An observation study in mice suggesting that the chromatin of macula densa cells undergoes loss of structural complexity that is most pronounced immediately after birth and remains during the first month of postnatal life. The findings add to the knowledge about the changes that take place in the kidney during postnatal development function and tissue structural organization, which are linked to changes in physiological function. ABSTRACT:Aim: To determine whether complexity of chromatin structure in kidney macula densa cells (MDC) decreases during postnatal development in mice.Methods: The levels of chromatin structural complexity were measured by determining fractal dimension of MDC nuclei. Kidney tissue was obtained from the total of 32 male Swiss albino mice divided into four age groups (n = 8): newborn (0 days), 10 days old, 20 days old and 30 days old. For a total of 640 MDC chromatin structures, fractal dimension, lacunarity, as well as parameters of Grey level co-occurrence matrix (GLCM) texture were determined.Results: Chromatin fractal dimension in animals aged 10 days, 20 days and 30 days was significantly lower (P < 0.05, P < 0.01 and P < 0.001, respectively), compared with newborn mice. This complexity reduction of chromatin architecture is in accordance with previously published studies, which detected generalized and sustained loss of both tissue and cell complexity during aging. The loss of complexity was texture-independent, since there was no statistically significant difference (P > 0.05) in both chromatin angular second moment and inverse difference moment between the age groups. Conclusion:Our results indicate that age-related nuclear intrinsic factors which do not influence chromatin texture may have an important role in MDC postnatal development.

The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular ␣1-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of ␣ 1-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals. Leydig cells; immobilization stress; corticosterone; adrenaline; testis; apoptosis THE LEVELS OF TESTOSTERONE (T) in circulation reflect the steroidogenic capacity of individual testicular Leydig cells and the total number of these cells per testes (2,3,10,16). Because Leydig cells from adult animals are fully differentiated and rarely proliferate or die under normal physiological conditions, their steroidogenic capacity is controlled predominantly by the status of the hypothalamo-pituitary-gonadal axis via GnRH-LH secretory pathway (10, 23). The sustained stress lowers circulating LH and androgen levels (13,40,42), and acute stress also lowers T levels without changing circulating LH levels (35, 42). Furthermore, the reciprocal changes in plasma corticosterone (CORT) and T in stressed rats (45) and increase in steroidogenic capacity of Leydig cells induced by suppression of CORT levels (19) suggested that this stress hormone directly inhibits T biosynthesis.Several hypotheses have also been introduced to explain the mechanism by which glucocorticoids directly inhibit androgenesis. The CORT-induced decline in steroidogenic capacity could reflect inhibition of the expression (48) and activity (47) of T-biosynthetic...

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.

Knowledge about the relationship between steroidogenesis and the regulation of the mitochondrial bioenergetics and dynamics, in steroidogenic cells, is not completely elucidated. Here we employed in vivo and ex vivo experimental models to analyze mitochondrial physiology in Leydig cells depending on the different LH-cAMP environments. Activation of LH-receptor in rat Leydig cells ex and in vivo triggered cAMP, increased oxygen consumption, mitoenergetic and steroidogenic activities. Increased mitoenergetic activity i.e., ATP production is achieved through augmented glycolytic ATP production and a small part of oxidative phosphorylation (OXPHOS). Transcription of major genes responsible for mitochondrial dynamics was upregulated for Ppargc1a (regulator of mitogenesis and function) and downregulated for Drp1 (main fission marker), Prkn, Pink1 and Tfeb (mitophagy markers). Leydig cells from gonadotropin-treated rats show increased mitogenesis confirmed by increased mitochondrial mass, increased mtDNA, more frequent mitochondria observed by a transmission electron microscope and increased expression of subunits of respiratory proteins Cytc/CYTC and COX4. Opposite, Leydig cells from hypogonadotropic-hypogonadal rats characterized by low LH-cAMP, testosterone, and ATP production, reduced markers of mitogenesis and mitofusion (Mfn1/2, Opa1) associated with reduced mtDNA content. Altogether results underline LH-cAMP signaling as an important regulator of mitochondrial physiology arranging mitochondrial dynamics, bioenergetic and steroidogenic function in Leydig cells.

Balkan universities use a substantial number of small mammals and amphibians in the teaching of physiology and pharmacology. This project investigated whether making computer-based alternatives readily available, and combining this availability with a staff development workshop focusing on methods of integrating such resources into undergraduate curricula, would have any effect on animal use. Teachers from 20 Institutes (from five Balkan countries) participated in the workshop. They presented information about animal use in teaching in their universities, and agreed to introduce at least one computer-based alternative into their teaching in the following year. They were surveyed by questionnaire before, during, and one year after, attending the workshop, in order to estimate any changes in animal use. The results showed a significant (P < 0.01) reduction in animal use and a high level of implementation of the alternatives provided at the workshop. Teachers recognised the potential benefits of using computers to support their teaching. They lacked knowledge about what computer-based alternatives are available and how to find information about them, including published evidence of their educational effectiveness. In this pilot study, a combination of staff development and making alternatives readily available to teachers had a significant impact on animal use in the teaching of physiology and pharmacology.