Insulin-like growth factor II and WT1 transcript localization in human fetal kidney and Wilms' tumor.

In situ hybridization was used to examine, in parallel, the localization of insulin-like growth factor II (Igf2) and WT1 transcripts in normal fetal kidney and Wilms' tumor. The expression of Igf2 and WT1 transcripts in the fetal kidney is almost complementary in both the epithelial and stromal cell lineages derived from the undifferentiated metanephrogenic blastema. The patterns of transcription of Igf2 in three Wilms' tumors appeared to be perturbed as compared to the normal fetal kidney. In these tumors Igf2 transcripts were detected in structures that are developmentally equivalent to the renal vesicle, which in the normal kidney do not contain Igf2 transcripts. These results suggest that Wilms' tumors arise from an alteration in the regulation of Igf2 mRNA synthesis.

Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young.

The neonatal IgG transporter FcRn consists of two chains, FcRn alpha and beta (also known as beta(2) microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG alpha and beta chain mRNA levels were investigated in the gut of brushtail possum (Trichosurus vulpecula) pouch young (PY) using Northern hybridisation. FcRn alpha transcripts were detected in the PY proximal intestine at all times examined, between days 1 and 195 of post-natal life, with increased levels detected from around day 110. The beta(2) microglobulin transcript levels in the PY proximal intestine were low to undetectable until day 110 of post-natal life and then increased dramatically after day 159. Both the FcRn alpha and beta gene transcripts were detected in a wide range of tissues in the adult possum (>365 days). Genomic sequences located 5' to the start of transcription of the FcRn alpha and beta(2) microglobulin genes were cloned and analysed for predicted cis-acting transcription control elements. Both the FcRn alpha and beta(2) microglobulin genomic sequences contained STAT5 binding motifs consistent with the transcription of both genes being modulated by prolactin. Using in situ hybridisation, the FcRn alpha and beta(2) microglobulin transcripts were localised to the epithelial cells of the PY intestine. However, no prolactin receptor transcripts were detected in the same epithelial cells suggesting that the observed changes in FcRn alpha and beta(2) microglobulin gene expression in the proximal intestine are not modulated directly by prolactin. The results are consistent with the hypothesis that changes in FcRn alpha and beta(2) microglobulin gene expression take place in the possum PY intestine to accommodate changes in maternal milk composition to meet the changing immunological demands of the PY.

Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MFalpha) leader and ovine follicle stimulating hormone (oFSH) alpha and beta subunit coding sequences. Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51.0 ng/ml and 17.5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the alpha and one copy of the beta subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay). It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive. Measurements of secreted roFSH alpha and beta subunit concentrations indicated less than 10% of alpha and 25-33% of beta subunits were stably dimerized. The receptor binding properties of the roFSH resemble those of native oFSH. In summary this paper reports the production, by P. pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.

The Australian brushtail possum (Trichosurus vulpecula) gonadotrophin alpha-subunit: analysis of cDNA sequence and pattern of expression.

A cDNA sequence from the gonadotrophin alpha-subunit mRNA of Australian brushtail possum (Trichosurus vulpecula) has been determined and analysed. Comparison with seven eutherian mammalian gonadotrophin alpha-subunit gene sequences revealed an average of 82.6% homology between the coding region nucleotide sequences and 88.8% identity between the predicted amino acid sequences. The predicted possum gonadotrophin alpha-subunit protein has ten evolutionarily conserved cysteine residues, two potential N-linked glycosylation sites and a putative enzyme recognition sequence which it has been suggested is required for sulphation of carbohydrate moieties. Comparison of the possum gonadotrophin alpha-subunit 3' untranslated region (UTR) sequence with the 3' UTRs of eutherian alpha-subunit transcripts revealed sequence homology. In particular, an 18 nucleotide imperfect palindromic sequence present in the possum 3' UTR, with the potential to form a hairpin loop, was found to be evolutionarily conserved and present in five out of seven eutherian alpha-subunit 3' UTR sequences. In situ hybridization localized the transcripts to a sub-population of anterior pituitary cells presumed to be gonadotrophs and thyrotrophs. In summary, these results indicate considerable conservation of the structure and function of the gonadotrophin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages.

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

cDNA sequence analysis, gene expression and protein localisation of the inhibin alpha-subunit of Australian brushtail possum (Trichosurus vulpecula).

An inhibin alpha-subunit cDNA sequence from the Australian brushtail possum (Trichosurus vulpecula) has been identified and analysed. The cDNA includes an open reading frame encoding a predicted precursor protein of 361 amino acids. The predicted protein sequence includes four possible proteolytic cleavage sites, 12 evolutionarily conserved cysteine residues and three potential N-linked glycosylation sites. The mature alpha-subunit is the carboxyl terminal fragment (alphaC) consisting of 131 amino acids. The full-length precursor protein shows a mean identity with eutherian homologues of 69.8%. The homology is not evenly distributed, with the putative alphaC fragment showing the highest level (79.7%). Using Northern hybridisation, an alpha-subunit transcript of approximately 1.6 kb was detected in adult possum ovary. Using in situ hybridisation and immunocytochemistry, inhibin alpha-subunit was localised exclusively to the granulosa cell layers of follicles. Hybridisation and immunostaining for the inhibin alpha-subunit were first observed in granulosa cells of primary follicles and the expression continued throughout all stages of follicular growth. Inhibin alpha-subunit mRNA and protein were also detected in cells of the corpus luteum. In summary, results indicate considerable conservation of the structure and possible function of the inhibin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages. The expression data suggest that, in the adult possum, inhibin may have a role in ovarian follicular growth from the primary stage of development.

Growth and paracrine factors regulating follicular formation and cellular function.

The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation and to report on the identity of growth and paracrine factors which might be involved in this process. Before follicular formation there is a massive and sustained colonisation of the fetal ovary by mesonephric cells, which become a precursor source of follicular cells. From within the ovarian medulla, somatic 'cell-streams' branch into the cortex around nests of oogonia and oocytes. These 'cell-streams', which contain elongated cells with either flattened or cuboidal shaped nuclei, express steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450(scc), and P450(aromatase) mRNA and/or protein. Follicles form from the association of an oocyte with the 'cell-stream' with either a single layer of flattened cells (i.e. type 1 follicle) or with a mixture of flattened and cuboidal cells (i.e. type 1a follicle). These newly-formed follicles have between 3 and 57 somatic cells (i.e. granulosa cells) and contain oocytes which vary in diameter between 23 and 52 microm. Newly formed and early growing follicles have been identified with growth factors or growth factor receptors in either the oocytes or granulosa cells. Many of the growth factors are from the TGFbeta superfamily and are expressed in a cell- and stage-specific manner.

The follicle-stimulating hormone beta-subunit gene of the common brushtail possum (Trichosurus vulpecula): analysis of cDNA sequence and expression.

Reverse transcription-PCR has been used to obtain a cDNA sequence from the follicle-stimulating hormone (FSH) beta-subunit gene of the Australian brushtail possum (Trichosurus vulpecula). Comparisons of the possum FSHbeta-mRNA coding region nucleotide sequence with that of six eutherian mammal homologues reveals a mean percent identity of 77.3% and 76.8% at the nucleotide and predicted amino acid-sequence levels respectively. Furthermore, the predicted amino acid sequence of the possum FSHbeta mature protein shows evolutionary conservation of twelve cysteine residues and two potential N-linked glycosylation sites. The protein lacks the CAGY motif present in most reported glycoprotein beta-subunit sequences. The translation termination codon and consensus polyadenylation sequence overlap, a feature observed in other mammalian FSHbeta genes. Northern hybridization of total RNA from adult female possum pituitary revealed three hybridizing transcripts of approximately 2.8, 1.2 and 0.5 kb which may arise from utilizing alternative polyadenylation signals. In situ hybridization localized the FSHbeta transcripts to a sub-population of anterior pituitary cells interpreted as being gonadotropes. In summary the results indicate considerable evolutionary conservation of the structure of the FSH beta-subunit gene between the marsupial and eutherian mammalian lineages.

Screening the foods of an endangered parrot, the kakapo (Strigops habroptilus), for oestrogenic activity using a recombinant yeast bioassay.

In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest. Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates. Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species. This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay. Low levels of oestrogenic activity were detected in one of the 'chick-raising' foods, but no oestrogenic activity was detected in the adult supplementary foods. The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined. Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestrogenic activity (approximately 10(-6) and 10(-4) of the activity of 17-beta-oestradiol, respectively).

Avian Clock gene polymorphism: evidence for a latitudinal cline in allele frequencies.

In comparison with most animal behaviours, circadian rhythms have a well-characterized molecular genetic basis. Detailed studies of circadian clock genes in 'model' organisms provide a foundation for interpreting the functional and evolutionary significance of polymorphic circadian clock genes found within free-living animal populations. Here, we describe allelic variation in a region of the avian Clock orthologue which encodes a functionally significant polyglutamine repeat (ClkpolyQcds), within free-living populations of two passerine birds, the migratory bluethroat (Luscinia svecica) and the predominantly nonmigratory blue tit (Cyanistes caeruleus). Multiple ClkpolyQcds alleles were found within populations of both species (bluethroat: 12 populations, 7 alleles; blue tit: 14 populations, 9 alleles). Some populations of both species were differentiated at the ClkpolyQcds locus as measured by F(ST) and R(ST) values. Among the blue tit, but not bluethroat populations, we found evidence of latitudinal clines in (i) mean ClkpolyQcds repeat length, and (ii) the proportions of three ClkpolyQcds genotype groupings. Parallel analyses of microsatellite allele frequencies, which are considered to reflect selectively neutral processes, indicate that interpopulation allele frequency variation at the ClkpolyQcds and microsatellite loci does not reflect the same underlying demographic processes. The possibility that the observed interpopulation ClkpolyQcds allele frequency variation is, at least in part, maintained by selection for microevolutionary adaptation to photoperiodic parameters correlated with latitude warrants further study.

Comparative in situ hybridization analysis of PAX2, PAX8, and WT1 gene transcription in human fetal kidney and Wilms' tumors.

Wilms' tumor (WT) is a childhood renal neoplasm with histological features resembling fetal kidney development. Two members of the paired box family of genes, PAX2 and PAX8, are expressed in WT and are potentially involved in its induction. A zinc finger gene, WT1, which is involved in WT induction, encodes a DNA binding protein, and like PAX2 and PAX8 proteins is a transcription factor with an important role in kidney development. We have compared the expression patterns of PAX2, PAX8, and WT1 in fetal kidney and WTs by in situ hybridization. The PAX2, PAX8, and WT1 genes were transcribed in the condensed mesenchyme and early stages of epithelial differentiation in fetal kidney. WT1 gene transcription was observed in the glomeruli of fetal kidney until a later stage in development than PAX genes. In WTs all three genes were expressed in the condensed blastema, but WT1 expression was not detectable in the epithelial structures in two WTs. No evidence of attenuation of PAX gene expression was found in WT. These results suggest that in some WTs the expression of WT1 is attenuated in structures that continued to express PAX genes. It is unlikely that both PAX2 and PAX8 genes would be mutated in WT. However, failure of PAX gene expression to attenuate in WTs may result from mutations involved in the onset of the tumor.

Localization of mRNA encoding c-kit during the initiation of folliculogenesis in ovine fetal ovaries.

The c-kit protein is a transmembrane tyrosine kinase receptor that binds the growth factor stem cell factor. Mutant alleles of the genes coding for both the receptor (c-kit) and its ligand (stem cell factor) affect gametogenesis, development of melanoblasts and some aspects of haematopoiesis. The aim of this study was to examine expression of the c-kit gene during folliculogenesis in fetal sheep ovaries using in situ hybridization. A 422 bp cDNA encoding the extracellular domain of the c-kit protein was amplified from sheep ovarian RNA using reverse-transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Riboprobes transcribed from the ovine cDNA encoding c-kit were used to detect the presence of mRNA encoding c-kit within the ovaries of fetal sheep on days 90, 100, 120 and 135 of gestation (term = 147 days). In day 90 and 100 fetal ovaries, mRNA encoding c-kit was not detected in association with oogonia during the period of meiosis (to prophase I) but was present in some of the isolated oocytes. In ovaries from day 90 to day 135, mRNA encoding c-kit was detected in the oocytes at every stage of follicular growth--primordial through to antral follicles. This pattern of localization is consistent with that demonstrated in mice.

Expression of the PAX2 gene in human fetal kidney and Wilms' tumor.

We have examined the pattern of expression of the human PAX2 gene in Wilms' tumors and human fetal kidney by Northern blot and in situ hybridization. Human PAX2 encodes a paired box-containing protein and has a high degree of homology with mouse and Drosophila paired box genes. In situ hybridization analysis reveals that PAX2 is expressed in nephrogenic structures in fetal kidney and also in Wilms' tumors. This pattern of expression suggests that PAX2 may have a role in differentiation of tissues in the kidney. In fetal kidney, PAX2 expression rapidly attenuates following the initial differentiation, but no evidence of attenuation was found in Wilms' tumors. The timing of PAX2 expression is restricted to fetal development, although high levels of expression were also observed in nephrogenic rests of residual normal juvenile kidney tissue adjacent to a Wilms' tumor. Nephrogenic rests are the presumptive precursors of Wilms' tumor but are not necessarily neoplastic. The failure of PAX2 expression to attenuate in Wilms' tumors and nephrogenic rests may be associated with events leading to the onset of Wilms' tumor. By somatic cell hybrid mapping, the PAX2 gene was localized to chromosome 10q22.1-q24.3, although this region has not previously been implicated in Wilms' tumor.

Stem cell factor and c-kit gene expression and protein localization in the sheep ovary during fetal development.

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor-ligand pair is essential to prevent apoptosis.

Control of early ovarian follicular development.

Early follicular growth refers to the development of an ovarian follicle from the primordial to early antral phase. In sheep and cows these phases of growth can be classified by the configuration of granulosal cells in the largest cross-section of the follicle as types 1 (primordial), 1a (transitory) 2 (primary), 3 and 4 (preantral) and 5 (early antral). Follicles classified as type 1 may be highly variable within each species with respect to number of granulosal cells and diameter of oocyte. Much of the variation in granulosal cell composition of type 1 follicles may occur at formation and this may account for the variability in granulosal cell composition throughout subsequent stages of growth. There appear to be important differences among species (for example sheep and cattle) in the number and function of granulosal cells relative to the diameter of the oocyte during the initiation of follicular growth. There is evidence that most, if not all, of the growth phases from types 1 to 5 are gonadotrophin-independent and that follicles develop in a hierarchical manner. In sheep, cows and pigs, numerous growth factor, growth factor receptor and gonadotrophin receptor mRNAs and peptides (for example c-kit, stem cell factor, GDF-9, beta B and beta A activin/inhibin subunit, alpha inhibin subunit, follistatin, FGF-2, EGF, EGF-R, TGF beta 1,2 and 3 FSH-R and LH-R) are expressed in a phase of growth (for example types 1-5)-specific and cell-specific manner. However, the roles of many of these factors remain to be determined.