M. A. Miglino

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.

Quantificação de células dos túbulos seminíferos e rendimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros

O presente estudo teve como objetivo avaliar o rendimento da espermatogenese de cutias criadas em cativeiro, por intermedio das razoes encontradas entre tipos celulares do epitelio seminifero. Os resultados apontaram que o rendimento da espermatogenese da cutia dos nove aos quatorze meses de idade nao chegou a um ponto de estabilizacao. O coeficiente de eficiencia de mitoses espermatogoniais nao aumentou com a idade. O rendimento meiotico, o rendimento geral da espermatogenese e o indice de celulas de Sertoli mostraram variacoes numericas em funcao da idade, entretanto, nao detectadas estatisticamente.

Canine Fibroblasts Expressing Human Transcription Factors: What is in the Route for the Production of Canine Induced Pluripotent Stem Cells

The aim of this study was to further clarify the mechanisms involved in inducing pluripotency using canine foetal fibroblast cells. The two pluripotency-related transcription factors, OCT4 and SOX2, coupled to a fluorescent reporter gene were transduced, individually or in combination, using a lentiviral system. Stable transgenic cell lineages were obtained and canine cells showed to be highly responsive to the integration and expression of human SOX2 and OCT4, also depending on the amount of virus used for incubation. Such positive results are essential for the establishment of pluripotency induction through the incorporation of known transcription factors into the genome of somatic cells.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryos initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring α-fetoprotein, α-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-α-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-α-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Bovine yolk sac: from morphology to metabolomic and proteomic profiles

In several species, placentation involves the presence of two different membranes responsible for maternal-fetal exchanges: the yolk sac and the chorioallantoic placenta. The yolk sac plays important roles in embryonic survival, mainly during the early stages of gestation. In bovine, it is a transitional membrane that is present until day 50-70 of pregnancy. Herein, we evaluated the morphological and molecular aspects of the yolk sac of bovine embryos during 24 to 52 days of gestation. A total of 69 embryos were allocated into three groups according to the crown-rump length and estimated ages. Yolk sac samples were then subjected to morphological and molecular analysis using mass spectrometry and nuclear magnetic resonance techniques. In contrast to alanine, which was observed only in Group I, during all gestational stages, we identified important metabolites such as aspartate, taurine, glycerophosphocholine, creatinine, creatine, hydrouracil, glutamate, glutamine, lactate, lysine, valine, myo-inositol, cadaverine, and choline. In addition, 314 random sequences of proteins were identified in the bovine yolk sac, and 47 of these were considered to be specific. Changes in alpha-fetoprotein and carcinoembryonic antigen concentrations during gestation were also evaluated. In conclusion, the majority of these proteins are related to the development of secondary metabolites that are involved in the activation of other proteins and metabolites, and in signaling pathways that are responsible for maternal-fetal exchanges, activation of programmed cell death mechanisms, and cellular differentiation, and also in proteins that are responsible for the yolk sac involution that is required to establish chorioallantoic placentation.

137 IRON TRANSFER ACROSS THE LLAMA PLACENTA (LAMA GUANICOE GLAMA)

The placenta of the llama has been described as epitheliochorial in type, but recent studies have not shown extensively the fetal nutrition aspects in this animal. In epitheliochorial placentation there is development of structures called areolae, as well as inter-microvillous attachment of the trophoblast, with irregular contact, to the uterine epithelium. This attachment is interrupted and the transfer of substances between the mother and the fetus takes place across the areolar cavity. These areolae appeared as small rounded or dome-shaped elevated areas of the chorioallantoic membrane over the narrow uterine gland openings. In order to detail their mechanisms of iron transfer in the llama placenta, we collected the samples of nine uteri between 28 to 36 weeks of pregnancy in association with fetal membranes. These samples were fixed in 4% paraformaldehyde in PBS, processed, and stained for light microscopy (HE, picrosirius, and Massons trichrome), histochemistry (Perls, acid phosphatase, and PAS reactions) and immunohistochemistry with rabbit anti pig uteroferrin antibody to confirm the iron transfer, because the uteroferrin is an iron transporter and a progesterone-induced hematopoietic growth factor. The trophoblast formed a columnar-type single layer that was comprised of cells of various sizes and shapes with basal nuclei, including the giant binucleate cells. The trophoblast formed chorionic projections which presented ramifications in number from 4 to 5. A great quantity of blood vessels were found in the materno-fetal interface, between the cells of uterine epithelium and around of the chorionic projections. A PAS-positive reaction was observed with diffuse cytoplasmic PAS staining at the apical region of the trophoblast at the materno-fetal interface as well as in the endometrial glands. Collagen fibers were observed in the mesenchyme and inside the chorionic projections. In the areolae we confirmed the positive reaction of the acid phosphatase enzyme that detects phagocytic activity. In the basal region of the uterine gland epithelium, which is columnar type, and in the gland lumina, this reaction demonstrated a strong positive stain. The Perls histochemical reaction that reveals ferric iron was positive in the areola, as well as in the uterine glands. The uteroferrin immunohistochemistry showed a strong stained in the areolae and in the epithelium and lumina of the uterine glands. Our findings suggest that the areola region and the endometrial glands play an important role in histiotrophic nutrition in llamas, and in fetal red blood cell formation by iron transfer from mother to the fetus. This work was supported by FAPESP, CNPq, CAPES, PRONEX, Brazil.