RESUMO
Guanosine 3',5'-cyclic monophosphate (cGMP), as a second messenger, plays potential roles in ovarian functions. To elucidate the role of phosphodiesterase (PDE) in cGMP signaling during ovarian follicular development, the present study was conducted to investigate ovarian cGMP level and cGMP-PDE activity by radioimmunoassay (RIA) in postnatal rats, immature rats during gonadotropin-primed follicular development, ovulation and luteinization, adult rats during normal estrous cycle, and aged rats that spontaneously developed persistent estrus (PE). All four rat models were confirmed by histological examination of one ovary, and the other ovary was used for RIA. In postnatal rats, cGMP level was high at birth and decreased dramatically by Day 5, and then, it increased maximally at Day 10 and declined at Day 21. However, cGMP-PDE activity did not significantly change during Days 1 to 10, but increased significantly on Day 21. In immature female rats, cGMP level markedly decreased upon treatment with equine chorionic gonadotropin (eCG), while cGMP-PDE activity did not show any significant changes; however, ovarian cGMP level and cGMP-PDE activity increased after injection of an ovulatory dose of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization. In adult rats during normal estrous cycle, cGMP level was high on proestrus and metestrus days, while cGMP-PDE activity was high on estrus day. In PE rats, ovarian cGMP level was similar to that in adult rats on estrus and diestrus days but lower than that on proestrus and metestrus days; ovarian cGMP-PDE activity was lower than that on estrus days but similar as the other estrous cycle days. In addition, there was a significant negative correlation between ovarian cGMP level and cGMP-PDE activity during normal estrous cycles in the adult rat (r = -0.7715, N = 16, P < 0.05), but not in the postnatal rat (r = -0.1055, N = 20, P > 0.05). Together, the results of our present study indicated that ovarian cGMP levels were not dependent on cGMP-PDE activity during early postnatal development, but highly dependent on cGMP-PDE activity in the adult rat. This implies that mechanisms of cGMP signaling involved in ovarian functions are stage-specific in the rat.
Assuntos
GMP Cíclico/metabolismo , Folículo Ovariano/fisiologia , Ovário/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Ativação Enzimática , Ciclo Estral , Feminino , Gonadotropinas/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Ovário/citologia , Ovário/efeitos dos fármacos , RatosRESUMO
DNA vaccination has been studied intensively as a potential vaccine technology. We evaluated the effect of an attenuated Salmonella choleraesuis-mediated inhibin DNA vaccine in rats. First, 15 rats were treated with different doses of an inhibin vaccine to evaluate vaccine safety. Next, 30 rats were divided into 3 groups and injected intramuscularly with the inhibin vaccine two (T1) or three times (T2) or with control bacteria (Con) at 4-week intervals. The inhibin antibody levels increased [positive/negative well (P/N) value: T1 vs Con = 2.39 ± 0.01 vs 1.08 ± 0.1; T2 vs Con = 2.36 ± 0.1 vs 1.08 ± 0.1, P < 0.05] at week 2 and were maintained at a high level in T1 and T2 until week 8, although a small decrease in T2 was observed at week 10. Rats in the T1 group showed more corpora lutea compared with the Con group (10.50 ± 0.87 vs 7.4 ± 0.51, P < 0.05). Estradiol (0.439 ± 0.052 vs 0.719 ± 0.063 ng/mL, P < 0.05) and progesterone (1.315 ± 0.2 vs 0.737 ± 0.11 ng/mL, P < 0.05) levels differed significantly at metestrus after week 10 between rats in the T1 and Con groups. However, there were no significant differences in body, ovary, uterus weights, or pathological signs in the ovaries after immunization, indicating that this vaccine is safe. In conclusion, the attenuated S. choleraesuis-mediated inhibin vaccine may be an alternative to naked inhibin plasmids for stimulating ovarian follicular development to increase the ovulation rate in rats.
Assuntos
Inibinas/genética , Inibinas/imunologia , Salmonella/genética , Vacinas Atenuadas/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Estradiol/sangue , Feminino , Imunização , Folículo Ovariano/imunologia , Folículo Ovariano/patologia , Ovulação , Progesterona/sangue , Ratos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversosRESUMO
The guinea pig is an excellent animal model for studying reproductive biology of adult humans and most domestic animals. Yet, whether this animal might serve as a good model for embryonic stage investigations and determinations of signals affecting or directing ovary development remains unknown. These questions were addressed by examining morphological evolution and the expression of biomarkers of cell proliferation and apoptosis in the ovaries of fetal and neonatal guinea pigs in the present study. Embryonic and neonatal guinea pigs at 30, 40, 50, 60, and 68 days postcoitum (dpc) and at 1 day postpartum (dpp) were evaluated, and the dynamic changes in follicles between 30 dpc and 1 dpp were observed. Results also showed that a critical period of follicular development in guinea pig embryos occurred at 40 to 50 dpc. Moreover, the proliferating-cell nuclear antigen, a cell proliferation marker, immunohistochemically stained healthy follicles, while caspase-3, an apoptosis marker, was mainly observed in atretic follicles. Together, these results demonstrate that cell proliferation and apoptosis contribute to follicular formation, development, and atresia in fetal and neonatal guinea pig ovaries. Furthermore, this study confirmed that the guinea pig is also an excellent animal model for studying reproductive biology in human and domestic animal embryos.
Assuntos
Apoptose/genética , Proliferação de Células , Desenvolvimento Embrionário/genética , Ovário/crescimento & desenvolvimento , Animais , Feminino , Feto/metabolismo , Cobaias , Humanos , Ovário/metabolismoRESUMO
Several studies have documented the process of early embryonic development in poultry; however, the molecular mechanisms underlying its developmental regulation are poorly understood, particularly in ducks. In this study, we analyzed differential gene expression of embryos 6 and 25 h following oviposition to determine which genes regulate the early developmental stage in ducks. Among 216 randomly selected clones, 39 protein-encoding cDNAs that function in metabolism, transcription, transportation, proliferation/apoptosis, cell cycle, cell adhesion, and methylation were identified. Additionally, the full-length cDNA of the Nanog gene, encoding a 302-amino acid protein, was obtained. Quantitative real-time polymerase chain reaction analyses were performed to detect expression levels of the selected genes during early and late embryonic stages, which revealed that these genes are expressed in a particular spatial and temporal pattern. These results indicate that these genes may play pivotal roles in the process of area pellucida formation through a complex and precise regulatory network during development in duck embryos.
Assuntos
Patos/genética , Biblioteca Gênica , Sequência de Aminoácidos , Animais , Patos/embriologia , Patos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Dados de Sequência MolecularRESUMO
The signal transducer and activator of transcription (STAT) genes are responsive to a wide range of cytokines, growth factors, and hormones, and thus control important biological processes. In humans, STAT4 mutations have been identified as genetic markers for rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren's syndrome, whereas little research has been conducted on bovine STAT4 mutations and their potential effects. Herein, 585 Chinese Holstein cows were used to investigate STAT4 mutations and their effects on milk performance traits. One haplotype block, containing g.95879G>A, g.96013G>C, was identified in intron 20 of the bovine STAT4 gene by restriction fragment length polymorphism-polymerase chain reaction and DNA sequencing. Two single nucleotide polymorphisms were significantly associated with milk yield at 305 days (P < 0.05), and with protein percentage (P < 0.05). Chinese Holstein cows with the haplotype GGGG had higher milk yields at 305 days and lower protein percentages. These results suggest that the 2 single nucleotide polymorphisms of STAT4 could be used as genetic markers for milk performance traits in Chinese Holstein cows.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Íntrons/genética , Leite/metabolismo , Polimorfismo Genético , Característica Quantitativa Herdável , Fator de Transcrição STAT4/genética , Animais , Bovinos , China , Feminino , Análise dos Mínimos Quadrados , Desequilíbrio de Ligação/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The insulin growth factor 1/phosphatase and tensin homologue deleted on chromosome 10/Akt/forkhead box (IGF-1/PTEN/Akt/FoxO) signaling pathway reportedly exhibits gastroprotective effects by reducing water immersion and restraint stress (WRS)-induced gastric mucosal cell apoptosis. We examined the expression and localization of IGF-1, PTEN, Akt, and FoxO proteins, caspase-3 activity, and the number of apoptotic cells in the duodenal mucosa of rats subjected to WRS to confirm whether the IGF-1/PTEN/Akt/FoxO signaling pathway has a role in the duodenal mucosa. The results indicated that WRS enhanced cell apoptosis in the duodenal mucosa. In addition, in normal rats, PTEN was found mainly in the cellular cytoplasm of the duodenal glands and lamina propria of villi. IGF-1 and total Akt were observed in the cellular cytoplasm of the duodenal glands. In addition, total Akt was found in the cellular cytoplasm of the myenteric plexus. FoxO3a and FoxO4 were primarily concentrated in the cellular cytoplasm of the lamina propria. Specifically, PTEN, FoxO3a and FoxO4 were also localized in the cellular cytoplasm of lamina propria of restituted villi in the duodenal mucosa of rat subjected to WRS. In addition, messenger RNA transcript levels of IGF-1, PTEN, Akt1, Akt2, FoxO3, and FoxO4 were upregulated in the duodenal mucosa, with a peak between the 4th and 8th day after 7 h of WRS. Furthermore, the results also suggested that Akt3 messenger RNA transcript levels in the duodenal mucosa of rats after WRS showed no significant differences compared with those in the non-WRS group. Collectively, our results implied that the IGF-1/ PTEN/Akt/FoxO signaling pathway was effective in regulating cellular apoptosis in the duodenal mucosa of rats after WRS.
Assuntos
Apoptose , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , Transdução de Sinais , Animais , Caspase 3/metabolismo , Citoplasma/metabolismo , Duodeno/patologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Imersão , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Microvilosidades/metabolismo , Especificidade de Órgãos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Restrição Física , Estresse Psicológico/metabolismoRESUMO
The melanocortin-4 receptor (MC4R) has important roles in regulating food intake, energy balance, and body weight in mammals. In pigs and cattle, MC4R mutations have been identified as genetic markers for growth and traits. Compared with abundant research conducted on other livestock species, little is known about mutations of the ovine MC4R gene. We investigated the effect of MC4R polymorphisms on birth weight and on 45-day weaning weight in 144 Hu sheep. Four single nucleotide polymorphisms (SNPs; g.1016 G/A, g.1240 T/C, g.1264 G/A, and g.1325 A/G) were identified in the 3ê-untranslated region of Hu sheep MC4R by PCR-single-strand conformation polymorphism and DNA sequencing. A haplotype block, containing g.1240 T/C, g.1264 G/A, and g.1325 A/G, was constructed within the Hu sheep MC4R gene. Four SNPs were found to be significantly associated with 45-day weaning weight, while the haplotype block was significantly associated with birth weight. Hu sheep with the genotypes GG in g.1016 G/A or with the genotype CCAAGG in the haplotype block, had higher 45-day weaning weights. We conclude that these 4 SNPs of the MC4R gene have potential as genetic markers for early growth traits in Hu sheep.
Assuntos
Peso ao Nascer/genética , Polimorfismo de Nucleotídeo Único , Receptor Tipo 4 de Melanocortina/genética , Carneiro Doméstico/genética , Animais , Sequência de Bases , Frequência do Gene , Marcadores Genéticos , Genótipo , Desequilíbrio de Ligação , Dados de Sequência Molecular , Análise de Sequência de DNA , Carneiro Doméstico/crescimento & desenvolvimento , DesmameRESUMO
The complement system helps in the direct lysis of invading pathogens and modulates phagocytic, humoral and cellular immune responses. Complement 4 is a critical component in complement activity and protection against many bacterial pathogens because it is essential to classical and lectin activation pathways. We used reverse transcription and PCR to investigate alternative splicing and expression of the complement component 4 (C4A) gene in Chinese Holstein cattle. The PCR products were cloned and sequenced. A novel splice variant involving intron 10 was identified, which we named C4A-AS. To examine how C4A gene activity is affected by bovine mastitis, six Chinese Holstein cattle were divided into healthy (non-mastitic) and Staphylococcus aureus-induced mastitic groups. Real-time quantitative PCR (qRT-PCR) revealed that the C4A-complete and C4A-AS transcripts are expressed at significantly different levels in healthy cows, while there were no significant differences in the mastitic group (P = 0.257). Expression of C4A-AS increased significantly when mastitis developed. We also examined the expression of C4A-complete and C4A-AS in several tissues (liver, heart, spleen, lung, kidney, tongue, and muscle). The two transcripts were expressed in all of these tissues but there were no significant differences in expression between healthy and mastitic cows. We therefore conclude that the C4A-complete transcript is the main transcript under normal physiological conditions, while C4A-AS is augmented when mastitis develops.