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Understanding the regulation of cellular AA uptake as protein supply changes is critical for predicting milk component yields because intracellular supplies partly regulate protein synthesis. Our objective was to evaluate cellular uptake and kinetic behavior of individual AA when cells are presented with varying extracellular AA supplies. Bovine primary mammary epithelial cells were grown to confluency and transferred to medium with an AA profile and concentration similar to that of plasma from dairy cows for 24 h. Treatments were 4 AA concentrations, 0.36, 2.30, 4.28, and 6.24 mM, which represented 16, 100, 186, and 271% of typical plasma AA concentrations, respectively, in lactating dairy cows. Twenty-four plates of cells (89.4 × 19.2 mm) were assigned to each treatment. Cells were first subjected to treatment medium enriched with 15N-labeled AA for 24 h and then incubated with treatment medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Intracellular free AA, intracellular protein-bound AA, and extracellular medium free AA were analyzed for concentrations and isotopic enrichment using gas chromatography-mass spectrometry. A dynamic, 12-pool model was fitted to the data for 14 AA to derive unidirectional uptake and efflux, protein turnover, transamination, oxidation, and synthesis. The derived concentration for half the maximal uptake (km) indicated no saturation of AA uptake at typical in vivo concentrations for 11 of the 14 AA. Arginine, Pro, and Val appeared to exhibit saturation kinetics. Net uptake of all essential AA except Phe was positive across treatments. Most nonessential AA exhibited negative net uptake values. Efflux of AA was quite high, with several AA exhibiting greater than 100% efflux of the respective influx. Intracellular pool turnover was rapid for most AA (e.g., 2 min for Arg), demonstrating plasticity in matching needs for protein translation to supplies. Intracellular AA concentrations increased linearly in response to treatment for most AA, whereas 9 AA exhibited quadratic responses. Amino acid uptake is responsive to varying extracellular supplies to maintain homeostasis. No saturation of uptake was evident for most AA, indicating that transporter capacity is likely not a limitation for most AA except possibly Arg, Val, and Pro in mammary epithelial cells.

Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with 15N-labeled AA for 24 h followed by removal of the 15N-labeled medium and incubation with medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective 15N and 13C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.

We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal–Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA–Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2–99.8% viability indicated low cell death rates. Proliferation (range, 1.02–1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis. Published version

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, −F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.

The handmade cloning and parthenogenesis as a control leads to the possibility of studying biological processes such as cell reprogramming, epigenetics, embryo genome activation and pre- and post-natal development. The objective of this study was to standardize the process the handmade cloning (HCM) and to compare the resultant embryo production with parthenogenesis and in vitro fertilization (IVF). The primary fibroblast culture was established from explants of ear, the manual cloning technique was standardized with this fibroblast and 10 processes to cloning, parthenogenesis and fertilization in vitro were performed, and the compared production and embryo quality was performed by an ANOVA. An in vitro culture of fibroblast cells was established with optimal characteristics, allowing the genetic material to be used in the process of cloning. The parthenote group without zona pellucida (ZP) exhibited a higher cleavage rate compared with the other groups of parthenotes with ZP and IVF embryo (p

La leptina es una hormona proteínica que participa en procesos fisiológicos relacionados con la regulación del metabolismo energético, reproducción, inmunidad, cáncer, lactancia, entre otros. Su receptor se expresa en casi la totalidad de los tejidos, presentando seis isoformas a partir de un mismo ARNm. La isoforma predominante en la glándula mamaria bovina es la Ob-Rb, a través de la cual se puede desencadenar una cascada de señalización por medio de las moléculas JAK-STAT, para inducir proliferación, diferenciación y apoptosis celular de acuerdo al estadio reproductivo de la hembra. En pequeños rumiantes, tales como ovejas y cabras, los niveles de leptina y su receptor varían considerablemente durante la preñez y lactancia: son altos al inicio de la gestación, tienden a descender hasta la mitad de esta etapa y permanecen bajos hasta la finalización de la lactancia. En la glándula mamaria de bovinos aún no se ha establecido un patrón de expresión de los receptores durante diferentes etapas reproductivas, estudio que se considera importante y necesario para comprender el efecto de la leptina sobre su receptor en glándula mamaria, especialmente durante la lactancia, período durante el cual sufre una remodelación celular del tejido glandular para sostener una alta producción láctea. En la presente revisión se plantea una hipótesis de cómo podría ser la vía de señalización desencadenada por el receptor de leptina en la glándula mamaria durante la lactancia.A leptina é um hormônio protéico envolvido em processos fisiológicos relacionados com a regulação do metabolismo energético, reprodução, imunidade, câncer, lactação, entre outros. Seu receptor é expresso em quase todos os tecidos e apresenta seis isoformas a partir do mesmo mRNA. A isoforma predominante na glândula mamária bovina é a Ob-Rb, através da qual é desencadeada uma cascata de sinalização através das moléculas JAK-STAT para induzir a diferenciação, proliferação e apoptose de acordo com o estágio reprodutivo da fêmea. Em pequenos ruminantes, como ovinos e caprinos, os níveis de leptina e seu receptor variam consideravelmente durante a gravidez e a lactação. São altos no início da gravidez, tendem a cair na metade dessa fase e permanecem baixos até o final da lactação. Ainda não foi estabelecido um padrão de expressão do receptor de leptina na glândula mamária de bovinos durante diferentes estágios reprodutivos. O estudo deste tema é importante e necessário para entender o efeito da leptina sobre o seu receptor na glândula mamária, especialmente durante a lactação, periodo no qual a glândula mamária sofre remodelação do tecido glandular com a finalidade de sustentar a alta produção de leite. A presente revisão apresenta uma hipótese sobre a via de sinalização que é desencadeada pelo receptor da leptina na glândula mamária durante a lactação.Leptin is a hormone-like protein involved in physiological processes related to the regulation of energy metabolism, reproduction, immunity, cancer, breastfeeding, among others. Leptin receptor is expressed in almost all tissues and generates six isoforms from a single mRNA. The predominant isoform in bovine mammary gland is the Ob-Rb, which triggers a signaling cascade through JAK-STAT molecules to induce proliferation, differentiation and apoptosis, according to the female reproductive stage. In small ruminants, such as sheep and goats, leptin and its receptor levels vary widely, being high at the beginning of pregnancy, tending to fall in mid-gestation, and remaining low until the end of lactation. An expression pattern of leptin receptor has not yet been established for the various reproductive stages in bovine mammary gland. Such study is considered important and necessary to understand the effect of leptin via its receptor in mammary gland, especially during lactation when the gland undergoes tissue remodeling to sustain high milk production. This review presents a hypothesis about the signaling pathway triggered by leptin receptor in the mammary gland during bovine lactation.

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50-90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation (r = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro.

The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

Los niveles de la hormona leptina y la expresión de sus re-ceptores en glándula mamaria bovina parecen ejercer una función en el desarrollo del tejido secretor durante la puber-tad. Se ha relacionado la presencia de estos receptores con mamogénesis y con regulación de síntesis de ácidos gra-sos. Existen estudios de la hormona y su receptor durante la pubertad y en el período posparto. También, se conoce acerca de presencia de los transcriptos de la hormona y de su receptor en glándula mamaria, en vacas secas preñadas, así como en el período de lactancia. Así, el objetivo de este estudio fue estudiar la expresión (transcripto y proteína) de los receptores de leptina, isoformas Ob-ra y Ob-rb, en glán-dula mamaria de vacas Holstein, durante la lactancia vs. el periodo seco. Se confirmó, por medio de las técnicas de RT-PCR y de Western blot, que los receptores de leptina se expresan en glándula mamaria. Los resultados de la RT-PCR mostraron expresión de ambas isoformas (ARNm) en los dos periodos, sin diferencia estadística; sin embargo, la proteína de la isoforma Ob-ra fue identificada solo en el periodo seco, mientras que la de la isoforma Ob-rb fue encontrada durante la lactancia y en el periodo seco, sin diferencia estadística, al comparar los niveles de expresión. Los datos muestran un perfil de expresión diferente en las vacas Holstein del estudio, en comparación con lo reportado en la literatura para otros rumiantes no especializados, en la producción de leche. Leptin hormone levels and expression of its receptors in mammary gland may be important regulators of secretory tissue during puberty. Receptor expression has been related with mammogenesis and with fatty acids synthesis. There are studies about the hormone and its receptors during puberty and postpartum. Also, the transcripts of leptin and receptors have been described in dry pregnant cows’ mammary gland as well as during lactating period. The objective of this study was to determine the expression level of leptin receptors (transcripts and proteins), isoforms Ob-ra and Ob-rb, in Holstein cows during lactation vs. dry periods. The presence of leptin receptors in mammary gland was confirmed by RT-PCR and Western blot technics. However, the Ob-ra was identified only in no lactating cows while isoform Ob-rb was found in both lactating and dry periods without statistical difference when levels of expression were compared. RT-PCR results indicated mRNA expression of both isoforms receptors with no significant difference when two periods were compared. Data suggest a differential profile of expression of leptin receptors in Holstein cows of this study compared to reports in no specialized milk producer ruminants. Incluye referencias bibliográficas

The porcine leptin receptor complementary DNA was cloned and sequenced and the leptin receptor gene expression evaluated in the porcine ovary. An open reading frame of 3498 nt cDNA was amplified from pig liver mRNA by RT-PCR. Sequence homology with the extracellular, transmembrane, and cytoplasmic domains of human, mouse, rat, sheep, and cow leptin receptors varied between 45% and 90%. Leptin receptor mRNA was present in porcine kidney, liver, spleen, lung, brain, testis, uterus, ovary, corpus luteum (CL), theca, and granulosa cells. The abundance of leptin receptor transcripts and protein varied during luteinization of granulosa cells in vitro and in the CL during the pig luteal phase. In the postovulatory CL, both mRNA and protein were low but detectable, maximal expression was observed in the midcycle CL, and lowest abundance occurred in regressed CL. Leptin receptor mRNA was present in granulosa cells at isolation and increased in abundance as the cells luteinized over 96 hr in culture. Leptin receptor protein was detectable after 12 hr of in vitro luteinization. We conclude that leptin receptor is expressed in granulosa and luteal cells, and varies during pig ovarian cell differentiation.

In Antioquia-Colombia, the primary milk production is mainly performed at the northern “altiplano” region using both manual and automatic milking systems. The product quality will define its commercialization directly to consumers after pasteurization or for processing and derivates production. In both cases, the initial microbial contamination is crucial for its final use. Colombian regulations, specific for regions, define the allowed values of colony forming units (CFU), so the producers not only can sell their production, but also obtain important price bonus. The objective of the present work was to study some factors that can affect the bulk milk CFU counts in northern Antioquia dairy farms. The dependent variable was measured, described with an unvaried analysis and associated with a logistic regression. CFU, and independent variables as milking system, altitude, milking time in churn (minutes), distance to bulk (m) and cow number were measured, analyzed and associated with univariated and logistic regression statistics. Results indicated that the main risk factor in the CFU variation was the time period that the milk remained out of the cold chain. Other critical factors were the number of milked cows, since it increases the milking time; equally, altitude where farms are located, possibly related to temperature and bacterial proliferation. En el Departamento de Antioquia, Colombia, la producción primaria de leche bovina se desarrolla, en su mayoría, en la región del altiplano norte, con manejos de ordeño de tipos manual y mecánico. La calidad del producto define la comercialización del mismo, ya sea para consumo directo, luego de pasteurización o para su proceso y posterior producción de productos derivados. En ambos casos, la contaminación microbiana inicial, se constituye en factor definitivo para el destino de la leche producida. Una normativa específica por zonas para Colombia define los valores de unidades formadoras de colonias (UFC) permitidas, para que los productores puedan no sólo vender su producción sino, también, obtener bonificaciones importantes, al momento de la venta. El objetivo de este trabajo fue estudiar algunos factores que pueden afectar los conteos de UFC en tanque final de hatos lecheros del norte de Antioquia. Se midieron, se describieron con análisis univariado y se asociaron con regresión logística la variable dependiente: UFC en tanque y las variables independientes: tipo de ordeño, altitud en metros, tiempo de la leche en las cantinas (en minutos), distancia al tanque (en metros) y el número de vacas en ordeño. Se encontró que un punto crítico dentro de la rutina de ordeño fue el tiempo transcurrido desde el ordeño hasta llegada al tanque, sin alcanzar la cadena de frío. Otro factor crítico fue el número de vacas, quizás porque esto estaría aumentando el tiempo que dura el ordeño. Igualmente, la altitud, por su posible relación con la temperatura y la proliferación de bacterias. Incluye referencias bibliográficas

Gonadal Sex Steroids: Production, Action and Interactions in Mammals

Objetivos. Describir la presencia de receptores de leptina (OBR) en el testículo en la llegada de la pubertad en terneros y estudiar la posible asociación entre la expresión de éstos y los niveles de leptina (L) y de testosterona. Materiales y métodos. Se utilizaron 6 terneros Holstein x Cebú a los cuales se les midió quincenalmente y durante 7 meses concentraciones de testosterona y de L por RIA. Fueron castrados unilateralmente según períodos peripuberales para estudiar por RT-PCR la expresión de isoformas del receptor de L. El análisis estadístico se realizó con el programa Statistica®. Resultados. La testosterona presentó niveles desde menores a 1ng/mL a los 6 y 6.5 meses de edad, hasta concentraciones de 5.3 ng/ml a los 12.5 meses antes de la llegada a pubertad, momento en el cual los animales tuvieron niveles de 4.01±1.8 ng/ml. Las concentraciones de L variaron entre 0.61 y 0.98 ng/ml, con una concentración en pubertad de 0.91±0.05 ng/ml. Dos isoformas, OBRi y OBRb, fueron encontradas y se correlacionaron significativamente entre ellas en la pubertad. Se hallaron correlaciones negativas entre OBRi-OBRb y niveles de testosterona y de L (p=0.08). Los niveles de testosterona y de L mostraron una correlación directa significativa. Conclusiones. Se sugiere un posible efecto directo de la leptina en gónadas de terneros hasta la llegada a pubertad. La correlación entre las isoformas de OBR y su asociación con las concentraciones de leptina y testosterona también sugiere la acción complementaria o conjunta de ambos receptores OBR en testículos de terneros peripuberales.

Brycon henni, is a native fish from Western Colombia is important as food and in sport fishing, and could be cultured in waters between 18 and 28 degrees C. A previous cytogenetic study in branchial cells indicated different chromosomal complements. Total blood samples were seeded in 4 ml Ham-F12 culture medium, supplemented with 0.5 ml Bovine Fetal Serum and 0.7 ml Phyitohemaglutinin M (Gibco) during 96 hr at 28 degrees C; an antimitotic factor (100 microl Colcemid 10%) was added. Cells were incubated in 10 ml KCl hypotonic solution (0.56%) for 24 min at 37 degrees C, fixed in methanol; acetic acid (3: 1) three times, separated by centrifugations, extended drip, and the chromosomes slides were stained with 5% Giemsa (5%). The best metaphases (6 males and 8 females) were photographed. There was a diploid number of 50 chromosomes: Metacentrics (26M), Submetacentrics (16SM) and Subtelocentrics (8ST). The fundamental number is 100 arms. The evolutionary tendency is type B with no supernumerary chromosomes; a first pair of big metacentric chromosomes is characteristic for Brycon, while no chromosomic sexual heteromorfism was detected.

La sabaleta Brycon henni, es un pez nativo del occidente colombiano importante para la seguridad alimentaria de las poblaciones ribereñas y para la pesca deportiva por sus característica combativas. El desarrollarse entre los 18 y 28ºC la convierte en una especie de cultivo promisoria. Un estudio citogenético a partir de células branquiales indicó diferentes complementos cromosómicos. Este trabajo aplicó la técnica de cultivo de linfocitos a muestras de sangre de adultos. Las muestras de sangre total fueron sembradas en 4 ml de medio de cultivo Ham-F12, suplementado con 0.5 ml de Suero Fetal Bovino y 0.7 ml del mitógeno Fitohemaglutinina M (Gibco®) durante 96 hr a 28ºC; 1.5 hr antes de la cosecha agregamos 100µl de Colcemid al 10% como factor antimitótico. Cada cultivo se incubó con 10 ml de solución hipotónica KCl 0.56% por 24 min a 37ºC, para continuar con tres fijaciones sucesivas, centrifugaciones y tinción Giemsa al 5%. Las mejores metafases fueron fotografiadas en microscopio, correspondientes a 6 machos y 8 hembras, indicando un número diploide de 50 cromosomas, clasificados en Metacéntricos (26M), Submetacéntricos (16SM) y Subtelocéntricos (8ST), para un número fundamental (NF) de 100 brazos. La tendencia evolutiva hallada fue de tipo B; no se encontraron cromosomas supernumerarios pero sí un primer par de cromosomas metacéntricos grandes, considerado marcador para el género Brycon, no determinante de heteromorfismo sexual. Estos resultados coinciden con los demás bricónidos investigados, en donde se podría considerar un ancestro común con un número cromosómico básico y cariotipos simétricos.Cytogenetic characterization of the neotropical fish Brycon henni (Pisces: Characidae). Brycon henni, is a native fish from Western Colombia is important as food and in sport fishing, and could be cultured in waters between 18 and 28ºC. A previous cytogenetic study in branchial cells indicated different chromosomal complements. Total blood samples were seeded in 4 ml Ham-F12 culture medium, supplemented with 0.5 ml Bovine Fetal Serum and 0.7 ml Phyitohemaglutinin M (Gibco®) during 96 hr at 28ºC; an antimitotic factor (100µl Colcemid® 10%) was added. Cells were incubated in 10 ml KCl hypotonic solution (0.56%) for 24 min at 37ºC, fixed in methanol; acetic acid (3: 1) three times, separated by centrifugations, extended drip, and the chromosomes slides were stained with 5% Giemsa (5%). The best metaphases (6 males and 8 females) were photographed. There was a diploid number of 50 chromosomes: Metacentrics (26M), Submetacentrics (16SM) and Subtelocentrics (8ST). The fundamental number is 100 arms. The evolutionary tendency is type B with no supernumerary chromosomes; a first pair of big metacentric chromosomes is characteristic for Brycon, while no chromosomic sexual heteromorfism was detected. Rev. Biol. Trop. 56 (4): 1619-1628. Epub 2008 December 12.

Cells of the ovarian follicle undergo extensive proliferation and differentiation from the time that the follicle escapes from the primordial state to its acquisition of ovulatory capacity. We examined the dynamic modification of the phosphorylation state of the histone H3 N-terminal tail in granulosa cells during follicular development. In rodent follicles, the granulosa cell H3 phosphorylation on Ser10 peaks during proestrus. This epigenetic mark is induced by both FSH and 17β-estradiol (E2), acting independently. E2-induced H3 phosphorylation fails to occur in mice with inactivated α-isoform of the nuclear estrogen receptor. E2 induction of histone phosphorylation is attenuated by cell cycle inhibition. Further, E2 induces the activity of the mitotic kinase, Aurora B, in a mammary tumor cell model where mitosis is estrogen receptor-α dependent. These results provide evidence for mitotic regulation in follicle development by estrogen and demonstrate a previously undiscovered mechanism for induction of cell proliferation in ovarian and mammary gland cells.

The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

The pattern of follicular growth was studied in 17 suckled zebu cows with average body condition and under extensive management in a tropical environment (23 degrees C, 78% humidity; 2200 mm annual rainfall; 1000 m altitude). The study covered the period from parturition to weaning at 12 months postpartum (PP). Data were collected by transrectal ultrasonography (7.5 MHz) at 48 h intervals, and progesterone (P4) measurements were performed by RIA. The sequential development of ovarian follicles greater than 4 mm was followed until regression or ovulation. Ovarian activity as characterized by growth and regression of follicles of 4 to 6 mm, with sporadic dominance, and a long interdominance interval was observed in every cow and from as early as 26 +/- 2 days PP. This follicular pattern was highly variable during the first 6 months: cows presented 2 to 20 follicular waves (FW) in which a dominant follicle (DF) grew to 8 +/- 1 mm with daily growth rates of 1.1 +/- 0.5 mm/day. The duration of dominance varied from 2 to 8 days and the interdominance time interval was 0 (overlapped waves) to 60 days. Neither behavioural oestrus nor ovulation was observed during this period. From 6 to 12 months PP, cows presented 7 to 20 FW, some with ovulation and/or corpora lutea (CL) formation. The ovulation was preceded by oestrus in some cases (43%). The mean (+/- sem) diameter of DF was 9 +/- 2.7 mm, their mean growth rate 1.4 +/- 0.2 mm/day, their duration of dominance was 2 to 8 days and the interdominance interval was 0 to 14 days. Progesterone concentrations (P4) from 1.0 to 13 ng/ml were found when a CL was present. Once cyclicity re-commenced at 217 to 278 days PP, the cows presented either normal (21 +/- 3 days), short (10 +/- 2 days), or long (50 +/- 4 days) cycles. The resumption of cyclicity was characterized by an increased frequency of emerging follicular waves. Under the conditions of this study, the suckled Bos indicus cows re-commenced ovarian follicular activity as early as described in B. taurus breeds, but the establishment of cyclicity was substantially later. These data add further to the panorama of postpartum reproductive physiology in tropical cattle.

Sirtuin 2 (SIRT2) is a member of a family of NAD+-dependent histone deacetylases (HDAC) that play diverse roles in cellular metabolism and especially for aging process. SIRT2 is located in the nucleus, cytoplasm, and mitochondria, is highly expressed in the central nervous system (CNS), and has been reported to regulate a variety of processes including oxidative stress, genome integrity, and myelination. However, little is known about the role of SIRT2 in the nervous system specifically during aging. Here, we show that middle-aged, 13-month-old mice lacking SIRT2 exhibit locomotor dysfunction due to axonal degeneration, which was not present in young SIRT2 mice. In addition, these Sirt2−/− mice exhibit mitochondrial depletion resulting in energy failure, and redox dyshomeostasis. Our results provide a novel link between SIRT2 and physiological aging impacting the axonal compartment of the central nervous system, while supporting a major role for SIRT2 in orchestrating its metabolic regulation. This underscores the value of SIRT2 as a therapeutic target in the most prevalent neurodegenerative diseases that undergo with axonal degeneration associated with redox and energetic dyshomeostasis. This study was supported by grants from the Spanish Institute for Health Carlos III and ‘Fondo Europeo de Desarrollo Regional (FEDER), Union Europea, una manera de hacer Europa’ [FIS PI11/01043, FIS PI14/00410], the Autonomous Government of Catalonia [SGR 2014SGR1430] to A.P., The Spanish Institute for Health Carlos III and ‘Fondo Europeo de Desarrollo Regional (FEDER), Union Europea, una manera de hacer Europa’ [Miguel Servet program CP11/00080, CPII16/00016, FIS PI15/ 00857] to S.F. and the Center for Biomedical Research on Rare Diseases (CIBERER) to M.R., and the Spanish Institute for Health Carlos III and FEDER funds from European Union (‘A way to build Europe’) [FIS grants PI14/01115, PI13/00584, and PI14/00328], Foundation La Marató de TV3 [345/C/2014], and the Autonomous Government of Catalonia [2014SGR168] to M.J., M.P.O., and R.P. The studies conducted at the Chromatin Biology Laboratory were supported by the Spanish Ministry of Economy and Competitiveness-MINECO [SAF2011-25860, SAF2014-55964R] to A.V.