Effect of exercise duration on toluene-induced locomotor sensitization in mice: a focus on the Renin Angiotensin System.

RATIONALE: Exercise attenuates addictive behavior; however, little is known about the contribution of exercise duration to this positive effect. The Renin Angiotensin System (RAS) has been implicated both in addictive responses and in the beneficial effects of exercise; though, its role in the advantageous effects of exercise on toluene-induced addictive responses has not been explored. OBJECTIVES: To evaluate the impact of different exercise regimens in mitigating the expression of toluene-induced locomotor sensitization and to analyze changes in RAS elements' expression at the mesocorticolimbic system after repeated toluene exposure and following voluntary wheel running in toluene-sensitized animals. METHODS: Toluene-induced addictive-like response was evaluated with a locomotor sensitization model in mice. Toluene-sensitized animals had access to running wheels 1, 2, 4 or 24 h/day for 4 weeks; thereafter, locomotor sensitization expression was evaluated after a toluene challenge. RAS elements (ACE and ACE2 enzymes; AT1, AT2 and Mas receptors) expression was determined by Western blot in the VTA, NAc and PFCx of toluene-sensitized mice with and without exercise. RESULTS: Individual differences in toluene-induced locomotor sensitization development were observed. Access to wheel running 1 and 2 h/day reduced but 4 and 24 h/day completely blocked locomotor sensitization expression. Repeated toluene exposure changed RAS elements' expression in the VTA, NAc and PFCx, while exercise mainly modified ACE and AT1 in air-exposed and toluene-sensitized mice. CONCLUSIONS: Inhalant-exposed animals show different sensitization phenotypes. Exercise duration determined its efficacy to attenuate the addictive-like response. Toluene exposure and exercise each modified RAS, the latter also modifying toluene-induced changes.

p-Chloroamphetamine decreases serotonin and induces apoptosis in granulosa cells and follicular atresia in prepubertal female rats.

Amphetamine derivatives negatively impact serotonin (5-HT) production, which triggers apoptosis in different tissues, depending on the receptor they bind. 5-HT in the ovary stimulates estradiol secretion, a survival factor of granulosa cells. The effect of amphetamine derivatives on the serotonergic system of the ovary and follicular development is unknown. Therefore, in this study, we investigated the effects of p-chloroamphetamine (pCA), derived from amphetamines, on estradiol production, follicular development, apoptosis of granulosa cells, and serotonin 5-HT7 receptor (R5-HT7) expression. Female rats (30 days old) were injected with 10 mg/kg of pCA intraperitoneally and were euthanized 48 or 120 h after treatment. The concentration of 5-HT in the hypothalamus decreased at 48 and 120 h after treatment and in the ovary at 120 h. The serum concentration of estradiol decreased at all times studied. Follicular atresia, TUNEL-positive (apoptotic) granulosa cells and Bax expression were elevated by pCA, but none of these effects was associated with R5-HT7 expression. These results suggest that pCA induces the dysregulation of the serotonergic system in the hypothalamus and the ovary, negatively impacting estradiol production and follicular development.

A new open-source hardware device to measure vertical sperm motility and concentration.

Sperm motility and concentration are commonly evaluated parameters in semen analysis. Those parameters are assessed objectively with commercial instrumentation such as computer-assisted sperm analysis systems (CASA) and hemocytometer. In CASA systems, sperm motility is assessed in the horizontal plane imposed by the stage of the microscope. Thus, there is lack of measurement of the vertical velocity of sperm. The female reproductive tract is a tridimensional space which the sperm traverse to reach the ovum, and there is a need for instruments measuring parameters more relevant to this real-world situation. In this report we describe the design, construction and use of an open-source hardware (OSH) device for evaluation of the vertical velocity of sperm, called UPSPERM. This device was also used to measure sperm concentration, and agreement with hemocytometer was evaluated. Bland-Altman analysis shows good agreement between these two methods of sperm counting. As a first application of UPSPERM, we evaluated the changes in boar sperm motility at distinct pH values between 7.0 and 8.0. The UPSPERM results showed that the vertical velocity of sperm was highest at pH 7.6 and 7.8. We propose that our UPSPERM offers a reliable and affordable option for obtaining measurements of vertical velocity and sperm concentration.

Impaired serotonin communication during juvenile development in rats diminishes adult sperm quality.

Spermatogenesis and steroidogenesis are testicular functions regulated by gonadotrophins as well as other factors, including serotonin. Testicular serotonin acts as an autocrine regulator of testosterone secretion, but studies on its role in spermatogenesis and sperm quality are scarce. Here, we analyzed the effects of intratesticular inhibition of serotonin synthesis on gonadotrophins, testosterone, and sperm quality. Both testicles of 30-day-old rats were injected once with saline solution (SS) or distinct concentrations of p-chloroamphetamine (PCA) (0.03, 0.06, or 0.12 mg). At 65 days of age, rats were euthanized and sperm density, motility, membrane integrity, mitochondrial function, and abnormalities were evaluated in gametes from the vas deferens. Inhibition of synthesis of intratesticular serotonin by PCA diminished the concentrations of testosterone and follicle-stimulating hormone (FSH) but luteinizing hormone (LH) levels were unaltered. Sperm density was not modified in animals injected with the different concentrations of PCA. In contrast, the percentage of sperm with abnormalities increased and the sperm membrane integrity decreased in animals injected at higher PCA concentrations. The functionality of sperm mitochondria in PCA-injected animals decreased only at the highest PCA dose. Our results indicate that testicular serotonin plays a role in testosterone synthesis and in the normal development of sperm, and blocking its effects disrupts the hormonal communication between the testis and hypophysis. ABBREVIATIONS: SS: saline solution; PCA: p-chloroamphetamine; FSH: follicle-stimulating hormone; LH: luteinizing hormone; TPH: tryptophan hydroxylase; MAO: monoamine oxidase; AC: absolute control group; PI: propidium iodide; FLICA: fluorescence inhibitor of caspase; 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; 17-KSR: 17-ketosteroid reductase; DHT: 5-dihydrotestosterone.

Fluoxetine treatment of prepubertal male rats uniformly diminishes sex hormone levels and, in a subpopulation of animals, negatively affects sperm quality.

Fluoxetine (Flx) is a selective serotonin reuptake inhibitor that alters the male reproductive system when administered at the adult stage or after maternal exposure. In the present study we evaluated the effects of Flx administration on reproductive parameters during juvenile-peripubertal development when treated male rats reached adulthood. Groups of rats were treated daily with Flx (5mgkg-1, i.p.) or saline (0.9% NaCl), or were left untreated. Rats were treated between 30 and 53 days of age and were killed at 65 days of age. Serotonin concentrations were determined in the hypothalamus, hypophysis and testis. Gonadotrophins, sex steroids and sperm quality (membrane integrity, sperm with functional mitochondria, sperm density, sperm motility and morphological abnormalities) were also evaluated. Flx did not affect bodyweight, but significantly diminished LH, FSH, progesterone and testosterone serum concentrations. After graphical analysis, a subgroup of rats was identified whose sperm quality parameters were greatly affected by Flx. In the present study we show that Flx administered to juvenile rats disrupts the hypothalamic-hypophyseal-testicular axis and its effects on sperm quality are not homogeneous in adults. In contrast, Flx altered concentrations of gonadotrophins and sexual steroids in all treated rats. These results suggest caution should be exercised in the prescription of Flx to prepubertal males.

Intrinsic and extrinsic apoptotic pathways are involved in rat testis by cold water immersion-induced acute and chronic stress.

Testicular apoptosis is activated by stress, but it is not clear which signaling pathway is activated in response to stress. The aim of this study was to investigate whether intrinsic, extrinsic, or both apoptotic signaling pathways are activated by acute and chronic stress. Adult male rats were subjected to cold water immersion-induced stress for 1, 20, 40, and 50 consecutive days. The seminiferous tubules:apoptotic cell ratio was assayed on acute (1 day) and chronic (20, 40, 50 days) stress. Apoptotic markers, including cleaved-caspase 3 and 8, the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were also determined after acute and chronic stress induction. Additionally, epididymal sperm quality was evaluated, as well as corticosterone and testosterone levels. An increase in tubule apoptotic cell count percentage after an hour of acute stress and during chronic stress induction was observed. The apoptotic cells rate per tubule increment was only detected one hour after acute stress, but not with chronic stress. Accordingly, there was an increase in Bax, cleaved caspase-8 and caspase-3 pro-apoptotic proteins with a decrease of anti-apoptotic Bcl-2 in both acutely and chronically stressed male testes. In addition, sperm count, viability, as well as total and progressive motility were low in chronically stressed males. Finally, the levels of corticosterone increased whereas testosterone levels decreased in chronically stressed males. Activation of the extrinsic apoptotic pathway was shown by cleaved caspase-8 increase whereas the intrinsic apoptotic pathway activation was determined by the increase of Bax, along with Bcl-2 decrease, making evident a cross-talk between these two pathways with the activation of caspase-3. These results suggest that both acute and chronic stress can potentially activate the intrinsic/extrinsic apoptosis pathways in testes. Chronic stress also reduces the quality of epididymal spermatozoa, possibly due to a decrease in testosterone.

Oxidative status in testis and epididymal sperm parameters after acute and chronic stress by cold-water immersion in the adult rat.

Stress is associated with detrimental effects on male reproductive function. It is known that stress increases reactive oxygen species (ROS) generation in the male reproductive tract. High ROS levels may be linked to low sperm quality and male infertility. However, it is still not clear if ROS are generated by stress in the testis. The objective of this study was to characterize the role of oxidative stress induced by cold-water immersion stress in the testis of adult male rats and its relation with alterations in cauda epididymal sperm. Adult male rats were exposed to acute stress or chronic stress by cold-water immersion. Rats were sacrificed at 0, 6, 12, and 24 hours immediately following acute stress exposure, and after 20, 40, and 50 days of chronic stress. ROS production increased only at 6 hours post-stress, while the activity and expression of antioxidant enzymes, lipid peroxidation (LPO), and sperm parameters were not modified in the testis. Corticosterone increased immediately after acute stress, whereas testosterone was not modified. After chronic stress, testicular absolute weight decreased; in addition, ROS production and LPO increased at 20, 40, and 50 days. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) decreased throughout the duration of chronic stress and the activity of catalase (CAT) decreased at 40 and 50 days, and increased at 20 days. The expression of copper/zinc superoxide dismutase (SOD1) and CAT were not modified, but the expression of phospholipid hydroperoxide glutathione peroxidase (GPx-4) decreased at 20 days. Motility, viability, and sperm count decreased, while abnormal sperm increased with chronic stress. These results suggest that during acute stress there is a redox state regulation in the testis since no deleterious effect was observed. In contrast, equilibrium redox is lost during chronic stress, with low enzyme activity but without modifying their expression. In addition, corticosterone increased while testosterone decreased, this decrease is related to the negative effects seen in sperm.

Confocal microscopy and image analysis indicates a region-specific relation between active caspases and cytoplasm in ejaculated and epididymal sperm.

Previously, it was suggested a relation between the presence of apoptosis markers with cytoplasm in mammalian sperm. In this work, flow cytometry, confocal microscopy and image analysis were used to analyze the relationship between active caspase-3 and -7 and intracellular esterases expression in ejaculated or epididymal ram sperm. Sperm obtained from ejaculates from the caput, corpus, or cauda of the epididymis were treated with an inhibitor of active caspase-3 and -7 and a marker of cytoplasmic esterases. Additionally, ejaculated sperm were incubated for one, two, or three hours before evaluation for active caspases. Sperm subpopulations positive for active caspases and/or intracellular esterases were detected by flow cytometry and confocal microscopy; however, image analysis of confocal images showed that the correlation between active caspases and cytoplasmic esterases in sperm is region-specific. Lower values of Spearman correlation coefficients were found when whole sperm or head sperm was analyzed; however, a high correlation was observed for midpiece sperm. Incubation of sperm for two or three hours promoted the autoactivation of caspases. It has been suggested that the presence of apoptotic markers in sperm are related with a process of abortive apoptosis and with errors during spermiogenesis. Our results permit us suggest that the origin of the relationship between active caspases and cytoplasmic esterases is due to differentiation errors occurring during spermiogenesis because the percentages of sperm with active caspases are not different in the caput, corpus, or cauda of the epididymis. In this study we demonstrate that existing sperm subpopulations can express active caspases and intracellular esterases and that the correlation between these molecules is high in midpiece sperm.

Viability tests, active caspase-3 and -7, and chromatin structure in ram sperm selected using the swim-up procedure.

Swim-up (SU) is a technique that permits the obtaining of motile sperm. Based on the sperm motility of neat ejaculates, we analyzed functional parameters, cytoplasmic esterases, and mitochondrial dehydrogenases of ram sperm using calcein acetomethylester and resazurin, respectively. Active caspase-3 and -7 and chromatin structure were evaluated in ram sperm before and after the SU process. There were no changes in any of the viability parameters after SU in neat semen samples with less or more than 25% motility. The percentage of active caspase-3 and -7 decreased after SU (68.8 +/- 4.6 vs 54.2 +/- 6.0), whereas a small but significant increase of chromatin structural abnormalities was observed (DNA fragmentation index [DFI], 287.3 +/- 3.1 vs 297.2 +/- 2.4). For the first time, the location of active caspase-3 and -7 was described for ram sperm. Notably, we found active caspases in the implantation fossa region. The presence of active caspases in neat ejaculates and the diminished presence of active caspases in SU-processed ejaculates suggest a role for caspases in motility and possibly in male fertility. The results of this study indicate that the evaluation of more than one cell-function marker is necessary to appropriately evaluate sperm quality. Furthermore, in semen samples with low motility, a lower percentage of sperm with active caspases is obtained after SU, although these sperm present increased values of DFI.

Función reproductiva de sementales ovinos importados de Nueva Zelanda durante su primera época reproductiva en México / Reproductive function in rams imported from New Zealand during their first reproductive season in Mexico

Importation of rams of high genetic value is a common practice in Mexico; nevertheless, reproductive variables of imported animals have not been followed along. The objective of this study was to evaluate the reproductive function of Suffolk rams along their first reproductive season in Mexico (October to February). The behavior of values of scrotal circumference, sperm count and morphology, and circulating testosterone in Suffolk ram during its first reproductive season in Mexico are in line with the ones reported for Suffolk rams in other latitudes; however, in Mexico such changes are lower in magnitude. Neuroendocrine-reproductive changes induced by environmental stimuli such as changes in duration of light-dark cycles have been broadly documented and are accepted as inducers of seasonal changes in the ram's behavior. In this work, both testosterone concentration and sperm production were observed in December. These results show that Suffolk ram imported to Mexico maintain their cyclic testicular function, although it has a lower magnitude than in other latitudes. Causes of this condition are unknown, but they may be related to changes in the duration of the light-dark cycles prevailing in Mexico.

La importación de sementales de alto valor genético ha sido una estrategia común en México. Sin embargo, debido a que no se le ha dado seguimiento al desempeño reproductivo de tales sementales se llevó a cabo el presente trabajo para realizarlo en sementales Suffolk durante su primera época reproductiva en México (octubre a febrero). El comportamiento de los valores de la circunferencia escrotal, la cuenta y la morfología espermática, así como las concentraciones circulantes de testosterona de los sementales Suffolk durante su primera época reproductiva en México, coinciden con las que se notifican en la literatura para esos animales en otras latitudes, aunque en México son de menor magnitud. Los cambios neuroendocrinos-reproductivos derivados de los estímulos ambientales, como las variaciones en la duración de los ciclos luz-oscuridad, han sido ampliamente documentados y se aceptan como responsables de los cambios estacionales en el comportamiento reproductivo de los carneros. En este trabajo, la mayor concentración de testosterona, así como la mayor producción de espermatozoides se observó en diciembre. Estos resultados indican que los sementales Suffolk importados son capaces de mantener su función testicular cíclica, aunque ésta se manifiesta con menor magnitud que en otras latitudes. Se desconocen las causas de este comportamiento, aunque pueden estar relacionadas con los cambios en la duración de los ciclos luz-oscuridad que ocurren en México.