RESUMO
The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/µl and linear (LP) at 30 ng/µl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/µl (N = 105), 30 ng/µl (N = 95) and 300 ng/µl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/µl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/µl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.
Assuntos
Animais Geneticamente Modificados , Técnicas de Cultura Embrionária/veterinária , Proteínas de Fluorescência Verde/metabolismo , Óvulo/fisiologia , Injeções de Esperma Intracitoplásmicas/veterinária , Suínos/embriologia , Animais , Fragmentação do DNA , Proteínas de Fluorescência Verde/genética , Marcação In Situ das Extremidades Cortadas , Partenogênese , Plasmídeos , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
The female germ line in mammals is subjected to massive cell death that eliminates 60-85% of the germinal reserve by birth and continues from birth to adulthood until the exhaustion of the germinal pool. Germ cell demise occurs mainly through apoptosis by means of a biased expression in favour of pro-apoptotic members of the BCL2 gene family. By contrast, the South American plains vizcacha, Lagostomus maximus, exhibits sustained expression of the anti-apoptotic BCL2 gene throughout gestation and a low incidence of germ cell apoptosis. This led to the proposal that, in the absence of death mechanisms other than apoptosis, the female germ line should increase continuously from foetal life until after birth. In this study, we quantified all healthy germ cells and follicles in the ovaries of L. maximus from early foetal life to day 60 after birth using unbiased stereological methods and detected apoptosis by labelling with TUNEL assay. The healthy germ cell population increased continuously from early-developing ovary reaching a 50 times higher population number by the end of gestation. TUNEL-positive germ cells were <0.5% of the germ cell number, except at mid-gestation (3.62%). Mitotic proliferation, entrance into prophase I stage and primordial follicle formation occurred as overlapping processes from early pregnancy to birth. Germ cell number remained constant in early post-natal life, but a remnant population of non-follicular VASA- and PCNA-positive germ cells still persisted at post-natal day 60. L. maximus is the first mammal so far described in which female germ line develops in the absence of constitutive massive germ cell elimination.
Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Óvulo/citologia , Roedores , Animais , Apoptose , Contagem de Células , Feminino , Atresia Folicular , Expressão Gênica , Genes bcl-2/genética , Idade Gestacional , Marcação In Situ das Extremidades Cortadas , Folículo Ovariano/embriologia , Ovário/química , Gravidez , Antígeno Nuclear de Célula em Proliferação/análise , América do SulRESUMO
Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species
Assuntos
Animais , Feminino , Folículo Ovariano/ultraestrutura , Microscopia Confocal , Ovário/ultraestrutura , Células Germinativas/ultraestrutura , Roedores/crescimento & desenvolvimento , Folículo Ovariano/citologia , Ovário/citologiaRESUMO
Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species
Assuntos
Animais , Feminino , Folículo Ovariano/ultraestrutura , Microscopia Confocal , Ovário/ultraestrutura , Células Germinativas/ultraestrutura , Roedores/crescimento & desenvolvimento , Folículo Ovariano/citologia , Ovário/citologiaRESUMO
Apoptosis-dependent massive germ cell death is considered a constitutive trait of the developing mammalian ovary that eliminates 65-85% of the germinal tissue depending on the species. After birth and during adult lifetime, apoptotic activity moves from the germ cell proper to the somatic compartment, decimating germ cells through follicular atresia until the oocyte reserve is exhausted. In contrast, the South American rodent Lagostomus maximus shows suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation, which finally exhausts the oocyte pool. The absence of follicular atresia in adult L. maximus might arise from a failure to move apoptosis from the germinal stratum to the somatic compartment after birth or being a constitutive trait of the ovarian tissue with no massive germ cell degeneration in the developing ovary. We tested these possibilities by analysing oogenesis, expression of germ cell-specific VASA protein, apoptotic proteins BCL2 and BAX, and DNA fragmentation by TUNEL assay in the developing ovary of L. maximus. Immunolabelling for VASA revealed a massive and widespread colonisation of the ovary and proliferation of germ cells organised in nests that disappeared at late development when folliculogenesis began. No sign of germ cell attrition was found at any time point. BCL2 remained positive throughout oogenesis, whereas BAX was slightly detected in early development. TUNEL assay was conspicuously negative throughout the development. These results advocate for an unrestricted proliferation of germ cells, without apoptosis-driven elimination, as a constitutive trait of L. maximus ovary as opposed to what is normally found in the developing mammalian ovary.
Assuntos
Apoptose , Proliferação de Células , Oócitos , Oogênese , Ovário/embriologia , Roedores/embriologia , Animais , Western Blotting , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário , Feminino , Imunofluorescência , Atresia Folicular , Idade Gestacional , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Oócitos/metabolismo , Oócitos/patologia , Ovário/metabolismo , Ovário/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Roedores/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Allocreadium lobatum Wallin, 1909 has been reported in cyprinid species of freshwater fish in Canada and in the United States. The population biology of A. lobatum in the host Semotilus atromaculatus Mitchill was studied from May through December 1991, in a USA creek. Overall prevalence (64%) and mean intensity (4.4 +/- 0.4) were greater than previously reported while abundance, reported for the first time, was 2.8 +/- 0.3. Several trends in A. lobatum population biology as a function of S. atromaculatus length were identified. Mean intensity and abundance of A. lobatum increased with host size and significant differences in prevalence and A. lobatum lengths were found to correlate with host lengths.