Investigating Chaperonin-Containing TCP-1 subunit 2 as an essential component of the chaperonin complex for tumorigenesis.

Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few definitive studies addressed the role of specific subunits in promoting the chaperonin's function in cancer. To this end, we investigated the activity of CCT2 (CCTß) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers.

Effects of dietary omega-3 PUFAs on growth and development: Somatic, neurobiological and reproductive functions in a murine model.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are relevant to fetal and infant growth and development. Objective: to assess whether long-term exposure to dietary ω-3 PUFA imbalance alters pre- and/or postnatal pups' development and reproductive function later in life. Mice dams were fed with ω-3 PUFA Control (soybean oil, 7%), Deficient (sunflower oil, 7%) or Excess (blend oil; 4.2% cod-liver+2.8% soybean) diet before conception and throughout gestation-lactation and later on, their pups received the same diet from weaning to adulthood. Offspring somatic, neurobiological and reproductive parameters were evaluated. Excess pups were lighter during the preweaning period and shorter in length from postnatal day (PND) 7 to 49, compared to Control pups (P<.05). On PND14, the percentage of pups with eye opening in Excess group was lower than those from Control and Deficient groups (P<.05). In Excess female offspring, puberty onset (vaginal opening and first estrus) occurred significantly later and the percentage of parthenogenetic oocytes on PND63 was higher than Control and Deficient ones (P<.05). Deficient pups were shorter in length (males: on PND14, 21, 35 and 49; females: on PND14, 21 and 42) compared with Control pups (P<.05). Deficient offspring exhibited higher percentage of bending spermatozoa compared to Control and Excess offspring (P<.05). These results show that either an excessively high or insufficient ω-3 PUFA consumption prior to conception until adulthood seems inadvisable because of the potential risks of short-term adverse effects on growth and development of the progeny or long-lasting effects on their reproductive maturation and function.

[Obesity and male fertility]. / Impacto de la obesidad en la función reproductiva masculina.

Obesity and male infertility have increased in the last decades; therefore, a possible association between these pathologies has been explored. Studies inform that obesity may affect fertility through different mechanisms, which alltogether could exert erectile dysfunction and/or sperm quality impairment. These include: 1) hypothalamic-pituitary-testicular (HPG) axis malfunction: obese hormonal profile is characterized by reduction of testosterone, gonadotrophins, SHBG and/or inhibin B concentrations (marker of Sertoli cells function) and hyperestrogenemy (consequence of aromatase overactivity ascribed to adipose tissue increase); 2) increased release of adipose-derived hormones: leptin increase could be responsible for some of the alterations on the HPG axis and could also exert direct deleterious effects on Leydig cells physiology, spermatogenesis and sperm function; 3) proinflammatory adipokines augmentation, higher scrotal temperature (due to fat accumulation in areas surrounding testes) and endocrine disruptors accumulation in adiposites, all of these responsible for the increase in testes oxidative stress and 4) sleep apnea, frequent in obese patients, suppresses the nocturnal testosterone rise needed for normal spermatogenesis. Finally, although controversial, all the above mentioned factors could comprise gametes quality; i.e. decrease sperm density and motility and increase DNA fragmentation, probably disturbing spermatogenesis and/or epididymal function. In summary, although obesity may impair male fertility by some/all of the described mechanisms, the fact is that only a small proportion of obese men are infertile, probably those genetically predisposed or morbidly obese. Nevertheless, it is likely that because the incidence of obesity is growing, the number of men with reduced fertility will increase as well.

Overweight and seminal quality: a study of 794 patients.

OBJECTIVES: To evaluate sperm quality, levels of markers of epididymal and accessory gland function, and T in semen from men grouped according to their body mass index (BMI). DESIGN: Blind prospective study. SETTING: Andrology and reproduction laboratory in Cordoba, Argentina (2006-2007). PATIENT(S): Seven hundred ninety-four men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): In semen samples, sperm quality (volume, density, motility, morphology, viability, hypoosmotic swell test, and nuclear maturity) and levels of neutral alpha-glucosidase, fructose, citric acid and T. RESULT(S): Multivariate analysis showed a negative association between BMI and motility, rapid motility and neutral alpha-glucosidase levels, and a positive association between BMI and seminal fructose levels. No associations were found among BMI and sperm concentration, the other parameters evaluated, or seminal T levels. CONCLUSION(S): Results found in our study support a deleterious effect of obesity on seminal quality, probably by alterations in the function of the epididymis (i.e., in epididymal maturation).

Developmental and neurobehavioral effects of perinatal exposure to diets with different omega-6:omega-3 ratios in mice.

OBJECTIVE: To investigate in mice the effect of diets enriched with soy or sunflower oil with different omega-6:omega-3 ratios on gestation, reproductive success, physical maturation, and the neurobiological development of the pups. METHODS: Dams were assigned, throughout gestation and lactation, to different groups: a commercial diet (CD), a soy oil-enriched diet (SOD), or a sunflower oil-enriched diet (SFOD). Measurements during gestation were dams' body weights and daily food intakes. Measurements in the offspring were physical parameters (body weight, body length, body mass index, fur appearance, pinna detachment, incisor eruption, eye opening, and puberty onset) and behavioral preweaning tests (surface righting reflex, negative geotaxis, and cliff avoidance). RESULTS: The SOD and SFOD dams became significantly heavier than the CD dams from gestational days 14 and 19, respectively, to parturition. There were no significant differences in gestational length or food consumption during pregnancy or lactation or in maternal weight during lactation. Diets did not modify litter size, sex ratio, survival index at weaning, or body weight. The SFOD and SOD offspring were significantly shorter than the CD offspring at weaning. The mean offspring physical scores of SOD and SFOD offspring were higher than CD offspring and simple reflexes were earlier in the SOD and SFOD groups. In SFOD offspring, puberty onset was significantly delayed, at postnatal days 26 and 27 in male and female offspring, respectively. CONCLUSION: This study suggests that the maintenance of an adequate omega-6:omega-3 ratio is necessary for the optimal growth and development of murine offspring. In populations that do not have sufficient provision of polyunsaturated fatty acids in the diet, their consumption would be advisable during gestation and lactation because these improve most neurodevelopmental outcomes included in this study.

Chronic administration of nonsteroidal-antiinflammatory drugs (NSAIDS): effects upon mouse reproductive functions.

Although nonsteroidal-antiinflamatory drugs (NSAIDs) are widely employed, reproductive side effects of prostaglandins long-term inhibition remain unknown. The objective of the present study was to evaluate the effects of chronic low/moderate NSAIDs doses upon mice reproductive functions. Male or female mice were injected (i.p. for 60 or 35 days respectively) with: ibuprofen doses A, B or C (0.56, 1.12 or 1.68 mg/100 g/day respectively) or piroxicam doses A, B or C (0.028, 0.056 or 0.084 mg/100 g/day respectively). Parameters evaluated were: a) in females, spontaneous and induced ovulation, oocyte maturity and spermatozoa migration through genital tract, b) in males, epididymal spermatozoa concentration, motility, viability, resistance to hypoosmotic shock, acrosomal status and membrane maturity and c) in both genders, in vitro and in vivo fertilization, reproductive hormones plasma levels and cyclooxigenase inhibition in reproductive tissues. In females ibuprofen (dose A) elicited a significant reduction in spontaneous and induced ovulation rates and piroxicam (dose A) diminished the concentration of spermatozoa found in the uterus after mating. Males treated with ibuprofen (dose B) showed a reduction in the in vitro fertilization ability. Our data reveal that chronic administration of ibuprofen or piroxicam can exert detrimental effects upon reproductive physiology, which depends on the doses and/or the drug employed.

Neutral alpha-glucosidase activity in mouse: a marker of epididymal function?

Neutral alpha-glucosidase (NAG) activity is considered a functional epididymal marker in several species. Unlike the rat, no NAG activity has been detected in mice. The aims of the present study were to evaluate NAG secretory activity (the supernatant of the incubated tissue) in mouse epididymis and to determine whether it could be used as a functional epididymal marker. Epididymides (whole or in parts) were incubated in the presence or absence of testosterone (10(-5) m) and secretory NAG activity was compared with known positive controls. Furthermore, we compared enzyme activity in epididymides from well-fed and undernourished mice (50% food restriction for 21 days), a model that alters the epididymal maturation processes. Spectrophotometric analysis revealed NAG activity in mouse epididymis (22.6 +/- 3.7 mU g(-1) tissue; n = 4), being higher in the caput. NAG activity was statistically higher in the caput than in the corpus and in the cauda. No significant differences existed between the caput NAG activity and complete epididymis NAG activity. In undernourished mice, we confirmed changes in epididymal maturation observed previously (i.e. increased number of immature spermatozoa and diminution of the sperm concentration). Concordantly, the epididymides of undernourished mice exhibited decreased enzyme secretory activity, which increased to values similar to those seen in controls following incubation in the presence of testosterone (22.5 +/- 2.6, 12.5 +/- 1.0 and 22.4 +/- 3.7 mU g(-1) tissue, n = 9 in control (n = 7), undernourished (n = 9) and undernourished + testosterone groups (n = 9), respectively). In conclusion, NAG activity was detected in mouse epididymis. Although the present study supports the possibility of using NAG as an epididymal marker, more studies are necessary to effectively prove that NAG activity can be used as an epididymal marker.

Stimulation by ghrelin of p42/p44 mitogen-activated protein kinase through the GHS-R1a receptor: role of G-proteins and beta-arrestins.

Results presented in this study indicate that in human embryonic kidney 293 cells (HEK 293), the ghrelin receptor growth hormone secretagogue receptor type 1a (GHS-R1a) activates the extracellular signal-related kinases 1 and 2 (ERK 1/2) via three pathways. One pathway is mediated by the beta-arrestins 1 and 2, and requires entry of the receptor into a multiprotein complex with the beta-arrestins, Src, Raf-1, and ERK 1/2. A second pathway is G(q/11)-dependent and involves a Ca(2+)-dependent PKC (PKCalpha/beta) and Src. A third pathway is G(i)-dependent and involves phosphoinositide 3-kinase (PI3K), PKCepsilon, and Src. Our current study reveals that G(i/o)- and G(q/11)-proteins are crucially involved in the beta-arrestin-mediated ERK 1/2 activation. These results thus support the view that the beta-arrestins act as both scaffolding proteins and signal transducers in ERK 1/2 activation, as reported for other receptors. The different pathways of ERK 1/2 activation suggest that binding to GHS-R1a activates ERK 1/2 pools at different locations within the cell, and thus probably with different physiological consequences.

Improving the predictive value of the hypoosmotic swelling test in humans.

Using a combined hypoosmotic swelling-eosin (HOS-E) technique in human semen samples, we evaluated the frequency of dead swollen spermatozoa (dHOS) after 10 and 30 minutes of incubation, the correlation between total HOS-reactive (tHOS) and viable HOS-reactive (vHOS) spermatozoa with other seminal parameters, and the possibility that dead spermatozoa react to HOS. We obtained the following results: [1] some dead spermatozoa swell under hypoosmotic conditions and [2] HOS-E results correlate strongly with other seminal parameters. We recommend that HOS be performed after 10 minutes of incubation because [1] the increase in the incubation time enhances the percentage of dHOS, [2] there are no differences in vHOS percentages between 10 and 30 minutes, and [3] correlation coefficients between vHOS and tHOS with other parameters are very similar at 10 or 30 minutes of incubation.