RESUMO

Abstract To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family.

Resumo Investigar a concentração ideal de andrógenos em cultura de folículos pilosos de carneiro Hetiano e detectar os efeitos da concentração de andrógenos na proliferação e apoptose de células foliculares, por meio de imunofluorescência e de determinação quantitativa, em tempo real, da fluorescência dos níveis de expressão gênica de proteína associada à queratina. Folículos pilosos foram isolados por microdissecção, e folículos de lã e pedaços de pele foram cultivados em várias concentrações de di-hidrotestosterona (DHT) em meio de cultura. Em seguida, medições diárias de crescimento longitudinal dos folículos capilares foram obtidas usando um micrômetro microscópico. Folículos de lã cultivados de Hetianos foram corados pelo método SACPIC para revelar a estrutura do folículo piloso, enquanto fatias de pele de carneiro foram usadas para observar a proliferação celular por imunocoloração e apoptose celular por meio do método TUNEL. Em âmbito da biologia molecular, a expressão gênica da proteína associada à queratina (Kap) foi estudada usando folículos capilares cultivados por vários dias, in vitro. Os efeitos das concentrações de andrógenos no crescimento e desenvolvimento dos folículos de lã de Hetianos foram estudados experimentalmente. Ensaios de proliferação de EdU revelaram que o andrógeno promoveu a proliferação celular dentro das papilas dérmicas do folículo piloso. A detecção de apoptose por TUNEL demonstrou que o tratamento com andrógeno poderia atrasar a apoptose celular. Os resultados da reação em cadeia da polimerase transcrição reversa quantitativa (qPCR) demonstraram que os padrões de expressão gênica da proteína de enxofre Kap1.1, KIF1.2, Kap2.12 e Kap4.2 foram significativamente maiores no grupo de ovinos Hetianos de montanha. Uma concentração de androgênio de 100 nM pode promover o crescimento de células foliculares de lã de Hetianos in vitro, resultando na superexpressão de alguns genes da família Kap.

Assuntos

RESUMO

To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family.

Assuntos

RESUMO

To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family.(AU)

Investigar a concentração ideal de andrógenos em cultura de folículos pilosos de carneiro Hetiano e detectar os efeitos da concentração de andrógenos na proliferação e apoptose de células foliculares, por meio de imunofluorescência e de determinação quantitativa, em tempo real, da fluorescência dos níveis de expressão gênica de proteína associada à queratina. Folículos pilosos foram isolados por microdissecção, e folículos de lã e pedaços de pele foram cultivados em várias concentrações de di-hidrotestosterona (DHT) em meio de cultura. Em seguida, medições diárias de crescimento longitudinal dos folículos capilares foram obtidas usando um micrômetro microscópico. Folículos de lã cultivados de Hetianos foram corados pelo método SACPIC para revelar a estrutura do folículo piloso, enquanto fatias de pele de carneiro foram usadas para observar a proliferação celular por imunocoloração e apoptose celular por meio do método TUNEL. Em âmbito da biologia molecular, a expressão gênica da proteína associada à queratina (Kap) foi estudada usando folículos capilares cultivados por vários dias, in vitro. Os efeitos das concentrações de andrógenos no crescimento e desenvolvimento dos folículos de lã de Hetianos foram estudados experimentalmente. Ensaios de proliferação de EdU revelaram que o andrógeno promoveu a proliferação celular dentro das papilas dérmicas do folículo piloso. A detecção de apoptose por TUNEL demonstrou que o tratamento com andrógeno poderia atrasar a apoptose celular. Os resultados da reação em cadeia da polimerase transcrição reversa quantitativa (qPCR) demonstraram que os padrões de expressão gênica da proteína de enxofre Kap1.1, KIF1.2, Kap2.12 e Kap4.2 foram significativamente maiores no grupo de ovinos Hetianos de montanha. Uma concentração de androgênio de 100 nM pode promover o crescimento de células foliculares de lã de Hetianos in vitro, resultando na superexpressão de alguns genes da família Kap.(AU)

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RESUMO

Non-viable eggs of Morelet's crocodile (Crocodylus moreletii) were collected from Gold Button (GBL) and New River lagoons (NRL) in northern Belize and screened for organochlorine (OC) compounds using gas chromatography (GC) with electron capture detection (ECD). All egg samples from both lagoons (n = 24) tested positive for one or more OCs. Primary contaminants were p,p-DDE and methoxychlor, detected in 100% and 29% of the eggs examined, respectively. Concentrations of individual OC contaminants ranged from 1 ppb (ng chemical/g egg) to > 0.5 ppm (microgram chemical/g egg). Total concentrations of OCs (sum of all OCs) for one egg collected from a nest at GBL reached as high as 0.7 ppm. Sediment samples from both lagoons also tested positive for OCs (lindane, aldrin, methoxychlor, heptachlor epoxide, p,p-DDT, among others). Nest media (soil and plant material) collected from crocodile nests at GBL were positive for p,p-DDT, methoxychlor, aldrin, endosulfan II, and endrin aldehyde. Based on the 24 egg samples analyzed to date, crocodiles from both lagoons are being exposed to OCs. Such exposure may present a health threat to populations of crocodiles in Central America.

Assuntos

RESUMO

Non-viable eggs of Morelet's crocodile (Crocodylus moreletti) were collected from Gold Botton (GBL) and New River lagoons (NRL) in northern Belize and screened for organochlorine (OC) compounds using gas chromatography (GC) with electron capture detection (ECD). All egg samples from both lagoons (n=24) tested positive for one or more OCs. Primary contaminants were p,p-DDE and methoxychlor, detected in 100 percent and 29 percent of the eggs examined, respectively. Concentrations of individual OC contaminants ranged from 1 ppb (ng chemical/g egg). Total concentrations of OCs (sum of all OCs) for one egg collected from a nest at GBL reached as high as 0.7 ppm. Sediment samples from both lagoons also tested positive for OCs (lindane, aldrin, methoxychlor, heptachlor epoxide, p, p-DDT, among others). Nest media (soil and plant material) collected from crocodile nests at GBL were positive for p, p-DDT, methoxychlor, aldrin, endosulfan II, and endrin aldehyde. Based on the 24 egg samples analyzed to date, crocodiles from both lagoons are being exposed to OCs. Such exposure may present a health threat to populations of crocodiles in Central America. (AU)

Assuntos

RESUMO

Organochlorine (OC) residues were recently detected in nonviable Morelet's crocodile (Crocodylus moreletii) eggs from northern Belize. To further the assessment of contaminant exposure in Belizean crocodiles, nonviable Morelet's crocodile eggs (n = 11) from southern Belize and American crocodile (Crocodylus acutus) eggs (n = 12) from the coastal zones of Belize were screened for 20 OCs. Results indicated p,p-DDE to be the most prevalent OC (96% occurrence) in eggs examined, with concentrations ranging from 5 to 372 ng/g. These concentrations are similar to those observed in crocodile eggs (10-180 ng/g) from northern Belize. A general trend toward higher DDE concentrations in Morelet's crocodile eggs (mean = 103 ppb) compared with American crocodile eggs (mean = 31 ppb) was observed. However, this trend may be due to site-specific contamination rather than differences in interspecific susceptibility to chemical exposure. Other OCs detected in crocodile eggs included the parent compound, p,p-DDT, and its metabolite, p, p-DDD.

Assuntos