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S Lambard, I Galeraud-Denis, PT Saunders and S Carreau

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.

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P.F. Whitelaw, C.D. Smyth, C.M. Howles and S.G. Hillier

ABSTRACT

Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.

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O Nakabayashi, H Kikuchi, T Kikuchi and S Mizuno

In birds, differentiation of embryonic gonads is not as strictly determined by the genetic sex as it is in mammals, and can be influenced by early manipulation with a sex steroid hormone. Thus administration of an aromatase inhibitor induces testis development in the genetic female, and administration of estrogen induces a left ovotestis in the genetic male embryo. Another feature of avian gonadogenesis is that only the left ovary develops in most species. Molecular mechanisms underlying these features at the level of gene expression have not been elucidated. In this paper, we present evidence that a gene for aromatase cytochrome P-450, an enzyme required for the last step in the synthesis of estradiol-17beta, is expressed in medullae of the left and right gonads of a female chicken embryo, but not in those of a male chicken embryo, and that an estrogen receptor gene is expressed only in epithelium (and cortex later, in the female) of the left, not the right, gonad of both sexes, but the expression in the male left gonad is temporary and restricted to an early stage of development. Differential expression of these two genes serves well to explain the above features of gonadal development in birds. Furthermore, in ovo administration of estradiol-17beta from the 5th to the 14th day of incubation does not cause expression of the estrogen receptor gene in the right gonad of chicken embryos of either sex, suggesting that the absence of expression of the estrogen receptor gene in the right gonad is not the result of down-regulation, but may be regarded as an important cause of the unilateral ovarian development.

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S A Gray, M A Mannan and P J O'Shaughnessy

ABSTRACT

The cytochrome P450 aromatase (P450arom) enzyme is required for bioconversion of androgen to oestrogen. In this study ovarian P450arom mRNA and enzyme activity have been measured during development in normal mice and hypogonadal (hpg) mice which lack circulating gonadotrophins. A semi-quantitative reverse transcription-PCR (RTPCR) technique was used to measure cytochrome P450arom mRNA levels and aromatase enzyme activity was measured directly. Using RT-PCR, P450arom mRNA was detectable in the adult mouse ovary and also in the uterus, kidney, brain and skeletal muscle but not in cardiac smooth muscle. In the normal mouse, P450arom mRNA was detectable in the ovary on the day of birth (day 1) and levels increased significantly up to day 15 with the most marked changes seen between days 1 and 5. Aromatase activity was also detectable at all ages in the ovary and increased significantly between days 1 and 7. In ovaries from [ill] mice, normal levels of P450arom mRNA were present on day 1 but there was no significant change in P450arom mRNA at later ages up to day 15. These results show that in the newborn mouse ovary, which contains only primordial follicles, there is a basal expression of P450arom mRNA which is not gonadotrophindependent. After 1 day, however, gonadotrophins are required for normal expression of ovarian P450arom and this coincides with development of primary and secondary follicles.

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J Levallet, H Mittre, B Delarue and S Carreau

Expression of cytochrome P450 mRNA in rat germ cells was characterized by reverse transcription PCR with various primers located at the 3'-end of the coding region. At least two unusual isoforms (Ex10-S and INT) of P450 aromatase (P450arom) mRNA were expressed. Analysis of their sequences demonstrated that an alternative splicing event occurred first at the exon-intron boundary of the GT consensus sequence of the last coding exon, and second in the internal 5' donor inside exon 9 used as a minor cryptic splicing site. These isoforms lacked the last coding exon which contained the heme-binding domain; in addition, for the Ex10-S transcript, the catalytic domain was also absent because of a frameshift in the open reading frame. The deduced amino acid sequences led to truncated P450arom polypeptides without the heme-binding domain, which were probably unable to convert androgens into estrogens. Adult rat germ cells are able to express P450arom mRNA, which is then translated into a biologically active enzyme which is involved in estrogen production. Moreover, for the first time, we report the existence of alternative splicing events of P45Oarom mRNA in pachytene spermatocytes and round spermatids, which probably cannot encode functional aromatase molecules.

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E L Yong, S G Hillier, M Turner, D T Baird, S C Ng, A Bongso and S S Ratnam

ABSTRACT

The co-ordinated biosynthesis of progesterone and oestradiol in the human ovary is critical for reproductive cyclicity and eventual pregnancy. The crucial regulatory enzymes for progesterone and oestradiol biosynthesis in granulosa cells are the cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LH surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gonadotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (dbcAMP). After the period of culture, total RNA was extracted from granulosa cells and Northern analyses were performed utilizing 32P-labelled cDNAs encoding P450arom and P450scc. Spent culture media were analysed for steroid and cAMP content.

Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone biosynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concentrations, LH generated a 30-fold higher level of cAMP than FSH. Furthermore, the differential effects of FSH and LH on P450 mRNA expression were reproduced by the presence of low and high concentrations of dbcAMP respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation in granulosa lutein cells. In contrast, it stimulated P450scc mRNA expression and progesterone synthesis in both mature granulosa and granulosa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom gene expression and oestradiol production, while LH/high cAMP levels maximally induced P450scc gene expression and function, in a development-related manner consistent with steroid production in vivo. These findings support the hypothesis that one set of genes (like P450arom) in human granulosa cells is regulated by FSH/low cAMP levels and another (like P450scc) by LH/high cAMP levels.

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X T Chang, T Kobayashi, H Kajiura, M Nakamura and Y Nagahama

ABSTRACT

A cDNA clone encoding the complete tilapia (a teleost fish, Oreochromis niloticus) cytochrome P450 aromatase (P450arom) was isolated from an ovarian follicle cDNA library. The deduced amino acid sequence (522 amino acid residues) had 72·2% and 59·5% homology with rainbow trout and catfish P450arom respectively, and about 50% homology with mammalian and avian P450arom. Expression of this cDNA in COS-7 cells produced a protein that converted exogenous testosterone to estrogens. Northern blots using a tilapia P450arom cDNA fragment and Western blots using an antiserum against a tilapia P450arom polypeptide fragment revealed a single P450arom mRNA (2·6 kb) and a single protein (59 kDa) in tilapia ovarian tissue respectively. These analyses also revealed that the levels of both P450arom mRNA and protein were low in early vitellogenic follicles, increased in midvitellogenic follicles, and declined to non-detectable levels in post-vitellogenic follicles. Changes in the ability of follicles to convert exogenous testosterone to estrogens (aromatase activity) were similar to those of P450arom mRNA and protein. These observations indicated that the capacity of tilapia ovarian follicles to synthesize estradiol-17β is closely related to the contents of P450arom mRNA and protein within them.

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M. Tanaka, T. M. Telecky, S. Fukada, S. Adachi, S. Chen and Y. Nagahama

ABSTRACT

The enzyme aromatase P-450 (P450arom) catalyses the conversion of androgen to oestrogen. A cDNA insert encoding P450arom was isolated from a rainbow trout (Oncorhynchus mykiss) ovary cDNA library. The insert was sequenced and found to contain an open-reading frame predicted to encode a protein of 522 amino acid residues. The deduced polypeptide is 52% homologous with human, mouse and rat P450arom and 53% homologous with that of chicken. The insert was confirmed to encode P450arom by introducing it into COS-1 monkey kidney tumour cells (COS-1 cells) and detecting the conversion of testosterone to oestradiol-17β by radioimmunoassay. The N-terminal region of the deduced polypeptide was 19 amino acids longer than that of the other four species, and was found by hydropathy plotting to be very hydrophobic.

Northern blot analysis revealed 2.6kb RNA transcripts which were present in the trout ovary during vitellogenesis and hybridized to the cDNA insert. In preparations from subsequent stages of ovarian development, no RNA transcripts hybridized to the probe. Since the RNA transcripts are present only during the stage of oestradiol-β production by the ovarian follicles, oestradiol-17β production may be regulated, in part, by the amount of P450arom mRNA present.

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T Kitano, K Takamune, T Kobayashi, Y Nagahama and SI Abe

The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.