.01); the lowest hippocampal concentration of allopregnanolone was found in adult rats. Female rats on proestrus morning and afternoon showed an hippocampal allopregnanolone concentration significantly higher than on diestrus or on estrus (p < 0.01), while rats on estrus showed hippocampal allopregnanolone concentration significantly lower than during other days of estrus cycle (p < 0.01). These data indicate differences in hippocampal concentration of allopregnanolone between prepubertal and adult rats and throughout estrous cycle in female rats. This finding suggest a putative role of neurosteroids in the modulation of behavioral changes occurring throughout reproductive life.

Evidence for a role for the neurosteroid allopregnanolone in the modulation of reproductive function in female rats.

The present study investigated the effect of allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one) or of passive immunoneutralization of brain allopregnanolone, the most potent steroid produced by neurons, on ovulation rate and sexual behavior in female rats. Allopregnanolone was injected intracerebroventricularly in rats on diestrus and proestrus and tests were done on estrus. The intracerebroventricular injection of allopregnanolone significantly decreased the number of oocytes collected on estrus (p < 0.01). To support a physiological involvement, antiserum to allopregnanolone was injected centrally to block the activity of the endogenous neurosteroid. When administered on diestrus and proestrus or only on proestrus, the antiserum was shown to be correlated with a significant increase (p < 0.01) in oocytes retrieved on estrus. In female rats treated with antiserum to allopregnanolone, the lordosis intensity was augmented significantly as compared to controls. Finally, the possible changes of medial basal hypothalamus concentration of allopregnanolone throughout the estrous cycle and at the time of ovulation were investigated. Hypothalamic extracts were eluted on high-pressure liquid chromatography and allopregnanolone concentration was measured by radioimmunoassay. Brain cortex was used as control tissue. Hypothalamic allopregnanolone concentration on proestrus morning and afternoon was found to be significantly lower than in the remaining phases of the estrous cycle (p < 0.01), while no significant changes were observed in brain cortex concentration of allopregnanolone. The present results suggest that hypothalamic allopregnanolone may be involved in the mechanism of ovulation, affecting hormonal and behavioral events.

Evidence for a role of neurosteroids in modulation of diurnal changes and acute stress-induced corticosterone secretion in rats.

The neurosteroid allopregnanolone has been shown to be a potent ligand of gamma-aminobutyric acid (GABA)-A receptors and enhances its receptor-mediated inhibitory events. Since central GABA plays a major inhibitory role, via GABA-A receptors, in hypothalamic-pituitary-adrenal (HPA) function in rats, the present study has evaluated the effect of passive immunoneutralization of allopregnanolone on diurnal changes in corticosterone secretion and acute stress-induced corticosterone secretion in rats. In the first protocol, four groups of male rats (prepubertal, fertile, castrated adult and aged) and three groups of female rats (prepubertal, fertile at different phases of the estrous cycle and aged) were studied. Rats were injected intracerebroventricularly (i.c.v.) with 10 microliters anti-allopregnanolone serum or 10 microliters normal rabbit serum (control) 24 h before exposure to an acute cold swimming stress, and sacrificed either before stress or after 5 min stress. In the second protocol, fertile male or female rats at diestrus II were injected i.c.v. with anti-allopregnanolone serum or normal rabbit serum and sacrificed on the following day at 10.00 or 18.00. Truncal blood samples were collected for measuring plasma corticosterone. Our results showed that there was no significant difference in basal plasma corticosterone levels between antiserum-treated and control rats of both sexes. However, in male rats, central injection of antiserum to allopregnanolone significantly potentiated plasma corticosterone response to stress in prepubertal and adult fertile rats as well as in castrated rats. Likewise, in female rats, the stress response of plasma corticosterone was enhanced by passive immunoneutralization of allopregnanolone in prepubertal and fertile rats throughout the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of mu-opioid receptors in the modulation of pituitary-adrenal axis in normal and stressed rats.

The availability of the most selective, high-affinity, natural opioid agonists for mu-receptors (dermorphin-DM) and delta-receptors (deltorphin-DT) has provided the possibility for in vivo studying of the role of acute and chronic activation of mu- and delta-opioid receptors on the functional activity of the hypothalamus-pituitary-adrenocortical (HPA) axis, both in basal conditions and in response to an acute stress in adult male rats. Plasma corticosterone (CS) and beta-endorphin-like-immunoreactivity (beta-EP-LI) levels were measured by specific radioimmunoassays before and after 5 and 30 minutes from the exposure to cold (3 +/- 0.5 C) water and forcing them to swim for 10 minutes (acute cold swimming stress). Acute administration of DM, the specific mu-receptor agonist, enhanced basal and stress induced plasma levels of CS and beta-EP-LI. These effects were antagonized by pretreatment with naloxone, specific mu-opioid receptor antagonist, but not by naltrindole, a delta-opioid receptor antagonist. Long-term administration of DM did not alter resting plasma levels of CS and beta-EP-LI, but significantly reduced stress-induced increase of these hormones. Both the acute and chronic administration of the DT, highly selective delta-opioid receptors agonist, failed to modify resting and stress induced hormone levels. Our present data show that DM throughout mu-opioid receptors, but not DT, modulates the response of HPA axis to acute stress in rats, increasing or decreasing the release of CS and beta-EP-LI when acutely or chronically administered, respectively.

Pituitary and ovarian interleukin-1 alpha content changes according to estrous cycle and acute stress exposure.

A complex interaction between the immune and neuroendocrine systems has been established. In particular, cytokines are known to be one of the mediators of the stress response, and modulate hormone secretion by acting in the brain, pituitary and gonads. The aim of the present study is to investigate whether pituitary and ovarian interleukin-1 alpha (IL-1 alpha) content changes according to the estrous cycle. In addition, the possible pituitary and ovarian IL-1 alpha changes in rats exposed to acute (5 min) or chronic intermittent (twice a day for 4 days) cold swimming stress were studied. The IL-1 alpha content of ovarian and pituitary homogenates was measured by a sensitive and specific radioimmunoassay. Immunoreactive IL-1 alpha (irIL-1 alpha) was detectable only in ovaries collected in rats at proestrus and estrus while not in those collected at diestrus I and II. The highest values were found at proestrus. No significant changes were found in ovarian irIL-1 alpha content in rats exposed to acute or chronic intermittent stress in comparison to control rats. In the pituitary, no difference in IL-1 alpha content was found throughout the estrous cycle. Acute stress induced a significant increase in pituitary irIL-1 alpha content only at proestrus (p < 0.01), however, no significant differences were found in comparison to control rats after chronic intermittent stress. The proestrus-related changes of ovarian IL-1 alpha may constitute a hormone-dependent signal within the ovary that is involved in the ovulatory process.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute stress- or lipopolysaccharide-induced corticosterone secretion in female rats is independent of the oestrous cycle.

The aim of the present study was to test whether oestrous cycle is associated with the hypothalamus-pituitary-adrenal (HPA) axis function. Thus, corticosterone secretion in rats was investigated following lipopolysaccharide (LPS), acute cold-swimming or ether stress or synthetic corticotrophin-releasing factor (CRF) administration throughout the oestrous cycle. Moreover, plasma corticosterone response to cold-swimming stress or LPS administration also was studied at different times of day on pro-oestrus of di-oestrus-I. The following observations were obtained: the morning plasma corticosterone levels in control rats did not differ with the stage of the oestrous cycle; plasma corticosterone levels increased significantly following LPS administration (2 mg/kg, ip) or following acute exposure to cold (4 degrees C)-swimming or ether stress. However, this increase in plasma corticosterone levels was not related to the stage of the oestrous cycle; synthetic CRF injection induced an increase in plasma corticosterone levels constant on di-oestrus-I and pro-oestrus; plasma corticosterone response to LPS administration or acute cold-swimming stress showed diurnal changes, with the lowest values at 18.00 h, which was independent of the oestrous cycle. By showing the unchanged corticosterone response to LPS, to acute stress and to exogenous CRF throughout the oestrous cycle, and the independence of the diurnal pattern of stress response on the oestrous cycle, the present study suggests that the oestrous cycle has no influence on the HPA activity under the present experimental conditions in rats.