Development and validation of a new monoclonal antibody to mammalian aromatase

Turner, Katie J.; MacPherson, Sarah E.; Millar, Michael; McNeilly, A. S.; Williams, Karin; Cranfield, Mark; Groome, NP; Sharpe, Richard M.; Fraser, H. M.; Saunders, Philippa T. K.

doi:10.1677/joe.0.1720021

Cited by 112 publications

(89 citation statements)

References 51 publications

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“…However, recently, aromatase has also been immunolocalised to the cytoplasm surrounding elongated spermatids (Turner et al 2002), and we and others have reported the presence of aromatase in human ejaculated spermatozoa (Aquila et al 2002, Lambard et al 2003.…”

Section: Introductionmentioning

confidence: 65%

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

Lambard¹,

Galeraud-Denis²,

Saunders³

et al. 2004

Journal of Molecular Endocrinology

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122

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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 (ER and ER ). 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, ER and ER were amplified from total RNA isolated from human germ cells and spermatozoa. We identified an ER 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 ER at ∼ 50 and ∼ 60 kDa, which could correspond to the long and short forms of ER formed from the use of alternative start sites. In human ejaculated spermatozoa, ER protein was not detected, even though we could amplify mRNA. Using Western blot analysis and a monoclonal antibody specific for ER , 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 ER 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.

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Section: Introductionmentioning

confidence: 65%

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

Lambard¹,

Galeraud-Denis²,

Saunders³

et al. 2004

Journal of Molecular Endocrinology

Self Cite

122

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“…Laser microdissection of preantral and antral follicles has recently confirmed these results [20]. Within the granulosa cell layer of antral follicles, aromatase expression is highest in the mural granulosa cells at the outer edge of the follicle when compared to granulosa cells closest to the antral cavity [14,21]. Moreover, aromatase is not expressed in cumulus granulosa cells [14,21].…”

Section: Mature Ovariesmentioning

confidence: 85%

“…Within the granulosa cell layer of antral follicles, aromatase expression is highest in the mural granulosa cells at the outer edge of the follicle when compared to granulosa cells closest to the antral cavity [14,21]. Moreover, aromatase is not expressed in cumulus granulosa cells [14,21]. The marked compartmentalization of aromatase expression within the granulosa layer is particularly striking in mature follicles from rats at proestrus (Fig.…”

Section: Mature Ovariesmentioning

confidence: 91%

Aromatase expression in the ovary: Hormonal and molecular regulation

2008

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Summary-Estrogens are synthesized by the aromatase enzyme encoded by the Cyp19a1 gene, which contains an unusually large regulatory region. In most mammals, aromatase expression is under the control of two distinct promoters a gonad-and a brain-specific promoter. In humans, this gene contains 10 tissue-specific promoters that are alternatively used in various cell types and tumors. Each promoter is regulated by a distinct set of regulatory sequences and transcription factors that bind to these specific sequences. The cAMP/PKA/CREB pathway is considered to be the primary signaling cascade through which the gonad Cyp19 promoter is regulated. Very interestingly, in rat luteal cells, the proximal promoter is not controlled in a cAMP dependent manner. Strikingly, these cells express aromatase at high levels similar to those found in preovulatory follicles, suggesting that alternative and powerful mechanisms control aromatase expression in luteal cells and that the rat corpus luteum represents an important paradigm for understanding alternative controls of the aromatase gene. Here, the molecular and cellular mechanisms controlling the expression of the aromatase gene in granulosa and luteal cells are discussed.

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“…Antisera produced in rabbits against recombinant bovine P450c17 (Peterson et al 2001) and recombinant human P450arom (Conley et al 1996) served as primary antibodies. Additionally, a murine monoclonal antibody against a synthetic peptide corresponding to amino acids 376-390 of human P450arom (Turner et al 2002) was tested (clone H4, purchased from Serotec GmbH, Dü sseldorf, Germany); the peptide sequence used for immunisation is completely conserved in bovine P450arom. Serum from a non-immunised rabbit or dilution buffer were used as negative controls.…”

Section: Immunohistochemical Staining Proceduresmentioning

confidence: 99%

Reciprocal expression of 17α-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis

Schuler¹,

Özalp²,

Hoffmann³

et al. 2006

Reproduction

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No definitive information is yet available on the steroidogenic capacity of the two morphologically distinct cell types forming the bovine trophoblast, the uninucleated trophoblast cells (UTCs) and the trophoblast giant cells (TGCs). Hence, in order to localise 17a-hydroxylase-C17,20-lyase (P450c17) on a cellular level and to monitor its expression as a function of gestational age, placentomes from pregnant (days 80 -284; n 5 19), prepartal (days 273 -282; 24 -36 h prior to the onset of labour; n 5 3) and parturient cows (n 5 5) were immunostained for P450c17 using an antiserum against the recombinant bovine enzyme. At all stages investigated, P450c17 was exclusively found in the UTCs of chorionic villi (CV), where staining was ubiquitous between days 80 and 160, but was largely restricted to primary CV and the branching sites of secondary CV between days 160 and 240. Thereafter, a distinct ubiquitous staining reoccurred in the UTCs of all CV in late pregnant, prepartal and parturient animals. Using an antiserum against human aromatase cytochrome P450 (P450arom), specific cytoplasmic staining was observed in TGCs. In placentomes from pregnant cows, staining intensity was higher in mature compared with immature TGCs and was more pronounced in the trophoblast covering big stem villi compared with the trophoblast at other sites of the villous tree. In placentomes of a parturient cow, specific staining was only found in mature TGCs that survived the normal, but substantial, prepartal decline in TGC numbers. These results clearly showed that bovine UTCs and TGCs exhibit different steroidogenic capacities, constituting a 'two-cell' organisation for oestrogen synthesis. P450c17 expression appears to be quickly down-regulated and P450arom is up-regulated when UTCs enter the TGC differentiation pathway.

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Development and validation of a new monoclonal antibody to mammalian aromatase

Cited by 112 publications

References 51 publications

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

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

Aromatase expression in the ovary: Hormonal and molecular regulation

Reciprocal expression of 17α-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis

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