"Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death"

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (,30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (similar to 30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Early embryonic death in mares: clinical and hormonal aspects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A Pollen Chase Experiment; Examining Varying Levels of Embryonic Inbreeding Depression

A pollen chase experiment was performed upon three Costa Rican populations of Witheringia solanacea to examine the breakdown of genetically enforced self incompatibility (SI) and the extent of embryonic inbreeding depression. Self-pollen was applied in the bud, with outcross pollen applied one day later, and outcross pollinations at both intervals as a control. A variety of responses were found among the populations. BOHS readily accepted self pollen and suffered from very low inbreeding depression. Monteverde and Las Cruces both have lower fruit set with self-pollination precedence indicating that bud pollinations can overcome the self-incompatibility response and that embryonic death due to inbreeding depression causes fruit failure. The treatment:control fruit set is higher for the Las Cruces plants indicating stronger SI response Self-precedence seeds from the Las Cruces plants are likely to be outcrossed. Self-precedence seeds from Monteverde are likely selfed.

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the α-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggests that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

Islet-brain1/JNK-interacting protein-1 is required for early embryogenesis in mice.

Islet-brain1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein that organizes the JNK, MKK7, and MLK1 to allow signaling specificity. Targeted disruption of the gene MAPK8IP1 encoding IB1/JIP-1 in mice led to embryonic death prior to blastocyst implantation. In culture, no IB1/JIP-1(-/-) embryos were identified indicating that accelerated cell death occurred during the first cell cycles. IB1/JIP-1 expression was detected in unfertilized oocytes, in spermatozoa, and in different stages of embryo development. Thus, despite the maternal and paternal transmission of the IB1/JIP-1 protein, early transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocytes.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

The role of the c-myc gene in pigment cell development and homeostasis

Résumé La dérégulation de c-Myc est un événement fréquent de la transformation cellulaire. Une régulation positive de cette oncoprotéine a été démontrée dans divers mélanomes cutanés primaires et métastatiques et est associée à un pronostic défavorable (Grover et al., 1996; Zhuang et al., 2008). c-Myc est considéré comme une molécule centrale impliquée dans plusieurs processus de l'homéostasie cellulaire. En raison de sa contribution importante dans la progression tumorale, la fonction de c-Myc a été étudiée intensément. Cependant nous connaissons peu le rôle de ce facteur de transcription dans l'embryogenèse et dans la spécification tissulaire. Un déficit total de c-Myc pendant l'embryogenèse conduit à la mort embryonnaire avant 10.5 jours de gestation. Cette mort est causée par de multiples imperfections du développement touchant la taille de l'embryon, le coeur, le péricarde, le tube neural et les cellules sanguines (Davis et al., 1993; Trumpp et al., 2001). Récemment, il a été montré que la plupart de ces anomalies sont secondaires et résultent d'une insuffisance du placenta dans les embryons c-myc-/- (Dubois et al., 2008). Sachant que c-Myc est important dans la maintenance des lignées de la crête neurale (Wei et al., 2007), nous nous sommes intéressés au rôle de c-Myc dans le développement des cellules pigmentaires et à leur homéostasie après la naissance. Un allèle floxé de c-myc (Trumpp et al., 2001) a été utilisé pour supprimer ce gène spécifiquement dans la lignée mélanocytaire à l'aide d'une souris transgénique Tyr::Cre (Delmas et al., 2003). L'ablation des deux allèles de c-myc dans les mélanocytes des souris c-myccKO conduit au phénotype de grisonnement des poils, observé directement après la naissance et associé à une diminution du nombre de mélanocytes dans le bulbe des follicules pileux. Les cellules pigmentaires restantes expriment les marqueurs mélanogéniques (Tyr, TRP-1, Dct and MITF) et semblent être fonctionnelles puisqu'elles peuvent produire et transférer la mélanine. De plus, la capacité de prolifération des mélanocytes déficients en c-Myc dans le bulbe des follicules pileux ne semble pas être affectée chez les nouveaux-nés. Les cellules souches mélanocytaires sont présentes, mais en nombre réduit, dans le bulge des follicules pileux à la fin de la morphogenèse chez les souris c-myccKO âgées de huit jours. Ces cellules sont maintenues sans changement durant le premier cycle pileux (vérifié à l'âge de trente jours), ce qui sous-entend que la fonction de c-Myc n'est pas nécessaire pour ce processus. Ceci explique pourquoi, en supposant que des cellules souches mélanocytaires fonctionnelles sont présentes dans la peau, nous n'observons pas de dilution de couleur de la robe liée à l'âge. Cependant, la présence de ces cellules souches mélanocytaires dans la peau c-myccKO ne suffit pas à assurer une quantité normale de mélanocytes différenciés dans le bulbe des follicules pileux. Cette population de cellules pigmentaires matures est sévèrement affectée par la suppression de c-Myc, ce qui contribue amplement au phénotype de grisonnement des poils. De plus, c-Myc paraît être important pour le développement des mélanocytes. Ainsi, le nombre de mélanoblastes diminue dans les embryons c-myccKO à partir du douzième jour de gestation. A treize jours de gestation, au stade où les mélanoblastes pénètrent dans l'épiderme et prolifèrent, les mélanoblastes déficients en c-Myc ne s'adaptent pas aux signaux de prolifération et se retrouvent en nombre réduit dans l'épiderme. Finalement, nous nous sommes intéressés, au rôle de N-Myc, un homologue proche de c-Myc, dans la lignée mélanocytaire. Nos expériences ont montré que. N-Myc était superflu pour le développement et l'homéostasie des mélanocytes, une seule copie du gène c-myc étant suffisante pour maintenir une pigmentation normale de la robe des souris c-mycc-myccKO/+~N_ myccKO/KO. Cependant, le rôle essentiel de N-Myc dans la maintenance des cellules mélanocytaires précurseurs apparaît lorsque c-Myc est absent, puisque la suppression simultanée des deux Myc résulte en une perte complète de la coloration de la robe. Ceci implique la présence d'un mécanisme compensatoire entre c- et N-Myc dans la lignée mélanocytaire, avec un rôle prédominant de c-Myc. Summary Deregulation of c-Myc is known to be a common event in cellular transformation. Upregulation of this oncoprotein was shown in a variety of primary and metastatic cutaneous melanomas and has been associated with a poor prognosis (Grover et al., 1996; Zhuang et al., 2008). c-myc is seen as a central molecule involved in many aspects of cellular homeostasis. c-Myc function has been intensively studied mostly because of its significant contribution to tumour progression. However little is known on the role of this transcription factor in embryogenesis and tissue specification. Complete loss of c-Myc during embryogenesis results in embryonic death before E10.5 due to multiple developmental defects including embryonic size, heart, pericardium, neural tube and blood cells (Davis et al., 1993; Trumpp et al., 2001). Recently it was discovered that most of these abnormalities are secondary and results of placental insufficiency in c-Myc-/- embryos (Dubois et al., 2008). Here, we focused on the role of c-Myc in pigment cell development and homeostasis after birth, knowing that c-Myc is important in the maintenance of neural crest lineages (Wei et al., 2007). A floxed allele of c-Myc (Trumpp et al., 2001) was used to specifically delete this gene in the melanocyte lineage using Tyr::Cre transgenic mice (Delmas et al., 2003). Removal of both c-Myc alleles in melanocytes of c-MyccKO mouse led to the grey hair phenotype which is seen directly after birth and was associated with a decrease in the melanocyte number in the bulb of the hair follicle. The remaining population of pigment cells express melanogenic markers (Tyr, TRP-1, Dct and MITF) and seem functionally normal since they can produce and transfer melanin. Furthermore proliferation capacity of c-Myc deficient melanocytes in the bulb of hair follicle seems not to be affected in newborn animals. Melanocyte stem cells (MSCs) are present but reduced in numbers in the bulge of the hair follicle at the end of morphogenesis in 8 days old c-MyccKO mice. These cells are maintained through the first hair cycle (as verified at P30) without any further changes, suggesting that c-Myc function is not required for this process. This explains why we did not detect any agerelated coat color dilution, assuming a presence of functional MSCs in the skin. Importantly, presence of MSCs in c-MyccKO skin was not sufficient for assuring a normal number of differentiated melanocytes in the bulb of the hair follicle. This population of mature pigmented cells is severely affected upon c-myc deletion thus largely contributing to the grey hair phenotype. Moreover, c-Myc appears to be important for melanocyte development. Thus, melanoblast number is affected in c-MyccKO embryos day 12 of gestation onwards. At E13.5, when melanoblasts enter the epidermis and proliferate, c-myc deficient melanoblasts failed to adapt to proliferation signals and are therefore reduced in number in the epidermis. Finally, we addressed the role of N-Myc, a closest homologue of c-Myc, in the melanocyte lineage. In these experiments, N-Myc was dispensable for melanocyte development and homeostasis, and even one copy of the c-myc gene was sufficient to maintain normal coat color pigmentation in c-mycc-mycCKO/+ ,N-myccKO/KO mice. However the crucial role of N-Myc in maintenance of melanocyte precursor cells became apparent when c-myc is eliminated since simultaneous deletion of both Myc results in complete loss of coat color pigmentation. This suggests compensatory mechanisms between c- and N-Myc with a predominant role of c-Myc in melanocyte lineage.

Equine conceptus development : a mini review

Many aspects of early embryonic development in the horse are unusual or unique; this is of scientific interest and, in some cases, considerable practical significance. During early development the number of different cell types increases rapidly and the organization of these increasingly differentiated cells becomes increasingly intricate as a result of various inter-related processes that occur step-wise or simultaneously in different parts of the conceptus (i.e., the embryo proper and its associated membranes and fluid). Equine conceptus development is of practical interest for many reasons. Most significantly, following a high rate of successful fertilization (71-96%) (Ball, 1988), as many as 30-40% of developing embryos fail to survive beyond the first two weeks of gestation (Ball, 1988), the time at which gastrulation begins. Indeed, despite considerable progress in the development of treatments for common causes of sub-fertility and of assisted reproductive techniques to enhance reproductive efficiency, the need to monitor and rebreed mares that lose a pregnancy or the failure to produce a foal, remain sources of considerable economic loss to the equine breeding industry. Of course, the potential causes of early embryonic death are numerous and varied (e.g. persistent mating induced endometritis, endometrial gland insufficiency, cervical incompetence, corpus luteum (CL) failure, chromosomal, genetic and other unknown factors (LeBlanc, 2004). However, the problem is especially acute in aged mares with a history of poor fertility in which the incidence of embryonic loss between days 2 and 14 after ovulation has been reported to reach 62-73%, and in which embryonic death is due primarily to embryonic defects rather than to uterine pathology (Ball et al., 1989; Carnevale & Ginther, 1995; Ball, 2000).

Características morfológicas da casca do ovo de avestruz

A análise morfológica da casca do ovo da avestruz é importante, pois diversos fatores morfológicos podem estar relacionados com a eclodibilidade do ovo desta espécie no Brasil. Para tanto foram analisadas 60 amostras de ovos aparentemente normais (ovos eclodidos e com morte embrionária). Foram efetuadas observações que permitiram estabelecer: a espessura média da casca do ovo, a porosidade da casca destes ovos, e correlacionar estas variáveis com a eclodibilidade. Os resultados foram analisados estatisticamente mediante o teste de Duncan, o que mostrou que ovos com menor porosidade tem também uma menor eclodibilidade. A porosidade média dos ovos eclodidos é de 19,87 poros/cm², e dos ovos com morte embrionária é de 16,78 poros/cm². Não houve diferença estatística entre a espessura da casca nas regiões estudada, nem interação entre viabilidade e as regiões.

Influência do momento da cobrição, em relação à ovulação, na fertilidade e na ocorrência de morte embrionária precoce em eqüinos

Verificou-se a influência do momento da cobrição, em relação à ovulação, na fertilidade e na ocorrência de morte embrionária precoce (MEP) em 405 éguas da raça Puro Sangue Inglês, acompanhadas por 629 ciclos estrais. As éguas foram divididas em cinco grupos, de acordo com o momento da cobrição: grupo I, composto por 79 éguas cobertas entre 48 e 36 horas antes da ovulação; grupo II, com 102 éguas cobertas 36 a 24 horas antes da ovulação; grupo III, com 166 éguas cobertas 24 a 12 horas antes da ovulação; grupo IV, com 185 éguas cobertas até 12 horas antes da ovulação; e grupo V, formado por 97 éguas cobertas até 12 horas após a ovulação. As ovulações foram determinadas por palpação retal e ultra-sonografia, realizadas a cada 12 horas. O diagnóstico de gestação foi feito 14 dias após a cobrição com auxílio de ultra-sonografia. Novo exame foi realizado aos 60 dias para verificar ocorrência de MEP. Maiores índices de prenhez foram observados nos grupos III e IV (P<0,06). Verificou-se comportamento quadrático da taxa de prenhez em função do momento da cobrição aos 14 dias (R²= 91,0%; P<0,05) e aos 60 dias de prenhez (R²= 89,2%; P<0,05). Não foi observada diferença entre os grupos para MEP pelo qui-quadrado, mas a análise de regressão revelou comportamento quadrático da variável (R²= 92,4%; P<0,05). O melhor momento para cobrição foram as 24 horas que antecederam a ovulação.

Uterine evaluation and gestation diagnosis in owl monkey (Aotus azarai infulatus) using the B mode ultrasound

Background Gynecological and obstetrical ultrasonography has become an indispensable tool in the routine management, health evaluation and research on captive non-human primates.Methods Ultrasound was used to evaluate the uterus and estimate the gestation of owl monkeys. Twelve couples were selected, where five were primiparous and seven multiparous females from the National Primate Center reproductive colony, Ananindeua-PA, Brazil. The procedures were carried out using the GE (R) Logiq 100 MP, equipped with a 7.5 MHz linear probe.Results the females showed a simple uterus, of elongated shape, regular outline and homogeneous echogenic texture. In the uterine measurements craniocaudal diameter, dorsoventral diameter and uterine volume (UV), significant differences were identified (P < 0.05) between ultrasound examinations of primiparous and multiparous females. The UV showed a positive correlation with the number of births. The gestational sac and the embryonic echo were visible between 28 and 38 days after mating. Between 48 and 68 days after mating, embryonic death was identified in all the gestations.Conclusions the chemical (use of tranquilizers) and husbandry factors (capture stress) may be related to the prenatal death. The establishing methods of conditioning the female to the ultrasonographic exam may offer a solution to this problem.

Vitamina E em dietas para reprodutoras de tilápia-do-nilo (Oreochromis niloticus)

Pós-graduação em Aquicultura - FCAV

Avaliação uterina e diagnóstico da gestação em macaco-da-noite (Aotus azarai infulatus) por ultra-som em modo-B

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)