Cell-type-specific determination of reactive oxygen species by flow cytometry.

BACKGROUND: Seminal leukocyte-generated reactive oxygen species may have a significant impact on sperm intracellular reactive oxygen species levels, therefore contributing to oxidative damage and consequent functional impairment of spermatozoa. This relationship may be utilized for male urogenital inflammation-driven oxidative stress diagnostics. OBJECTIVE: To obtain seminal cell-specific, reactive oxygen species-related fluorescence intensity cut-off values to differentiate leukocytospermic samples displaying reactive oxygen species overproduction (oxidative burst) from normozoospermic seminal samples. MATERIAL AND METHODS: Ejaculates gained by masturbation were obtained from patients in the framework of andrology consultations. The results published in this paper were generated from samples for which the attending physician requested spermatograms and seminal reactive oxygen species laboratory tests. Routine seminal analyses were performed according to World Health Organization guidelines. Samples were divided into normozoospermic "non-inflamed," and leukocytospermic groups. The semen was stained by 2',7'-dichlorodihydrofluorescein diacetate and the reactive oxygen species-related fluorescence signal and the percentage of reactive oxygen species-positive spermatozoa within the living population were quantified by flow cytometry. RESULTS: Reactive oxygen species-related mean fluorescence intensity was higher in both spermatozoa and leukocytes from leukocytospermic samples than in those from normozoospermic samples. Mean fluorescence intensity in spermatozoa was positively and linearly correlated with mean fluorescence intensity measured in leukocytes in both groups. DISCUSSION: The capacity of spermatozoa to generate reactive oxygen species is at least three log lower than that of granulocytes. The question is whether the reactive oxygen species-producing machinery of spermatozoa is capable of causing autologous oxidative stress or whether leukocytes are the predominant source of seminal oxidative stress. Based on our observations, the reactive oxygen species production of leukocytes may have a significant impact on the overall reactive oxygen species levels measured in spermatozoa. CONCLUSION: Reactive oxygen species-overproducing leukocytospermic and normozoospermic seminal samples can reliably be differentiated based on reactive oxygen species mean fluorescence intensity measurement.

Colocalized neurotransmitters in the hindbrain cooperate in adaptation to chronic hypernatremia.

Chronic hypernatremia activates the central osmoregulatory mechanisms and inhibits the function of the hypothalamic-pituitary-adrenal (HPA) axis. Noradrenaline (NE) release into the periventricular anteroventral third ventricle region (AV3V), the supraoptic (SON) and hypothalamic paraventricular nuclei (PVN) from efferents of the caudal ventrolateral (cVLM) and dorsomedial (cDMM) medulla has been shown to be essential for the hypernatremia-evoked responses and for the HPA response to acute restraint. Notably, the medullary NE cell groups highly coexpress prolactin-releasing peptide (PrRP) and nesfatin-1/NUCB2 (nesfatin), therefore, we assumed they contributed to the reactions to chronic hypernatremia. To investigate this, we compared two models: homozygous Brattleboro rats with hereditary diabetes insipidus (DI) and Wistar rats subjected to chronic high salt solution (HS) intake. HS rats had higher plasma osmolality than DI rats. PrRP and nesfatin mRNA levels were higher in both models, in both medullary regions compared to controls. Elevated basal tyrosine hydroxylase (TH) expression and impaired restraint-induced TH, PrRP and nesfatin expression elevations in the cVLM were, however, detected only in HS, but not in DI rats. Simultaneously, only HS rats exhibited classical signs of chronic stress and severely blunted hormonal reactions to acute restraint. Data suggest that HPA axis responsiveness to restraint depends on the type of hypernatremia, and on NE capacity in the cVLM. Additionally, NE and PrRP signalization primarily of medullary origin is increased in the SON, PVN and AV3V in HS rats. This suggests a cooperative action in the adaptation responses and designates the AV3V as a new site for PrRP's action in hypernatremia.