RESUMO
The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.
Assuntos
Progesterona/farmacologia , Progestinas/farmacologia , Receptores de Progesterona/efeitos dos fármacos , Toxocara canis/efeitos dos fármacos , Animais , Sequência de Bases , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Cães , Feminino , Citometria de Fluxo , Intestinos/parasitologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Camundongos , Microscopia Confocal , Movimento/efeitos dos fármacos , Reação em Cadeia da Polimerase , Receptores de Progesterona/análise , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Toxocara canis/crescimento & desenvolvimento , Toxocara canis/fisiologiaRESUMO
We evaluated the direct effects of progesterone on the morphology, maturation and behavior of Haemonchus contortus larvae in vitro. The presence and location of possible progesterone receptors in these larvae were also determined. The addition of 8ng/mL of progesterone to larval cultures over 10days reduced larval enlargement, while the addition of 160ng/mL of the hormone increased the enlargement. Up to 62% and 65% of the H. contortus larvae molted from third-stage larvae (L3) to fourth-stage larvae (L4) when cultured in RPMI-1640 media without hormone for 5 and 10days, respectively. The addition of different progesterone concentrations (1, 8, 16, 80 and 160ng/mL) to the larval cultures significantly inhibited the molting process within the same periods. The addition of 8ng/mL or higher progesterone concentrations to the cultures significantly increased larval motility (p<0.05) compared with unstimulated larvae. Flow cytometry showed the expression of progesterone receptors (P4-R) in 15% of the cells from newly isolated H. contortus larvae. When the larvae were cultured for 5days in the presence of the hormone, the percentage of P4-R+ cells remained the same. In contrast, unstimulated larvae showed a significant reduction in the number of P4-R+ cells. Using confocal microscopy, a greater concentration of P4-Rs was immunolocated in the anterior portion of the alimentary tract of the larvae, suggesting that the cells in this region are targeted by the hormone. The results of the present study show that H. contortus larvae have possible P4-Rs and respond to this hormone by inhibiting their molting process, thereby suggesting the participation of progesterone in the larval arrest phenomenon.
Assuntos
Haemonchus/efeitos dos fármacos , Progesterona/administração & dosagem , Progestinas/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Haemonchus/genética , Haemonchus/crescimento & desenvolvimento , Haemonchus/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Muda/efeitos dos fármacos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
The in vitro effect of prolactin (PRL) on the growth and motility of Toxocara canis larvae was assessed. Additionally, the expression and location of prolactin receptors (PRL-Rs) were determined in the larvae. Larvae of T. canis were incubated with different concentrations of PRL for different periods of time. The stimulated larvae accelerated their enlargement and increased their motility. The mean percentage of PRL-R+ cells in non-stimulated larvae, measured by flow cytometry was 7.3±0.3%. Compared with non-stimulated larvae, the mean fluorescence intensity (p<0.05) increased in larvae incubated with 40ng/mL of PRL for 10 days. A 465-bp length fragment was amplified from larvae gDNA by PCR. The sequence of this fragment showed 99% similarity with the gene fragment that codes for the PRL-R of the domestic dog. A high concentration of PRL-Rs was immune-located in the posterior region of the larval intestine; therefore, the intestinal cells in this region were most likely the targets for this hormone. Based on these results, PRL-Rs were identified in T. canis larvae, and the in vitro stimulation with PRL increased the number of these receptors, accelerated the growth and modified the activity of larvae. All of the above suggest that T. canis larvae are evolutionarily adapted to recognize the PRL of their definitive host and furthermore might explain the reactivation of tissue-arrested larvae during the gestation of bitches, which does not occur in gestating females of other species.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Prolactina/farmacologia , Receptores da Prolactina/metabolismo , Toxocara canis/efeitos dos fármacos , Toxocara canis/fisiologia , Toxocaríase/parasitologia , Animais , Hormônios/farmacologia , Técnicas In Vitro , Larva , Toxocara canis/genética , Toxocara canis/crescimento & desenvolvimentoRESUMO
Summary The objective of the present study was to evaluate the viability of frozen embryos obtained from various private farmers in a culture medium for 4 h. Forty-seven embryos were used that had been previously graded as good and fair. These embryos were evaluated using stereoscopic microscopy by experienced clinicians prior to freezing. Embryos were divided in two groups: the non-cultured group, made up of six good quality embryos, and five fair; and the cultured group that consisted of 20 good quality embryos and 16 fair. Fifty-four per cent of the good quality embryos achieved a favourable development during culture whereas just 42% of embryos determined to be fair were observed to have adequate development. This evaluation was undertaken by serial photographs obtained at the onset of culture and 4 h later. This finding was corroborated by a more specific technique: terminal deoxynucleotide transferase dUTP nick end labelling-bromodeoxyuridine (TUNEL-BrdU). These results are indicative of the necessity of tight quality controls for commercially produced frozen embryos, as once thawed it is unlikely that clinicians will examine them to determine their physiological status prior to transfer.
Assuntos
Apoptose , Blastocisto/citologia , Proliferação de Células , Criopreservação , Meios de Cultura/química , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia EletrônicaRESUMO
This study describes the structural and ultrastructural characteristics of gonadal sex differentiation and expression of Vasa, a germline marker, in different developmental stages of embryos and newborn fry of the barred splitfin Chapalichthys encaustus, a viviparous freshwater teleost endemic to Mexico. In stage 2 embryos, the gonadal crest was established; gonadal primordia were located on the coelomic epithelium, formed by scarce germ and somatic cells. At stage 3, the undifferentiated gonad appeared suspended from the mesentery of the developing swimbladder and contained a larger number of germ and somatic cells. At stages 4 and 5, the gonads had groups of meiotic and non-meiotic germ cells surrounded by somatic cells; meiosis was evident from the presence of synaptonemal complexes. These stages constituted a transition towards differentiation. At stage 6 and at birth, the gonad was morphologically differentiated into an ovary or a testis. Ovarian differentiation was revealed by the presence of follicles containing meiotic oocytes, and testicular differentiation by the development of testicular lobules containing spermatogonia in mitotic arrest, surrounded by Sertoli cells. Nuage, electron-dense material associated with mitochondria, was observed in germ cells at all gonadal stages. The Vasa protein was detected in all of the previously described stages within the germ-cell cytoplasm. This is the first report on morphological characteristics and expression of the Vasa gene during sexual differentiation in viviparous species of the Goodeidae family. Chapalichthys encaustus may serve as a model to study processes of sexual differentiation in viviparous fishes and teleosts.
Assuntos
Ciprinodontiformes/embriologia , Morfogênese , Processos de Determinação Sexual , Animais , Biomarcadores/metabolismo , Ciprinodontiformes/anatomia & histologia , Ciprinodontiformes/fisiologia , Embrião não Mamífero/anatomia & histologia , Desenvolvimento Embrionário , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Masculino , Caracteres SexuaisRESUMO
In sheep embryos, steroidogenic activity has been reported as taking place during the period of sexual differentiation. In the case of mouse embryos, the sporadic detection or absence of steroidogenic enzymes suggests that the ovary is inactive. The purpose of this work was to establish if mouse undifferentiated gonads express steroidogenic enzymes in a similar way as in sheep embryos. To know this, we analyzed the mRNA expression pattern of 3ß-Hsd1 and P450arom as well as protein expression pattern of 3ß-HSD1 and Testosterone in normal undifferentiated and differentiated gonads from both male and female mice embryo. Our data indicate that there is expression of 3ß-Hsd1 in XX gonads during gonad differentiation period. Nevertheless the Testosterone which would indicate steroidogenic activity is not produced. Besides, the absence of P450arom indicates that the production of Estradiol as observed in the ovaries of sheep does not occur. The detection of 3ß-Hsd1 in the early stages of ovarian development, as well as the absence of Testosterone suggests that XX gonads are not steroidogenic and that 3ß-Hsd1 enzyme may play a different role than in the steroidogenesis process.
Assuntos
3-Hidroxiesteroide Desidrogenases/biossíntese , Aromatase/biossíntese , Ovário/enzimologia , Diferenciação Sexual/fisiologia , Testículo/enzimologia , 3-Hidroxiesteroide Desidrogenases/genética , Animais , Aromatase/genética , Estradiol/biossíntese , Estradiol/genética , Estradiol/metabolismo , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Ovário/citologia , Ovário/crescimento & desenvolvimento , RNA Mensageiro/genética , Diferenciação Sexual/genética , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testosterona/biossíntese , Testosterona/genética , Testosterona/metabolismoRESUMO
The aim of the present study was to evaluate a culture system as a non-invasive approach intended for assessing the viability of recently thawed embryos prior to transfer. Embryos (n=51) were collected seven days after insemination out of 20 cows that had been treated to synchronize estrus and induce superovulation. Embryos were classified as good, fair, and poor and frozen. All embryos were cultured in McCoy medium. Morphology was monitored for a period of 24h to register the development stage every 30 min for the first 2h, and every hour thereafter. A sample of four embryos of each classification was separated at 4h, another four at 12h, and the remaining seven at 24h and the degree of apoptosis was determined for all the embryos using the TUNEL technique. Embryos of good and fair quality did not undergo major detrimental changes in development even after 7h of incubation, whereas poor quality embryos experienced changes as early as 2h after incubation. Good quality embryos invariably had fewer numbers of apoptotic cells than those of fair and poor quality suggesting that embryo culture can be a useful method to assess viability and to confirm the quality of thawed embryos previously stored in liquid nitrogen prior to transfer.
Assuntos
Criopreservação , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos , Animais , Apoptose/fisiologia , Bovinos , Contagem de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Feminino , Congelamento , Gravidez , Controle de Qualidade , Superovulação/fisiologia , Fatores de TempoRESUMO
The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, Mexico. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual-purpose cattle farmers. Forty-three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t-test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel-positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.
Assuntos
Bovinos/embriologia , Criopreservação/veterinária , Embrião de Mamíferos/fisiologia , Preservação de Tecido/veterinária , Animais , Criopreservação/métodos , Transferência Embrionária/veterinária , Embrião de Mamíferos/ultraestrutura , Feminino , Marcação In Situ das Extremidades Cortadas/veterinária , Gravidez , Preservação de Tecido/métodosRESUMO
SOX9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that SRY, the testis determining gene, turns SOX9 on in male embryos because it is turned off in female embryos. Spatial expression of SRY follows a cranio-caudal pattern. Here, we asked if SOX9 is expressed in the same cell lineage and with a similar pattern as SRY. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of SOX9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first SOX9-positive cells appear. Although SOX9-positive cells are located among loose mesenchymal cells by stages of 8-14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first SOX9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of SOX9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that SOX9 is expressed only in loose mesenchymal cells in both sexes and that expression of SOX9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of SOX9 and SRY pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges.
Assuntos
Genitália/citologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Células de Sertoli/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Agregação Celular , Linhagem da Célula , Feminino , Genitália/embriologia , Genitália/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOX9 , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Sex determination is controlled either by genetic or environmental factors. In mammals Sry initiates determination but no homologue of this gene exists in non-mammalian species. Other genes of the mammalian sex-determining pathway have been identified in gonads of different vertebrates. Sox9, Dax1, and Dmrt1 are expressed at the onset of gonadal development in birds and reptiles. In the sea turtle Lepidochelys olivacea, a species with temperature sex determination (TSD), Sox9 is expressed in undifferentiated gonads at male- (MPT) or female-promoting temperatures (FPT). At MPT, Sox9 remains expressed in male gonads, but at FPT it is downregulated coinciding with the onset of the ovarian morphologic differentiation and female sex determination. At MPT however, male sex is determined early than at FPT in still undifferentiated gonads suggesting that other genes maintain Sox9 expression in testis. Here we used RT-PCR to study the expression profiles of Dax1, Dmrt1, and Sox9 in gonads of embryos of L. olivacea incubated at MPT or at FPT. The profiles were correlated with sex determination during and after the temperature-sensitive period (TSP). Dax1 maintained similar levels at both temperatures during the TSP. The Dax1 expression level increased significantly in ovaries compared to testes at stage 27, once they were morphologically distinct. The expression levels of Dmrt1 were higher at MPT than at FPT at all stages, in contrast with Sox9 levels which were similar at both temperatures at stages 23-25. Together, current results suggest that, whereas Dax1 is not involved in TSD in L. olivacea, upregulation of Dmrt1 and downregulation of Sox9 may play a role in male and female sex determination, respectively.