Philipa Saunders - Academia.edu (original) (raw)
Papers by Philipa Saunders
The Biochemist, 2009
Androgens are the major drivers of masculinization and male fertility, and disruption of androgen... more Androgens are the major drivers of masculinization and male fertility, and disruption of androgen signalling has been implicated in the most common male congenital abnormalities, such as hypo spadias (opening of the urethra in a position not at the tip of the penis) and cryptorchidism (testes in the abdomen not the scrotum), as well as with an increased risk of developing testicular cancer or having a low sperm count in adulthood1.
Journal of Endocrinology, 2020
The endometrium is a complex multicellular tissue that is exquisitely sensitive to the actions of... more The endometrium is a complex multicellular tissue that is exquisitely sensitive to the actions of sex steroids synthesised in the ovary (endocrine system). Recent studies have highlighted a previously under-appreciated role for local (intracrine) metabolism in fine-tuning tissue function in both health and disease. In this review we have focused on the impact of oestrogens and androgens on endometrial function summarising data from studies on normal endometrial physiology and disorders including infertility, endometriosis and cancer. We consider the evidence that expression of enzymes including aromatase, sulphatase and AKR1C3 by endometrial cells plays an important role in tissue function and malfunction and discuss results from studies using drugs targeting intracrine pathways to treat endometrial disorders. We summarise studies exploring the spatial and temporal expression of oestrogen receptors (ERalpha/ESR1, ERbeta/ESR2 and GPER) and their role in mediating the impact of endoge...
Reproduction Abstracts, 2014
Smoking by women during pregnancy can result in a 30–48% reduction in sperm count and testis size... more Smoking by women during pregnancy can result in a 30–48% reduction in sperm count and testis size in the exposed offspring in adulthood, probably because of a decrease in the number of Sertoli cells. Until recently Sertoli cell proliferation was thought to be androgen independent because fetal Sertoli cells do not express the androgen receptor, but new evidence suggests that androgens may play the lead role in regulating Sertoli cell proliferation in fetal (Tan et al. 2005) and early neonatal life (Atanassova et al. 2005). We hypothesize that a unifying mechanism via which fetal exposure to environmental chemicals could affect sperm count in adulthood, is through interference with androgen production/action. To test this hypothesis, we have compared the effects of exposing fetal male rats from E13.5 onwards to 7,12-diemethyl benz[a]anthracene (DMBA) or it’s metabolite DMBA-DHD (as candidate molecules present in cigarette smoke), or n-dibutyl phthalate (DBP; a plasticizer shown to lower fetal testosterone levels), on Sertoli cell number, proliferation, apoptosis and functional development, using a combination of in vitro and in vivo studies. We have also used the AR antagonist, Flutamide, and tissue from mice, in which ablation of androgen action has been induced by transgenesis (ARKOs).Any effects that androgens do have on Sertoli cell proliferation/number must be indirect, so our studies are also directed at identifying the pathways via which any such effects could be mediated, such as local production of Insulin-Like Growth Factor 1 (IGF1) which has already been shown to increase Sertoli cell proliferation. We have demonstrated that fetal exposure to DMBA, DBP or Flutamide reduces expression of IGF1 mRNA, although preliminary results have so far shown no change in Sertoli cell number in treated animals and Sertoli cell proliferation indices are still under investigation
Journal of andrology
We have examined the location of the DAZL protein in fetal and adult rodents and human specimens ... more We have examined the location of the DAZL protein in fetal and adult rodents and human specimens and found that there is a shift from a predominantly nuclear to a predominantly cytoplasmic distribution of the protein in human testis. In rat testis and human ovary, however, the protein is predominantly, if not exclusively, cytoplasmic throughout germ cell development. One possible explanation for this could be that the DAZ protein is responsible for the nuclear localization of DAZL in human males. We have tested this hypothesis by examining the testis of marmosets, which lack the Daz genes and have found that the DAZL protein is both nuclear and cytoplasmic in spermatogonia, and by analyzing testis sections from DAZ-deleted patients in whom the cytoplasmic location of DAZL is evident in remaining germ cells. Transfection experiments indicate that the differences in DAZL expression between rodents and humans are not caused by the amino acid differences between the 2 proteins, and that...
Nature, Jan 4, 1997
RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins conta... more RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the different...
Nature Reviews Genetics, 2002
Molecular Biology of the Cell, 2007
Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs... more Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide ⌬-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.
BMC Developmental Biology, 2007
Background: Germ cells arise from a small group of cells that express markers of pluripotency inc... more Background: Germ cells arise from a small group of cells that express markers of pluripotency including OCT4. In humans formation of gonadal compartments (cords in testis, nests in ovary) takes place during the 1st trimester (6-8 weeks gestation). In the 2nd trimester germ cells can enter meiotic prophase in females whereas in males this does not occur until puberty. We have used qRTPCR, Westerns and immunohistochemical profiling to determine which of the germ cell subtypes in the human fetal gonads express OCT4, DAZL and VASA, as these have been shown to play an essential role in germ cell maturation in mice.
Journal of andrology
The aim of the present study was to assess whether proteins secreted by the seminiferous tubules ... more The aim of the present study was to assess whether proteins secreted by the seminiferous tubules (ST) can be detected in testicular interstitial fluid (IF) and testicular (TV), spermatic (SV), and peripheral venous (PV) plasma from adult rats. An antiserum was raised against seminiferous tubule-conditioned medium (STCM) prepared from adult rats and used in conjunction with Western blot analysis to screen IF and blood samples resolved by one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of IF and PV were analyzed from control adult rats and rats exposed to scrotal heating (43 degrees C for 30 minutes) 24 hours earlier to ascertain whether damage to spermatogenesis would affect 'leakage' of proteins from the seminiferous tubules. In all control rats, the STCM antiserum specifically detected three proteins in testicular IF with molecular weights of 24, 16, and 14 kDa, respectively. Heat treatment increa...
Journal of andrology
There is considerable variation, both within and between species, in reports of nuclear steroid r... more There is considerable variation, both within and between species, in reports of nuclear steroid receptor localizations in the male reproductive tract. In this study, androgen receptor (AR) and estrogen receptors ERalpha and ERbeta were visualized by immunohistochemistry in adult male mice reproductive tracts, including testes, efferent ductules; initial segment, caput, corpus, and cauda epididymides; and vas deferens. Antibody specificity was demonstrated by Western blot and antibody competition. In testis, AR was expressed in Leydig cells, Sertoli cells, and most peritubular cells, but not in germ cells; Sertoli cells showed more intense staining in stages VI-VII; ERalpha was present in Leydig and some peritubular cells; ERbeta was in Leydig, some peritubular, all Sertoli and germ cells except in spermatids and meiotic spermatocytes. In efferent ductules, AR was strongly expressed in ciliated and nonciliated epithelial cells and in stromal cells; ERalpha was strongly expressed in c...
Testicular cancer is more common in individuals with disor- ders of the male reproductive tract. ... more Testicular cancer is more common in individuals with disor- ders of the male reproductive tract. It has been suggested that inappropriate exposure to estrogens during fetal life may have an impact on maturation of testicular germ cells that are the cells of origin of the majority of testis cancers. The aim of the present study was to establish whether human
Steroids, 2002
Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER␣, NR3A1) and bet... more Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER␣, NR3A1) and beta (ER, NR3A2) have been identified. ER mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, and gonads in both males and females, as well as in cancers of the breast and prostate. In many tissues the pattern of expression of ER is distinct from that of ER␣. A number of variant isoforms of the wild type beta receptor (ER1), have been identified. In the human these include: (1) use of alternative start sites within the mRNA leading to translation of either a long (530 amino acids, hER1L) or a truncated form (487aa hER1s); (2) deletion of exons by alternative splicing; (3) formation of several isoforms (ER2-5) due to alternative splicing of exons encoding the carboxy terminus (F domain). We have raised monoclonal antibodies specific for hER1 as well as to three of the C terminal isoforms (2, 4 and 5). Using these antibodies we have found that ER2, 4 and 5 proteins are expressed in nuclei of human tissues including the ovary, placenta, testis and vas deferens.
Estrogen action is dependent upon the presence of specific ligand- activated receptors in target ... more Estrogen action is dependent upon the presence of specific ligand- activated receptors in target tissues. The aim of the present experi- ments was to compare the spatial and temporal pattern of expression of estrogen receptor b (ERb) with that of ERa in full thickness en- dometrial samples (from the superficial to the basal zone) obtained from both women and rhesus
The FASEB Journal, 2009
Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert th... more Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogenesis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM-specific AR-knockout (PTM-ARKO) mice in which gross reproductive development was normal, but all PTM-ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild-type littermates. These changes were not explained by any deficits in testosterone, luteinizing hormone, or follicle-stimulating hormone concentrations. SC function was impaired in PTM-ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen-dependent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen-driven stromal-epithelial interactions underpinning the regulation of spermatogenesis; PTM-ARKO mice will enable identification of the new molecular pathways involved.
Reproduction, 2005
An increase in scrotal temperature can lead to the production of poor quality spermatozoa and inf... more An increase in scrotal temperature can lead to the production of poor quality spermatozoa and infertility. In the present study we have used mice to examine the impact of mild, scrotal heat stress (42 degrees C for 30 min) on numbers of spermatozoa as well as on the integrity of their DNA. Spermatozoa recovered from the epididymides hours (1 to 24) or days (7 to 32) after treatment were analysed using COMET and sperm chromatin structure (SCSA) assays. The treatment induced a stress response in both the testis and the epididymis that was associated with reduced expression of the cold inducible RNA binding protein (Cirp) and an increase in germ cell apoptosis (Apotag positive cells). Although spermatozoa present in the epididymis at the time of heating contained correctly packaged DNA, its integrity was compromised by heat stress. In addition, although some germ cells, which were present within the testis at the time of heat stress, were removed by apoptosis, many germ cells completed their development and were recovered as motile spermatozoa with damaged DNA. In conclusion, these data demonstrate that scrotal heat stress can compromise the DNA integrity of spermatozoa and this may have clinical implications for patients undergoing IVF and intra-cytoplasmic sperm injection (ICSI).
Proceedings of the National Academy of Sciences, 2004
Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen... more Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre͞loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Mü llerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte͞spermatid development͞ survival critically depends on androgens.
PLoS ONE, 2011
Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic... more Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1-7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45⁻) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.
PLoS ONE, 2010
Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be i... more Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis.
Nature, 2011
Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphi... more Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0-D1-D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards β2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an 'innate HLA sensor' domain. Second, whereas the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA-B*5701…
The Biochemist, 2009
Androgens are the major drivers of masculinization and male fertility, and disruption of androgen... more Androgens are the major drivers of masculinization and male fertility, and disruption of androgen signalling has been implicated in the most common male congenital abnormalities, such as hypo spadias (opening of the urethra in a position not at the tip of the penis) and cryptorchidism (testes in the abdomen not the scrotum), as well as with an increased risk of developing testicular cancer or having a low sperm count in adulthood1.
Journal of Endocrinology, 2020
The endometrium is a complex multicellular tissue that is exquisitely sensitive to the actions of... more The endometrium is a complex multicellular tissue that is exquisitely sensitive to the actions of sex steroids synthesised in the ovary (endocrine system). Recent studies have highlighted a previously under-appreciated role for local (intracrine) metabolism in fine-tuning tissue function in both health and disease. In this review we have focused on the impact of oestrogens and androgens on endometrial function summarising data from studies on normal endometrial physiology and disorders including infertility, endometriosis and cancer. We consider the evidence that expression of enzymes including aromatase, sulphatase and AKR1C3 by endometrial cells plays an important role in tissue function and malfunction and discuss results from studies using drugs targeting intracrine pathways to treat endometrial disorders. We summarise studies exploring the spatial and temporal expression of oestrogen receptors (ERalpha/ESR1, ERbeta/ESR2 and GPER) and their role in mediating the impact of endoge...
Reproduction Abstracts, 2014
Smoking by women during pregnancy can result in a 30–48% reduction in sperm count and testis size... more Smoking by women during pregnancy can result in a 30–48% reduction in sperm count and testis size in the exposed offspring in adulthood, probably because of a decrease in the number of Sertoli cells. Until recently Sertoli cell proliferation was thought to be androgen independent because fetal Sertoli cells do not express the androgen receptor, but new evidence suggests that androgens may play the lead role in regulating Sertoli cell proliferation in fetal (Tan et al. 2005) and early neonatal life (Atanassova et al. 2005). We hypothesize that a unifying mechanism via which fetal exposure to environmental chemicals could affect sperm count in adulthood, is through interference with androgen production/action. To test this hypothesis, we have compared the effects of exposing fetal male rats from E13.5 onwards to 7,12-diemethyl benz[a]anthracene (DMBA) or it’s metabolite DMBA-DHD (as candidate molecules present in cigarette smoke), or n-dibutyl phthalate (DBP; a plasticizer shown to lower fetal testosterone levels), on Sertoli cell number, proliferation, apoptosis and functional development, using a combination of in vitro and in vivo studies. We have also used the AR antagonist, Flutamide, and tissue from mice, in which ablation of androgen action has been induced by transgenesis (ARKOs).Any effects that androgens do have on Sertoli cell proliferation/number must be indirect, so our studies are also directed at identifying the pathways via which any such effects could be mediated, such as local production of Insulin-Like Growth Factor 1 (IGF1) which has already been shown to increase Sertoli cell proliferation. We have demonstrated that fetal exposure to DMBA, DBP or Flutamide reduces expression of IGF1 mRNA, although preliminary results have so far shown no change in Sertoli cell number in treated animals and Sertoli cell proliferation indices are still under investigation
Journal of andrology
We have examined the location of the DAZL protein in fetal and adult rodents and human specimens ... more We have examined the location of the DAZL protein in fetal and adult rodents and human specimens and found that there is a shift from a predominantly nuclear to a predominantly cytoplasmic distribution of the protein in human testis. In rat testis and human ovary, however, the protein is predominantly, if not exclusively, cytoplasmic throughout germ cell development. One possible explanation for this could be that the DAZ protein is responsible for the nuclear localization of DAZL in human males. We have tested this hypothesis by examining the testis of marmosets, which lack the Daz genes and have found that the DAZL protein is both nuclear and cytoplasmic in spermatogonia, and by analyzing testis sections from DAZ-deleted patients in whom the cytoplasmic location of DAZL is evident in remaining germ cells. Transfection experiments indicate that the differences in DAZL expression between rodents and humans are not caused by the amino acid differences between the 2 proteins, and that...
Nature, Jan 4, 1997
RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins conta... more RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the different...
Nature Reviews Genetics, 2002
Molecular Biology of the Cell, 2007
Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs... more Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide ⌬-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.
BMC Developmental Biology, 2007
Background: Germ cells arise from a small group of cells that express markers of pluripotency inc... more Background: Germ cells arise from a small group of cells that express markers of pluripotency including OCT4. In humans formation of gonadal compartments (cords in testis, nests in ovary) takes place during the 1st trimester (6-8 weeks gestation). In the 2nd trimester germ cells can enter meiotic prophase in females whereas in males this does not occur until puberty. We have used qRTPCR, Westerns and immunohistochemical profiling to determine which of the germ cell subtypes in the human fetal gonads express OCT4, DAZL and VASA, as these have been shown to play an essential role in germ cell maturation in mice.
Journal of andrology
The aim of the present study was to assess whether proteins secreted by the seminiferous tubules ... more The aim of the present study was to assess whether proteins secreted by the seminiferous tubules (ST) can be detected in testicular interstitial fluid (IF) and testicular (TV), spermatic (SV), and peripheral venous (PV) plasma from adult rats. An antiserum was raised against seminiferous tubule-conditioned medium (STCM) prepared from adult rats and used in conjunction with Western blot analysis to screen IF and blood samples resolved by one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of IF and PV were analyzed from control adult rats and rats exposed to scrotal heating (43 degrees C for 30 minutes) 24 hours earlier to ascertain whether damage to spermatogenesis would affect 'leakage' of proteins from the seminiferous tubules. In all control rats, the STCM antiserum specifically detected three proteins in testicular IF with molecular weights of 24, 16, and 14 kDa, respectively. Heat treatment increa...
Journal of andrology
There is considerable variation, both within and between species, in reports of nuclear steroid r... more There is considerable variation, both within and between species, in reports of nuclear steroid receptor localizations in the male reproductive tract. In this study, androgen receptor (AR) and estrogen receptors ERalpha and ERbeta were visualized by immunohistochemistry in adult male mice reproductive tracts, including testes, efferent ductules; initial segment, caput, corpus, and cauda epididymides; and vas deferens. Antibody specificity was demonstrated by Western blot and antibody competition. In testis, AR was expressed in Leydig cells, Sertoli cells, and most peritubular cells, but not in germ cells; Sertoli cells showed more intense staining in stages VI-VII; ERalpha was present in Leydig and some peritubular cells; ERbeta was in Leydig, some peritubular, all Sertoli and germ cells except in spermatids and meiotic spermatocytes. In efferent ductules, AR was strongly expressed in ciliated and nonciliated epithelial cells and in stromal cells; ERalpha was strongly expressed in c...
Testicular cancer is more common in individuals with disor- ders of the male reproductive tract. ... more Testicular cancer is more common in individuals with disor- ders of the male reproductive tract. It has been suggested that inappropriate exposure to estrogens during fetal life may have an impact on maturation of testicular germ cells that are the cells of origin of the majority of testis cancers. The aim of the present study was to establish whether human
Steroids, 2002
Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER␣, NR3A1) and bet... more Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER␣, NR3A1) and beta (ER, NR3A2) have been identified. ER mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, and gonads in both males and females, as well as in cancers of the breast and prostate. In many tissues the pattern of expression of ER is distinct from that of ER␣. A number of variant isoforms of the wild type beta receptor (ER1), have been identified. In the human these include: (1) use of alternative start sites within the mRNA leading to translation of either a long (530 amino acids, hER1L) or a truncated form (487aa hER1s); (2) deletion of exons by alternative splicing; (3) formation of several isoforms (ER2-5) due to alternative splicing of exons encoding the carboxy terminus (F domain). We have raised monoclonal antibodies specific for hER1 as well as to three of the C terminal isoforms (2, 4 and 5). Using these antibodies we have found that ER2, 4 and 5 proteins are expressed in nuclei of human tissues including the ovary, placenta, testis and vas deferens.
Estrogen action is dependent upon the presence of specific ligand- activated receptors in target ... more Estrogen action is dependent upon the presence of specific ligand- activated receptors in target tissues. The aim of the present experi- ments was to compare the spatial and temporal pattern of expression of estrogen receptor b (ERb) with that of ERa in full thickness en- dometrial samples (from the superficial to the basal zone) obtained from both women and rhesus
The FASEB Journal, 2009
Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert th... more Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogenesis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM-specific AR-knockout (PTM-ARKO) mice in which gross reproductive development was normal, but all PTM-ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild-type littermates. These changes were not explained by any deficits in testosterone, luteinizing hormone, or follicle-stimulating hormone concentrations. SC function was impaired in PTM-ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen-dependent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen-driven stromal-epithelial interactions underpinning the regulation of spermatogenesis; PTM-ARKO mice will enable identification of the new molecular pathways involved.
Reproduction, 2005
An increase in scrotal temperature can lead to the production of poor quality spermatozoa and inf... more An increase in scrotal temperature can lead to the production of poor quality spermatozoa and infertility. In the present study we have used mice to examine the impact of mild, scrotal heat stress (42 degrees C for 30 min) on numbers of spermatozoa as well as on the integrity of their DNA. Spermatozoa recovered from the epididymides hours (1 to 24) or days (7 to 32) after treatment were analysed using COMET and sperm chromatin structure (SCSA) assays. The treatment induced a stress response in both the testis and the epididymis that was associated with reduced expression of the cold inducible RNA binding protein (Cirp) and an increase in germ cell apoptosis (Apotag positive cells). Although spermatozoa present in the epididymis at the time of heating contained correctly packaged DNA, its integrity was compromised by heat stress. In addition, although some germ cells, which were present within the testis at the time of heat stress, were removed by apoptosis, many germ cells completed their development and were recovered as motile spermatozoa with damaged DNA. In conclusion, these data demonstrate that scrotal heat stress can compromise the DNA integrity of spermatozoa and this may have clinical implications for patients undergoing IVF and intra-cytoplasmic sperm injection (ICSI).
Proceedings of the National Academy of Sciences, 2004
Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen... more Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre͞loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Mü llerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte͞spermatid development͞ survival critically depends on androgens.
PLoS ONE, 2011
Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic... more Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1-7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45⁻) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.
PLoS ONE, 2010
Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be i... more Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis.
Nature, 2011
Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphi... more Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0-D1-D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards β2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an 'innate HLA sensor' domain. Second, whereas the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA-B*5701…