A Xenopus distal-less gene in transgenic mice: conserved regulation in distal limb epidermis and other sites of epithelial-mesenchymal interaction.

In this paper, we show the conserved regulation of the homeodomain gene Distal-less-3 (Dlx-3) by analyzing the expression of a promoter from the Xenopus ortholog, Xdll-2, in transgenic mice. A 470-bp frog regulatory sequence confers appropriate expression on a lacZ reporter gene in the ectodermal component of structures derived from epithelial-mesenchymal interactions. Remarkably, this includes structures absent in Xenopus, such as the hair follicle and mammary gland, suggesting that conserved regulatory elements can be used to control the formation of structures peculiar to individual species. In addition, expression of Dlx-3 in developing limbs is highest at the most distal portion. This pattern is duplicated by the Xenopus promoter, indicating that this DNA may include sequences responsive to conserved proximodistal patterning signals in the vertebrate limb.

Avaliação comparativa da suplementação de ácidos graxos saturados e insaturados sobre o desempenho produtivo, qualidade oocitária e embrionária, e metabolismo de vacas leiteiras no período de transição e início de lactação

Objetivou-se avaliar a suplementação dietética de ácidos graxos saturados e insaturados sobre o metabolismo, e desempenhos produtivo e reprodutivo no período de transição e início de lactação de vacas leiteiras. Foram utilizadas 36 vacas da raça holandesa distribuídas aleatoriamente para receber uma das três dietas experimentais. No período pré-parto as dietas foram: Controle (CON), sem adição de gordura e 2,8% de extrato etéreo baseado na matéria seca; Gordura Saturada (SAT), com inclusão de 2,4% de MAGNAPAC® (Tectron Ltda.) com 4,7% de EE baseado na MS; Gordura Insaturada (INS), com inclusão de 11% de grão de Soja, com 4,7% de EE baseado na MS. No período pós-parto, Controle (CON), sem adição de gordura e com 2,8% de EE baseado na MS; Gordura Saturada (SAT), com inclusão de 2,6% de MAGNAPAC® (Tectron Ltda.) com 5,0% de EE baseado na MS; Gordura Insaturada (INS), com inclusão de 13% de Grão de Soja, com 5% de EE baseado na MS. As dietas foram fornecidas 35 dias da data prevista do parto até 90 dias de lactação. No período pré-parto foi utilizada silagem de milho como volumoso, em uma relação volumoso:concentrado de 70:30, enquanto que no período pós-parto foram utilizados 5% de feno de tifton e 45% de silagem de milho como fontes de volumoso, com uma relação entre volumoso:concentrado de 50:50. A produção de leite foi mensurada diariamente durante todo o período experimental. As amostras utilizadas para análise da composição e o perfil de ácidos graxos do leite foram coletadas semanalmente, sendo provenientes das duas ordenhas diárias. As amostras de sangue para análise dos metabólitos sanguíneos foram coletadas semanalmente. Do dia 14 ao dia 90 pós-parto foi realizado avaliação da dinâmica folicular por ultrassonografia. Nos dias 30, 60 e 90 foram realizadas aspirações foliculares, com posterior fertilização in vitro dos oócitos. Todas as variáveis mensuradas foram avaliadas pelo procedimento PROC MIXED do SAS (2004) utilizando-se os seguintes contrastes ortogonais: Controle vs Fontes de Lipídeo (C1); Fonte de ácidos graxos saturados x Fonte de ácidos graxos insaturados (C2). Foi utilizado nível de 5% de significância. No período pós-parto, a suplementação de lipídeos aumentou as concentrações de AGNE quando comparada a dieta CON. O tratamento INS reduziu as concentrações de proteínas totais e de BHB quando comparado ao SAT. Houve interação entre tempo e dieta paras as variáveis colesterol total, LDL e BHB. Houve redução da produção de leite corrigida para 3,5% de gordura, na produção total de gordura e de proteína, e no teor de gordura do leite quando comparado o tratamento INS com o SAT. A suplementação de lipídeo reduziu as concentrações do somatório dos ácidos graxos saturados (Σ A.G. Saturados), dos ácidos graxos com menos de 16 carbonos (>C16), e da relação entre ácidos graxos saturados com insaturados (Σ SFA/(MUFA+PUFA)); e aumentou as concentrações de ácidos graxos acima de 16 carbonos (>C16), de ácidos graxos insaturados com 18 carbonos, da somatória dos ácidos graxos insaturados e dos ácidos graxos poli-insaturados (Σ A.G. Poli-insaturados). O tratamento INS aumentou a concentração de ácidos graxos poli-insaturados totais (Σ A.G. poli-insaturados), e reduziu o total de ácidos graxos de 16 carbonos (C16) em relação ao tratamento SAT. Houve redução no número de folículos classe 1, e folículos totais (NC1 e NTFol) com suplementação de lipídeo. O tratamento SAT aumentou o número de folículos classe 5 (NC5), em relação ao INS. Não houve alteração na qualidade oocitária e embrionária com a suplementação de lipídeo e entre as duas fontes de lipídeo. A suplementação de lipídeos insaturados através da suplementação via grão de soja cru e integral, quando comparada à suplementação de lipídeos saturados, para vacas no período de transição e início de lactação, não interferiu na dinâmica folicular e qualidade oocitária e embrionária; e reduziu o desempenho produtivo, devido às reduções na produção de gordura do leite a na produção de leite corrigida para 3,5% dos animais suplementados

Desenvolvimento de protocolos para IATF com 7 dias de permanência do CIDR® em fêmeas Nelore

O objetivo do experimento I foi avaliar a redução do tempo de permanência do dispositivo de P4 de 9 para 7 dias sob parâmetros reprodutivos de vacas Nelore. Foram utilizadas 674 vacas lactantes entre 40-60 dias pós parto que receberam no início do protocolo (d0) BE + CIDR. No momento da retirada do CIDR foi administrado PGF2 α, ECP e eCG. A IATF ocorreu 55 e 48 horas após a retirada do dispositivo nos tratamentos 7d-CIDR e 9d-CIDR, respectivamente. Dez dias após a IA foi realizada colheita de sangue para dosagem de P4 sérica e confirmação da ovulação. Vacas tratadas com 7d-CIDR apresentaram menor (p < 0,01) folículo ovulatório em relação ao 9d-CIDR. No entanto, a concentração de P4 pós-IA, taxas de ovulação, detecção de estro e prenhez não foram influenciadas pelo tempo de permanência do CIDR. Assim, o uso do CIDR por 7 dias promoveu desempenho reprodutivo semelhante em vacas Nelore comparado ao protocolo com 9 dias. O experimento II teve o objetivo de avaliar os efeitos da reutilização do CIDR por até 35 dias de uso em vacas e 42 dias em novilhas Nelore. Utilizou-se 749 vacas lactantes 40-60 dias pós parto e 92 novilhas púberes. No d0 os animais receberam BE + CIDR novo (CIDR1) ou previamente usado por 7 (CIDR2), 14 (CIDR3), 21 (CIDR4), 28 (CIDR5) e 35 (CIDR6) dias. No momento da retirada do CIDR (d7) foi administrado PGF2 α, ECP, eCG e exame de US para mensuração do maior folículo (FD), além de colheita de sangue para dosagem de P4. A IATF ocorreu 55 horas após a retirada do dispositivo. O diâmetro do FD foi maior (p < 0,01) de acordo com o maior número de usos do CIDR nas vacas, a concentração de P4 reduziu nos CIDRs reutilizados porém se mantiveram acima de 1,5 ng/ml e a taxa de prenhez não foi afetada pela reutilização do dispositivo por até 5 vezes em vacas e o sexto uso em novilhas. O protocolo com 7 dias de permanência permite a reutilização do CIDR por até 6 vezes mantendo a mesma eficiência reprodutiva. No experimento III o objetivo foi avaliar se a aplicação do eCG dois dias antes da retirada do dispositivo aumenta o tamanho do FO, CL e taxa de prenhez. Foram utilizadas 681 vacas lactantes 40-60 dias pós parto e 182 novilhas púberes. Os animais foram distribuídos em dois tratamentos com aplicação de eCG no quinto (5d-eCG) ou sétimo dia (7d-eCG). No d0, os animais receberam BE + CIDR e no dia 7 o CIDR foi retirado e administrado PGF2 α e ECP. Dez dias após a IA foi realizada US para mensuração do CL e colheita de sangue para dosagem de P4. A IATF ocorreu 55 horas após a retirada do dispositivo. O tratamento 5d-eCG aumentou (p < 0,01) o FO nas vacas em relação ao grupo 7deCG e o mesmo ocorreu nas novilhas. Em vacas, a concentração de P4 pós IA foi mais alta (p = 0,04) no 5d-eCG. Em novilhas o diâmetro do CL pós-IA foi maior (p < 0,01) no 5d-eCG. No entanto, a antecipação da aplicação do eCG foi eficiente em aumentar o folículo ovulatório no momento da IATF, mas não aumentou a taxa de prenhez

A deletion mutation in GDF9 in sisters with spontaneous DZ twins

A loss of function mutation in growth differentiation factor 9 (GDF9) in sheep causes increased ovulation rate and infertility in a dosage-sensitive manner. Spontaneous dizygotic (DZ) twinning in the human is under genetic control and women with a history of DZ twinning have an increased incidence of multiple follicle growth and multiple ovulation. We sequenced the GDF9 coding region in DNA samples from 20 women with DZ twins and identified a four-base pair deletion in GDF9 in two sisters with twins from one family. We screened a further 429 families and did not find the loss of function mutation in any other families. We genotyped eight single nucleotide polymorphisms across the GDF9 locus in 379 families with two sisters who have both given birth to spontaneous DZ twins (1527 individuals) and 226 triad families with mothers of twins and their parents (723 individuals). Using case control analysis and the transmission disequilibrium test we found no evidence for association between common variants in GDF9 and twinning in the families. We conclude that rare mutations in GDF9 may influence twinning, but twinning frequency is not associated with common variation in GDF9.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries. (C) 2004 Wiley-Liss, Inc.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Patched1 functions as a gatekeeper by promoting cell cycle progression

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G(1)-S phase and G(2)-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a gatekeeper. In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

Testicular germ cell tumors exhibit evidence of hormone dependence

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone-dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin-releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real-time RT-PCR analysis was used to determine the expression profiles of hormone pathway-associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real-time RTPCR analysis of follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone-related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model. (c) 2005 Wiley-Liss, Inc.

Retromer controls epithelial cell polarity by trafficking the apical determinant crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.

Phonophoresis and topical drug delivery

In this study, investigations into phonophoresis were conducted by employing 3 distinct in vitro models. The aim of the first model was to evaluate the effect of ultrasound on the migration rate of different classes of molecules through agar gel. The derived data suggested that small, relatively hydrophobic molecules are more susceptible to ultrasound-enhanced diffusion through the water-filled channels of the agar gel. The application of heat alone increased drug migration by a similar magnitude as the ultrasound, indicating that ultrasonic heating directly increases the thermodynamic potential for diffusion. In the second experimental system, whole rat skin was pre-sonicated and then examined for changes in its barrier properties. At high intensities (1 to 2W cm-2), ultrasonic waves irreversibly compromised the barrier properties of the skin, following the general patterns described in the literature reports. At low intensities (< 1W cm-2), ultrasound discharged sebum from the sebaceous glands so as to fill much of the hair follicle shafts. This entirely novel phenomenon is probably produced by the mechanical effects of the beam. The deposition of sebaceous lipids within the hair follicle shafts can mean that this absorption pathway is blocked for hydrophilic molecules that penetrate via this route. Consequently, this phenomenon can be utilised as a probe to measure the relative follicular contribution to total penetration for these molecules. In the final phonophoresis model, modified Franz cells were employed in order to assess the ultrasound effect on the concurrent transdermal permeation of various molecules through whole rat skin. For the most lipophilic agent tested, the rate-limiting step of absorption was partitioning from the stratum corneum into the viable epidermis. Sonication did not accelerate this step.

Molecular biology and biochemistry of brain tumours

ßElucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the ß-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Primer induced restriction analysis (PIRA) of a rarer and alternative polymorphism in the GHRH-R receptor, in which Thr replaces Ala at codon 57, in human GH-secreting pituitary tumours was investigated. Whilst the rarer form correlated with an increased response of the pituitary cells to GHRH in vitro, allele distribution studies revealed that it is unlikely that the polymorphism contributes to increased risk of developing GH-secreting tumours and therefore acromegaly. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type. Since this gene product is involved with development, these results suggest that p-catenin mutations may contribute to the initiation and subsequent growth of congenital adamantinomatous craniopharyngiomas.

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

The endocrine basis for reproductive life-history trade-offs during the previtellogenic resting stage in the yellow fever mosquito, Aedes Aegypti

Juvenile hormone (JH) is the central hormonal regulator of life-history trade-offs in many insects. In Aedes aegypti, JH regulates reproductive development after emergence. Little is known about JH's physiological functions after reproductive development is complete or JH's role in mediating life-history trade-offs. By examining the effect of hormones, nutrition, and mating on ovarian physiology during the previtellogenic resting stage, critical roles were determined for these factors in mediating life-history trade-offs and reproductive output. The extent of follicular resorption during the previtellogenic resting stage is dependent on nutritional quality. Feeding females a low quality diet during the resting stage causes the rate of follicular resorption to increase and reproductive output to decrease. Conversely, feeding females a high quality diet causes resorption to remain low. The extent of resorption can be increased by separating the ovaries from a source of JH or decreased by exogenous application of methoprene. Active caspases were localized to resorbing follicles indicating that an apoptosis-like mechanism participates in follicular resorption. Accumulations of neutral lipids and the accumulation of mRNA's integral to endocytosis and oocyte development such as the vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) were also examined under various nutritional and hormonal conditions. The abundance of mRNA's and neutral lipid content increased within the previtellogenic ovary as mosquitoes were offered increasing sucrose concentrations or were treated with methoprene. These same nutritional and hormonal manipulations altered the extent of resorption after a blood meal indicating that the fate of follicles and overall fecundity depends, in part, on nutritional and hormonal status during the previtellogenic resting stage. Mating female mosquitoes also altered follicle quality and resorption similarly to nutrition or hormonal application and demonstrates that male accessory gland substances such as JH III passed to the female during copulation have a strong effect on ovarian physiology during the previtellogenic resting stage and can influence reproductive output. Taken together these results demonstrate that the previtellogenic resting stage is not an inactive period but is instead a period marked by extensive life-history and fitness trade-offs in response to nutrition, hormones and mating stimuli.

Sex steroid feedback regulation of pituitary gonadotropins during early secondary oocyte growth in coho salmon (Oncorhynchus kisutch) – a potential target of endocrine disruption.

Thesis (Ph.D.)--University of Washington, 2016-06