Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar.

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.

CD44 exon 6 expression as a possible early prognostic factor in primary node negative breast carcinoma.

Breast cancer is the most common female malignancy affecting approximately one woman in eight. Many attempts have been made to define markers which may have potential clinical applications in diagnosis as well as therapy. New isoforms of CD44 with alternative spliced exons have recently been described. We studied the expression of CD44 exon 6 using a semi-quantitative RT-PCR reaction on a panel of 25 normal breast specimens, 10 mammary fibroadenomas, eight cystic samples and 52 primary breast tumors. Significant correlation was found between CD44 exon 6 expression and the overall survival of the N-M-population, P = 0.032, (logrank test by Mantel's method). The same result was also observed for the disease-free survival, P = 0.000002 (logrank test by Mantel's method). CD44 exon 6 expression, as detected by our RT-PCR-based method, might be a useful prognostic indicator of metastasis in breast cancer. However, these preliminary results need to be confirmed by later retrospective and prospective studies on a larger number of patients.

'Dosimetric breast size': a new and useful parameter for the prediction of local recurrence after breast conservative treatment.

To evaluate the risk of local recurrence following breast-conserving therapy for breast cancer, we measured the distance between each entry point of the irradiation on the surface of the breast in line with the axis of the external and internal tangential fields (dosimetric breast size). 652 breast cancer patients were retrospectively analysed, with a median age of 51 years and a median follow-up of 99 months (range 84-192). There were 50 local recurrences, 44 isolated and 6 associated with nodal recurrence or metastases. The global rates of local recurrences at 5 and 10 years were 5.3% and 9%, respectively (Kaplan-Meier analysis). Following a Cox's multivariate analysis, the only significant and independent parameters related to local recurrence were quality of excision, age at diagnosis and dosimetric breast size. For a small dosimetric breast size (< or = 10 cm), the rate of local recurrence was 14.1 compared with 11.8 for medium dosimetric breast size (> 10 cm-< or = 12 cm) and 5.2 for large dosimetric breast size (> 12 cm). If the analysis was restricted to only those with complete excision, then the relative risk for a patient with a small dosimetric breast size was three times that for a large breast size.

Radical surgery and conservative treatment of ductal carcinoma in situ of the breast.

70 cases of strictly intraductal breast carcinoma were treated from January 1975 to December 1987. 34 patients underwent radical modified mastectomy, and 36 patients had local excision (2), lumpectomy (26) or quadrantectomy (8), with a complementary irradiation in 34/36 cases (with boost in 32). The main histological subtype is comedocarcinoma (25/70). One local relapse (3%) is noted in radical surgery group at 55 months. 3 local relapses (9%) are noted in conservative treatment group, respectively at 27, 48 and 52 months. The obvious factor influencing the local recurrence is the inefficient surgical excision. Since breast screening programs may lead to early duct carcinoma in situ identification, our results suggest that appropriate conservative surgery associated to radiation therapy could be an adequate alternative to mastectomy in the treatment of this in situ lesion.

Association between breast cancer and family malignancies.

In a case-control study, the relationship between a family history of cancer of the breast, ovary, colon, uterus or prostate and the risk of breast cancer was investigated. The data consisted of family histories from 495 breast cancer cases and 785 controls aged 20-56 years. A positive association was found between the occurrence of breast cancer and a history of breast cancer in the families of the subjects affected. This relationship increased linearly with both the degree of kinship of the affected relatives and with their number. The risk of breast cancer associated with other types of cancer in the family was not significantly different from unity.

[Specific expression of human pS2 gene in breast cancer]. / Expression spécifique du gène humain pS2 dans les cancers du sein.

The hormone-dependence of some human breast cancers is well recognized. However, the molecular mechanisms responsible for the growth stimulation of these cancers by oestrogens are still poorly understood. With the hope of elucidating these mechanisms, we have recently cloned and studied the structure-function relationship of the human oestrogen and progestin receptors, and also undertaken a study aimed at characterizing genes whose expression is controlled by oestrogens in hormone-dependent breast cancers. We review here our findings concerning one of these genes and its expression products, the pS2 gene. We discuss also whether a systematic determination of pS2 gene expression in breast cancer biopsies could be useful to establish a new biochemical classification of these cancers which may be useful to improve the diagnosis of hormone-dependent cancers.

Solid state fermentation reactors: from lab scale to pilot plant.

Relatively many workers in the world are studying different aspects in SSF processes but few are working on reactor design and scale-up. From about 10 years, we are developing reactors from lab scale to pilot plant, based on the same technology, reactor design and flowsheet to allow fermentation with a deep layer (up to 1 m in the pilot plant). These reactors have all a forced aeration and the possibility or not to agitate. Regulations of temperature and water content of the culture are monitored by a special device.

Identification of 3 beta-hydroxy-5,7-androstadien-17-one as a secretory product of the fetal horse gonad in vivo and in vitro.

Isolation of 3 beta-hydroxy-5,7-androstadien-17-one, as a major component of steroids extracted from vein blood of the fetal gonads of the horse, supports the proposed role for the compound as a precursor for equilin formation in the placenta of the mare. The 5,7-diene was extracted from blood collected from gonadal veins of fetal ovaries and testes in situ, and from a fetal testis connected to an artery in the neck region of the mare. Perfusion of fetal gonads in the laboratory was carried out to allow longer periods of collection. In addition, isolated cell preparations from a fetal testis were incubated for 4-8 h in tissue culture to investigate steroid secretion in vitro. Final purification of neutral steroids in the extracts was carried out by high performance liquid chromatography, and identification was made by u.v. and mass spectrometry. The presence of 3 beta-hydroxy-5,7-androstadien-17-one in extracts from all sources provided evidence for its secretion in vivo and in vitro. Other 5,7-dienes, which were less polar than the C19 compound, were noted in extracts of media but not identified. These data support the view that a 5,7-diene pathway is involved in the biosynthesis of 3 beta-hydroxy-5,7-androstadien-17-one in the fetal horse gonad.

Metabolism of oestrone and oestradiol-17beta to conjugated steroids by the accessory sex glands of the male pig.

Oestrogens are secreted in large amounts by boar testes and are known to have a synergistic effect with testosterone on the production of large volumes of seminal plasma. Thus, oestrogens play a role in regulating the large accessory sex glands in the boar. Since testosterone metabolites (e.g. 5alpha-dihydrotestosterone) account for much of its action in target tissues we have looked at the metabolism of oestrogens in the accessory sex glands of the male pig. Tissues from seminal vesicles and bulbourethral glands of 6-week-old castrate and intact males, and 12-week-old castrate animals, were incubated with (3)H-labelled oestrone and oestradiol-17beta. Aliquots of spent culture medium and of methanolic tissue extracts were taken to measure radioactivity, prior to separation of unconjugated and conjugated steroids on Waters C(18) Sep-Pak cartridges. About one-third of the radioactivity appeared as conjugates in the media from both glands with each oestrogen. Subsequently, sulphoconjugated steroids and glucuronidates were recovered in series from C(18) cartridges after solvolysis and enzyme hydrolysis respectively. Furthermore, about one-third of the conjugated fraction in each case remained unhydrolysed after these treatments. In conclusion, it is clear that a study of the actions of oestrogens on these glands must consider the dynamics of metabolism of the oestrogens presented to them by the testes and would include conjugation of steroids by the glands themselves.

Postnatal decline in gonadal secretion of dehydroepiandrosterone and 3 beta-hydroxyandrosta-5,7-dien-17-one in the newborn foal.

Dehydroepiandrosterone (DHEA) and 3 beta-hydroxyandrosta-5,7-dien-17-one (7-dehydro-DHEA) are secreted in large quantities by the remarkably hypertrophied fetal gonads of both sexes in the pregnant mare. Their secretion serves as the fetal component of a feto-placental unit for oestrogen production in equine pregnancies. They are secreted in large amounts but show a decline in late pregnancy when the fetal gonads regress and levels of oestrogens in the mare fall as a consequence. We have examined the levels of these precursor steroids in the newborn foal in the first days after birth. DHEA and 7-dehydro-DHEA were measured in peripheral plasma in a direct RIA with a DHEA antibody which cross-reacts with 7-dehydro DHEA (> 150%). Subsequent studies were performed with solid-phase extraction, separation of unconjugated from conjugated steroids, and HPLC fractionation followed by RIA. Detection on HPLC at 254 and 280 nm was compared with results from RIA. It was concluded that DHEA is the major steroid produced by the gonads at birth. The concentrations are highly variable in the first day of postnatal life (70.45 +/- 63.06 ng/ml, n = 52) and decline rapidly to < 2 ng/ml (n = 6) at 96 h after birth. At this time the sulphate form is also seen, with an increasing ratio of DHEAS/DHEA as the value for total DHEA falls. The mechanism and significance of the apparent abrupt decline in gonadal steroidogenesis in the newborn foal remain unknown.

Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo.

19-Hydroxytestosterone and 19-hydroxyandrostenedione have been identified as secretory products of the testes in the mature male domestic pig. Their isolation and identification were made by reverse-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry (CGC-MS) of extracts from testicular vein blood and media of incubations with Leydig cells. Blood was collected from veins on the surface of the testes of anaesthetized boars. Collagenase-dispersed Percoll-purified cells (> 90% pure) were incubated (20 x 10(6) cells/flask) with androstenedione (8.75 mumol/l) or [3H]androstenedione (5 x 10(6) c.p.m.) for < 60 min. Steroids were recovered from plasma or media by solid-phase extraction and the unconjugated fractions chromatographed isocratically in two solvent systems (acetonitrile:water, 37:63 (v/v) and methanol:water, 70:30 (v/v)) before CGC-MS analysis. 19-Hydroxytestosterone was present in greater quantities than 19-hydroxyandrostenedione in testicular vein blood; it was also seen as a quantitatively significant metabolite of unlabelled and radioactive androstenedione in the incubation studies. The demonstration of the secretion of 19-hydroxyandrogens from porcine testes thus raises questions concerning the physiological significance of a testicular, rather than an adrenal, secretion of these compounds.

Reproductive factors and breast cancer risk. Effect of age at diagnosis.

The data from a French case-control study of 495 patients with breast cancer and 542 control subjects interviewed in five French public hospitals, were analyzed to assess the effect of reproductive factors (age at menarche, age at first full-term pregnancy, the time interval between these two ages, and parity) on the risk of breast cancer. Age at menarche, age at first full-term pregnancy, the time interval between these two ages, and parity appeared to have a limited influence on breast cancer risk. However, the relationship between these factors and the risk of breast cancer varied according to the age at breast cancer diagnosis. In the youngest group of women, the most consistent effects came from factors occurring early in life (menarche, first full-term pregnancy, and consequently the time interval between these two events). These factors had a null or weak effect on the oldest group of women. The protective effect of high parity was confined to the oldest group of women.

Expression of CD44 spliced variants with both exons v9 and v10 in primary N(-)M(-) breast carcinomas correlates with metastasis.

We analyzed the representation of CD44 isoforms with both exons v9 and v10 among CD44 total amount and also examined correlation between their expression, clinical parameters and survival. We used a semi-quantitative RT-PCR reaction and a panel of 25 normal breast specimens, 10 mammary fibroadenomas, 8 cystic samples and 52 primary breast tumors. CD44 expression level was statistically higher in malignant tumors than in normal breast tissues (p = 0.038) or in fibroadenomas (p = 0.047) and correlated with histological grading, p = 0.047. Ratios CD44 variants with both exons v9 and v10/ total CD44 were similar in normal breast tissues and fibroadenomas but lower in the cystic samples. In primary N(-)M(-) breast tumors, unfavourable outcome and relapse were correlated with low ratios.

Variations in the risk of breast cancer associated with a family history of breast cancer according to age at onset and reproductive factors.

In a case-control study of 495 breast cancer patients and 785 controls between 20 and 56 years of age, the risk of breast cancer associated with a family history of breast cancer was studied according to age and reproductive factors. The familial risk of breast cancer was not significantly modified by age at onset, age at menarche, number of children, age at first full-term pregnancy, menstrual cycle length or age at menopause. However, the familial risk significantly increased with the number of abortions (p < 0.05) and seemed to decrease after a natural menopause (p = 0.08). These results suggest that a familial predisposition to breast cancer exerts the same influence during the first six decades of life, except maybe when there are isolated or repeated events such as abortions or artificially imposed menopause, in which case the risk is apparently greater.

Oral contraceptives and breast cancer: a French case-control study.

The relationship between the risk of breast cancer and oral contraceptive use was investigated in a case-control study conducted in France between 1983 and 1987 in five public hospitals. Some 464 cases aged 25 to 56 years and 542 matched controls were interviewed about their history of the use of oral contraceptives (OC). Results are given for the entire population and for the subgroup of 358 and 379 premenopausal cases and controls. The multivariate relative risk estimate, for ever user, was 1.5 (p less than 0.01) in the whole group as well as in the premenopausal subgroup (p less than 0.02). However, there was no evidence that the effect varied appreciably according to duration of use, age at first use, use before first full-term pregnancy (FFTP) and time since first or last use. The risk was not altered for any particular brand of OC. We conclude that, because of the widespread attention given to the relationship between OC use and breast cancer, information bias might be responsible for part of the excess in risk observed among OC ever users.

PIP: The relationship between the risk of breast cancer and oral contraceptive (OC) use was investigated in a case-control study conducted in France between 1983-87 in 5 public hospitals. Some 464 cases ages 25-56 years and 542 matched controls were interviewed about their history of OC use. Results are provided for the entire population and for the subgroups of 358 and 379 premenopausal cases and controls. The multivariate relative risk estimate, for ever-use, was 1.5 (p0.01) in the entire group as well as in the premenopausal subgroup (p0.02). However, there was no evidence that the effect varied appreciably according to the duration of use, age at 1st use, use prior to 1st fullterm pregnancy, and time since 1st or last use. The risk was not altered for any particular OC brand. The authors conclude that because of the widespread attention given to the relationship between OC use and breast cancer, information bias might be responsible for a part of the excess of risk observed among OC ever-users.

Variation in the interaction between familial and reproductive factors on the risk of breast cancer according to age, menopausal status, and degree of familiality.

BACKGROUND: Studies have found that reproductive factors might have a variable effect on the occurrence of breast cancer (BC) according to the existence or not of a family history of BC. The effect of a family history of BC on the risk of BC may also vary according to the age at diagnosis and the degree of kinship. This may confound the relation between familial risk and reproductive factors. A combined analysis was performed to study the interaction between familial risk and reproductive factors according to degree of familiality, age at interview and menopausal status. METHODS: The present analysis included 2948 cases and 4170 controls in seven case-control studies from four countries. The combined relative risks were estimated using a Bayesian random-effects logistic regression model. RESULTS: The main effects of reproductive life factors on the risk of BC are in agreement with previous studies. Two-way interactions between subject's age or menopausal status and a family history of BC were not significant. Although the three-way interaction between age, familial risk and parity was not significant, familial risk seemed to be increased slightly for women with high parity compared with women with low parity in the older age group, and seemed to be slightly decreased for women with high parity compared with women with low parity in younger women. The subject's age also appeared to have an effect on the interaction between familial risk and the age at first childbirth (P = 0.1). CONCLUSIONS: A possible influence of reproductive and menstrual factors on familial risk of BC has been suggested previously and was also evident in the present study. Three-way interactions between age, family history and parity or age at first childbirth might exist and they merit further investigation.

Identification of 5 alpha-androstane-3 beta,17 beta-diol and 3 beta-hydroxy-5 alpha-androstan-17-one sulfates as quantitatively significant secretory products of porcine Leydig cells and their presence in testicular venous blood.

By means of high performance liquid chromatography and gas chromatography-mass spectrometry it has been found that 5 alpha-androstane-3 beta,17 beta-diol sulfate and 3 beta-hydroxy-5 alpha-androstan-17-one sulfate (epiandrosterone) are major secretory steroids of the mature boar testes. These same compounds were similarly identified in culture media when porcine Leydig cells were incubated with androstenedione as substrate. In addition, they were seen as the principal secretory products when [3H]androstenedione and [3H]testosterone were used as substrates; and their presence was greatly reduced by an inhibitor of 5 alpha-reductase (N,N-diethyl,4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide). Greater quantities of 5 alpha-androstanediol than epiandrosterone were noted in all instances. These findings provide further evidence of the versatile activity of the boar testes in steroidogenesis.

Hydroxylation of 19-norandrogens by porcine Leydig cells.

The metabolism of 19-norandrogens by porcine Leydig cells was investigated. Non-radioactive 19-norandrostenedione (19-Nor A) and [3H]19-nortestosterone (19-Nor T) were used as substrates in incubations with cell preparations from mature male pigs. Steroid products were separated by reversed-phase HPLC and material in selected peaks was rechromatographed before attempts to identify them by GC-MS. Both 11 beta- and 6 beta-hydroxylated derivatives of 19-Nor A were found and a third product (11-oxo-19-Nor A) was tentatively identified. The profile of radioactive metabolites from [3H]19-Nor T also favours the view of a capacity for hydroxylation of 19-norandrogens by porcine Leydig cells. The significance of these findings together with our earlier report of direct 11 beta-hydroxylation of C19 steroids by such cells remains to be examined.

Estrogen causes cell death of estrogen receptor stably transfected cells via apoptosis.

We examined the effect of estrogen on the growth of estrogen receptor (ER) stably transfected cells (Rat1 + ER). 17-Beta-estradiol (E2, 10 nM) inhibited approximately 35-50% of Rat1 + ER growth after 3 d of treatment. The half-maximal growth inhibition occurred at 0.5-0.75 nM of E2 concentration and was saturated above 10 nM. This E2-induced antiproliferative effect was mediated through the ER since E2 did not cause any change in ER-negative parental Rat1 cells. Cells started to detach from plates and the adherent cells exhibited nuclear condensation. Apoptotic cell populations showed a 25% increase at 2 d of E2 treatment over controls that were quantified by fluorescence-activated cell sorter analysis. This indicates that E2 induced apoptosis in Rat1 + ER cells.

Echographic study of follicular maturation and ovulation during the normal menstrual cycle.

Follicular development was followed by echography during 18 menstrual cycles in 10 women volunteers. Results were correlated with changes in serum hormone levels, basal body temperature curves, and cervical mucus changes. The average follicular growth as measured from the time of echographic visualization was 0.3 cm/day. The diameter of the follicle measured the day before disappearance was 2.7 +/- 0.3 cm. In all cases echography was shown to indicate accurately the time of ovulation. Given its inoffensive nature, the usefulness of echography in determining the physical event of ovulation is demonstrated. Possible applications in the surveillance of ovulation induction methods and in artificial insemination are discussed.

PIP: Echography of the ovarian follicle was compared with indirect tests of ovulation detection, such as basal body temperature, serum hormone levels, and cervical mucus changes, to analyze its effectiveness as an ovulation detection device. Follicular maturation was studied in 10 normal women volunteers, aged 24-28 years, with regular menstrual cycles and not taking oral contraceptives. In all, 18 cycles were studied. Average follicular growth by echographic visualization was .3 cm/day; the diameter of the follicle the day before disappearance was 2.7+ or -.3 cm. The echographic report in all ways correlated with other ovulation detection methods, and since it is noninvasive and unoffensive, the use of echography for ovulation surveillance in particular cases, such as artificial insemination and ovulation induction experiments, is recommended.