Biomedical subjects

P T K Saunders

Publications and source records attributed to P T K Saunders.

11 recordsLinked to original sources

PubMed2006

The tumour-suppressor protein ASPP1 is nuclear in human germ cells and can modulate ratios of CD44 exon V5 spliced isoforms in vivo.

The ASPP1 (Apoptosis Stimulating Protein of p53) protein is an important tumour-suppressor. We have detected a novel protein interaction between the human ASPP1 (hASPP1) protein and the predominantly nuclear adaptor protein SAM68. In the human testis, full-length endogenous hASPP1 protein is located in the nucleus like SAM68, predominantly within meiotic and postmeiotic cells. Mouse ASPP1 (mASPP1) protein is mainly expressed in the brain and testis. The interaction with nuclear SAM68 is likely to be restricted to human germ cells, since endogenous mASPP1 protein is exclusively cytoplasmic. The C-terminal region of hASPP1 efficiently targeted a fused GFP molecule to the nucleus, whereas the N-terminus of hASPP1 targeted GFP to the cytoplasm. In the context of the full-length molecule this cytoplasmic targeting sequence is dominant in HEK293 and Saos-2 cells, since full-length hASPP1-GFP is almost exclusively cytoplasmic. Despite its predominantly cytoplasmic location, we show that ASPP1-GFP expression in HEK293 cells can regulate the ratio of alternative spliced isoforms derived from a pre-mRNA regulated downstream of cytoplasmic signalling pathways, and our data suggest that ASPP1 may operate in this case downstream or parallel to RAS signalling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Oestrogen receptor beta and neoadjuvant therapy with tamoxifen: prediction of response and effects of treatment.

In order to elucidate the relative importance of oestrogen receptor (ER)alpha, ERbeta and an ERbeta variant (ERbeta2/betacx) in the response of breast cancers to tamoxifen, tumour levels of each receptor were assessed in 36 patients before and after 3 months of neoadjuvant treatment with tamoxifen (20 mg daily). All patients were postmenopausal women presenting with large ERalpha-positive breast cancers. Clinical response to treatment was assessed by tumour volume changes as determined from sequential ultrasounds and pathological response by comparison of the tumour morphology before and after treatment. Of 33 cases, 23 (70%) were classified as having a clinical response and 16 (48%) as having a response pathologically. All tumours stained positively for ERalpha and ERbeta and 15 out of 33 (45%) for ERbeta2/betacx. There were no significant differences in quantitative expression of any receptor between tumours that subsequently responded and that did not, whether response was assessed clinically or pathologically. Tamoxifen treatment was associated with a decrease in ERalpha, but an increase was the most frequent change (17 out of 33) in ERbeta, and no consistent change was evident in staining of the ERbeta2/betacx variant. In summary, ERbeta1 and ERbeta2/betacx variant protein are detected in ERalpha-positive breast tumours but their expression is not associated with a response to tamoxifen. Differential changes in ERalpha and ERbeta were seen with treatment.

Aged↗

PubMed2005

Androgen regulation of stage-dependent cyclin D2 expression in Sertoli cells suggests a role in modulating androgen action on spermatogenesis.

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI-VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.

Animals↗

PubMed2005

Gonadal effects of a mouse Denys-Drash syndrome mutation.

Gonadal effects of the Denys-Drash syndrome (DDS) mutation Wt1(tmT396 )were examined in chimaeric and heterozygous mice. Since the only heterozygote was 41,XXY, Sertoli cell function was assessed by comparison with age-matched control XXY testes. Control XXY Sertoli cells showed immuno-expression of WT1 and androgen receptor (AR) indistinguishable from wild-type (40,XY), but expressed anti-Mullerian hormone (AMH). In contrast, DDS Sertoli cells showed only faint immuno-expression of WT1 and did not express AR or AMH. While XY<-->XY DDS chimaeras were male, XX<-->XY chimaeras were predominantly female. In the rare XX<-->XY DDS males the Sertoli cell lineage was largely derived from Wt1 mutant XY cells. We conclude that DDS mutant cells can form Sertoli cells, that the dominant mutation does not cause male sex reversal in mice but distorts the sex ratio of XX<-->XY chimaeras, and that there may be a link between WT1, AMH and AR expression by Sertoli cells in vivo.

Animals↗

PubMed2004

Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors.

It is now well established that oestrogens play a part in germ cell function. These hormones are synthesised by the cytochrome P450 aromatase (P450 arom) and act via two kinds of receptor (ERalpha and ERbeta). Although the presence of aromatase and oestrogen receptors in mammalian testis is now well documented, the localisation of these proteins in human germ cells is not yet clear. The primary purpose of the current study was to look for the expression of aromatase and oestrogen receptors in human germ cells. Human immature germ cells were collected from semen samples with an excess of rounds cells (>20%) and purified spermatozoa were obtained after sedimentation on a discontinuous PureSperm gradient. Expression of aromatase and oestrogen receptors was determined by RT-PCR with specific primers, and by Western blot using monoclonal antibodies. RT-PCR products for aromatase, ERalpha and ERbeta were amplified from total RNA isolated from human germ cells and spermatozoa. We identified an ERalpha isoform variant that lacks exon 4 in human germ cells and visualised P450 arom as a single band of 49 kDa in germ cells, as we have already reported for human ejaculated spermatozoa. By Western blot, we identified two proteins for ERbeta at approximately 50 and approximately 60 kDa, which could correspond to the long and short forms of ERbeta formed from the use of alternative start sites. In human ejaculated spermatozoa, ERbeta protein was not detected, even though we could amplify mRNA. Using Western blot analysis and a monoclonal antibody specific for ERalpha, we detected two proteins in human immature germ cells: one of the expected size (66 kDa) and a second one of 46 kDa. In mature spermatozoa, only the 46 kDa band was observed and we speculate it may be related to the ERalpha isoform lacking exon I. In conclusion, we have identified P450 arom and ER proteins (full-length and variant) in human germ cells. Further studies are now required to elucidate the mechanism of action of oestrogens on human male germ cells, in terms of both genomic and 'non-genomic' pathways.

Adult↗

PubMed2003

Absence of mDazl produces a final block on germ cell development at meiosis.

The autosomal gene DAZL is a member of a family of genes (DAZL, DAZ, BOULE), all of which contain a consensus RNA binding domain and are expressed in germ cells. Adult male and female mice null for Dazl lack gametes. In order to define more precisely the developmental stages in germ cells that require Dazl expression, the patterns of germ cell loss in immature male and female wild-type (+/+, WT) and Dazl -/- (DazlKO) mice were analysed. In females, loss of germ cells occurred during fetal life and was coincident with progression of cells through meiotic prophase. In males, testes were recovered from WT and DazlKO males obtained before and during the first wave of spermatogenesis (days 2-19). Mitotically active germ cells were present up to and including day 19. Functional differentiation of spermatogonia associated with detection of c-kit positive cells did not depend upon expression of Dazl. RBMY-positive cells (A, intermediate, B spermatogonia, zygotene and preleptotene spermatocytes) were reduced in DazlKO compared with WT testes. Staining of cell squashes from day 19 testes with anti-gamma-H2AX and anti-SCP3 antibodies showed that germ cells from DazlKO males were unable to progress beyond the leptotene stage of meiotic prophase I. It was concluded that in the absence of Dazl, germ cells can complete mitosis, and embark on functional differentiation but that, in both sexes, progression through meiotic prophase requires this RNA binding protein.

Animals↗

PubMed2003

Germ cell-somatic cell interactions during spermatogenesis.

The adult testis has two essential functions, namely the synthesis and secretion of the steroid hormone testosterone and the production of mature spermatozoa. Germ cells undergo mitosis, meiosis and condensation (spermiation) in close association with Sertoli cells and in defined associations with each other within the seminiferous epithelium. Normal spermatogenesis and fertility are dependent upon paracrine interactions between the somatic cells (Sertoli, Leydig and peritubular) and the germ cells, and upon endocrine support from the pituitary gland. Evidence for paracrine interactions between somatic and germ cells has been gained from observations on the patterns of expression of proteins, studies on isolated cell populations, germ cell or Leydig cell ablation with toxicants, mouse mutants and transgenics, and more recently from germ cell transplantation.

Animals↗

PubMed2002

Expression of oestrogen receptor beta (ERbeta1) protein in human breast cancer biopsies.

Oestrogen action is mediated via specific receptors that act as ligand-activated transcription factors. A monoclonal antibody specific to the C-terminus of human oestrogen receptor beta has been characterized and the prevalence of expression of oestrogen receptor beta protein investigated in a well defined set of breast cancers. Reverse transcription-polymerase chain reaction analysis of RNA from tissue biopsies detected oestrogen receptor beta in all samples examined. The anti-oestrogen receptor beta antibody cross reacted specifically with both long (approximately 59 Kd) and short (approximately 53 Kd) forms of recombinant oestrogen receptor beta. Western blot analysis of breast tumours contained both forms of oestrogen receptor beta protein although in some samples lower molecular weight species (32--45 Kd) were identified. Fifty-one breast cancer biopsies were examined using immunohistochemistry; 41 (80%) were immunopositive for oestrogen receptor alpha, 48 (94%) were immunopositive for oestrogen receptor beta and 38 (74.5%) co-expressed both receptors. Expression of oestrogen receptor beta was exclusively nuclear and occurred in multiple cell types. There was no quantitative relationship between staining for the two ERs although in tumours in which both receptors were present immunoexpression of oestrogen receptor alpha was invariably more intense. The significance of oestrogen receptor beta protein expression in breast cancers to therapy remains to be determined but the availability of a well characterized antibody capable of detecting oestrogen receptor beta in archive material will facilitate the process.

Antibodies, Monoclonal↗

PubMed2002

Testicular changes during infantile 'quiescence' in the marmoset and their gonadotrophin dependence: a model for investigating susceptibility of the prepubertal human testis to cancer therapy?

BACKGROUND: Inexplicably, boys treated with some therapies for cancer at age 2-10 years, a time of supposed 'testicular quiescence', are at risk of low sperm counts/infertility in adulthood. Our aims were to use the marmoset as a surrogate for man to establish testicular cell function/activity during 'quiescence' between the neonatal period and puberty, and to test if any cell activity could be suppressed by prior treatment with a GnRH antagonist. METHODS AND RESULTS: Based on immunoexpression studies, functional development of Sertoli cells (SGP-2, androgen receptor) and Leydig cells (3 beta-hydroxysteroid dehydrogenase) was detectable at an age (35 weeks) when the testis is considered to be quiescent, and in advance of the pubertal rise in blood testosterone levels (50-60 weeks). Other changes at 35 weeks were the appearance of focal seminiferous tubule lumens and proliferating germ cells [indicated by immunoexpression of proliferating cell nuclear antigen (PCNA)]. Treatment from 25 to 35 weeks with GnRH antagonist largely (>85%) prevented these changes. However, the PCNA-labelling index of spermatogonia in GnRH antagonist-treated animals did not differ from controls (41.3 versus 43.6%) though total spermatogonia volume per testis was reduced by 41%. Some protein markers (inhibin-alpha, estrogen receptor-beta) showed little change with age or treatment. Beyond 35 weeks, GnRH antagonist-treated animals showed a delay in the pubertal rise in plasma testosterone levels. CONCLUSIONS: These findings reinforce the view that the 'childhood' testis is not quiescent. This may explain the damaging effects of some cancer therapies on subsequent fertility of boys and raises the issue of protective intervention. The present studies suggest that GnRH antagonist-based intervention might be only partially successful. Identification of the factors regulating spermatogonial development in the infant marmoset may aid in the design of such strategies.

Aging↗

PubMed2002

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.

Aging↗

PubMed2002

Development and validation of a new monoclonal antibody to mammalian aromatase.

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.

Animals↗