Persistent factor VIII inhibitors and orthopaedic complications in children with severe haemophilia A.

Persistence of inhibitors against factor VIII (FVIII) may be a risk factor that increases physical disability in haemophilia A (HA) patients. This study aimed to evaluate prevalence of FVIII inhibitors in previously treated children with severe HA and the impact of persistent inhibitors on knee joint status and lumbar bone mineral density (BMD). Fifty children with severe HA, FVIII <1%; aged 5-16 years were enrolled in this study; they received plasma-derived FVIII on-demand treatment for 50-250 exposure days (EDs). Inhibitors were checked at basal visit and were followed up for 1 year, using Bethesda assay. Cross-sectional clinical scoring and radiological evaluation of the knee joint (by Arnold-Hilgartner staging and Pettersson score), along with lumbar BMD by Dual Energy X-ray Absorptiometry (DEXA) were performed. Patients with persistent inhibitors for 1 to 5 years, median 2.5 years, were 10 (20%). Six had high titre and none of them had completely normal knees, seven had advanced knee arthropathy and six had low lumbar BMD in comparison to 2 and 8 of the 40 patients without inhibitors respectively (P < 0.05). Persistence of inhibitors for more than 2 years without immuno-prophylaxis was a risk factor for joint damage. Low lumbar BMD was found in 88.9% of patients with stages four and five knee arthropathy and in 66.7% of patients with positive hepatitis C. Severe HA children in this Egyptian study had a relatively low prevalence of persistent FVIII inhibitors, which, if not treated, may increase the risk of knee arthropathy and lumbar osteopenia.

Skin responses to topical dehydroepiandrosterone: implications in antiageing treatment?

BACKGROUND: Although low dehydroepiandrosterone (DHEA) is suspected to have a role in skin ageing, little information is available on the mechanisms potentially involved. OBJECTIVES: To obtain information on androgen receptor (AR) and procollagen expression in ageing skin during DHEA treatment. METHODS: A placebo-controlled, randomized, prospective study was performed with 75 postmenopausal women aged 60-65 years. The women were treated twice daily for 13 weeks with 3·0 mL of placebo or 0·1%, 0·3%, 1% or 2% DHEA cream applied on the face, arms, back of hands, upper chest and right thigh where 2-mm biopsies were collected before and after treatment. RESULTS: Although the overall structure of the epidermis was not significantly affected at the light microscopy level, AR expression examined by immunocytochemistry was markedly increased by DHEA treatment. In the dermis, the expression levels of procollagen 1 and 3 mRNA estimated by in situ hybridization were increased by DHEA treatment. In addition, the expression of heat shock protein (HSP) 47, a molecule believed to have chaperone-like functions potentially affecting procollagen biosynthesis, was also found by immunocytochemistry evaluation to be increased, especially at the two highest DHEA doses. CONCLUSION: These data suggest the possibility that topical DHEA could be used as an efficient and physiological antiageing skin agent.

Evaluation of the oral health situation of a group of Egyptian haemophilic children and their re-evaluation following an oral hygiene and diet education programme.

Haemophilic children in Egypt have received minimal dental intervention and their dental needs required assessment. The purpose of this study was to assess the oral health needs of a sample (n = 60) of Egyptian haemophilic children (6-12 years), so as to develop, implement and evaluate an oral hygiene education programme over an 8-month period on the experimental group (n = 30) vs. the control group (n = 30). The oral hygiene index simplified (OHI-S) index was used for baseline data and at the end of the study, while DMFS and defs were used to collect caries experience baseline data on each subject. The results showed that the DMFT and deft were significantly higher than those of the non-haemophilic population in Egypt and also higher than those of haemophilic children in developed countries and that the decayed component represented most of the index values. At phase I, the mean value of the OHI-S of experimental and the control groups was 2.67 +/- 0.45 and 2.53 +/- 0.53, respectively, but the difference was not significant (P > 0.05), both values were in the 'fair' category (1.3-3.0). At phase II, the end of the 8 months follow-up period and after the application of a strict oral care programme in the experimental group, there was a significant decrease from 2.67 to 1.20 (P < 0.001), a shift of values occurred from the 'fair' category to the 'good' category (0.1-1.2) while there was no significant difference in the control group. It can be concluded that professional plaque control, education and access to oral hygiene aids is paramount to improve oral health of these children.

Prospective evaluation of high-cost management of severe chronic ITP in children and adolescents<16 years.

Chronic ITP rarely presents with severe bleeding episodes (SBE). Number and duration of SBE were evaluated in relation to the cost of management. Out of 157 chronic ITP patients attending our institution from 1994 to 2003, 37 patients, <16 years with persistent thrombocytopenia (>6 months), suffering from SBE or platelet count<10x10(9)/L were prospectively randomized to receive either intravenous immunoglobulins (IVIG), anti-D immunoglobulin (anti-D) or high-dose methyl prednisolone (HDMP). Sixty-one patient-years were followed, during which 351 SBE were documented. The high-cost management (IVIG and anti-D) showed insignificantly better platelet recovery, less frequent SBE with shorter duration per patient, higher rate of CR, and less splenectomy in contrast to the steroid groups. The effectiveness of high-cost management compared with methyl prednisolone could not be documented in this study.

Gender differences and effects of sex steroids and dehydroepiandrosterone on androgen and oestrogen alpha receptors in mouse sebaceous glands.

BACKGROUND: It is generally believed that the sebaceous gland is an intracrine organ which synthesizes its own active hormones to meet its local needs. OBJECTIVES: To understand further the mechanisms of sex steroid action in mouse sebaceous glands. METHODS: We have used immunocytochemistry to examine the expression of oestrogen receptor alpha (ERalpha) and androgen receptor (AR) in mouse sebaceous glands. RESULTS: In intact males AR is exclusively localized in the nuclei of basal and mature sebocytes, while in females it is present at a lower level in both the nuclei and the cytoplasm. Three weeks following gonadectomy (GDX), a marked decrease of AR labelling is observed in male sebocytes, while no change occurs in female sebocytes. Treatment of GDX animals with dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) increases AR expression, while 17beta-estradiol (E2) decreases the stimulatory effect of DHT and DHEA. ERalpha is detected only in basal sebocytes of intact females but not in males. Following treatment with E2, ERalpha expression becomes visible in GDX males while DHT and DHEA inhibit the effect of E2. CONCLUSIONS: The present data show gender differences and demonstrate that DHT, E2 and DHEA exert specific effects on the expression of AR and ERalpha in mouse sebocytes.

Effects of dehydroepiandrosterone, Premarin and Acolbifene on histomorphology and sex steroid receptors in the rat vagina.

To assess the specific estrogenic and/or androgenic effects of a potential novel hormone replacement therapy, we have examined the morphology of the rat vagina 9 months after ovariectomy (OVX) and treatment of OVX animals with dehydroepiandrosterone (DHEA), conjugated estrogens Premarin and the selective estrogen receptor modulator Acolbifene. OVX led to atrophy and inflammatory changes while Acolbifene reduced the inflammation incidence and induced mucification of the vaginal epithelium. Premarin induced a typical keratinized stratified squamous epithelium while DHEA induced stimulation of the vaginal epithelium, with mucous cells typical of an androgenic effect, combined with increased collagen fiber compactness of the lamina propria. On the other hand, after OVX, the vaginal muscle layer decreased by 46%, an effect which was 41 and 100% reversed by DHEA and Premarin, respectively. The present data show particularly interesting effects of DHEA on the three layers of the vaginal wall, namely a highly mucified epithelium, an increased muscularis thickness and increased collagen fiber compactness in the lamina propria. DHEA exerts both androgenic and estrogenic effects on the vaginal mucosa, thus providing a more physiological replacement therapy.

Prostate cancer susceptibility genes: lessons learned and challenges posed.

In most developed countries, prostate cancer is the most frequently diagnosed malignancy in men. The extent to which the marked racial/ethnic difference in its incidence rate is attributable to screening methods, environmental, hormonal and/or genetic factors remains unknown. A positive family history is among the strongest epidemiological risk factors for prostate cancer. It is now well recognized that the role of candidate genetic markers to this multifactorial malignancy is more difficult to identify than the identification of other cancer susceptibility genes. Indeed, despite the localization of several susceptibility loci, there has been limited success in identifying high-risk susceptibility genes analogous to BRCA1 or BRCA2 for breast and ovarian cancer. Nonetheless, three strong candidate susceptibility genes have been described, namely ELAC2 (chromosome 17p11/HPC2 region), 2'-5'-oligoadenylate-dependent ribonuclease L (RNASEL), a gene in the HPC1 region, and Macrophage Scavenger Receptor 1 (MSR1), a gene within a region of linkage on chromosome 8p. Additional studies using larger cohorts are needed to fully evaluate the role of these susceptibility genes in prostate cancer risk. It is also of interest to mention that a significant percentage of men with early-onset prostate cancer harbor germline mutation in the BRCA2 gene thus confirming its role as a high-risk prostate cancer susceptibility gene. Although initial segregation analyses supported the hypothesis that a number of rare highly penetrant loci contribute to the Mendelian inheritance of prostate cancer, current experimental evidence better supports the hypothesis that some of the familial risks may be due to inheritance of multiple moderate-risk genetic variants. In this regard, it is not surprising that analyses of genes encoding key proteins involved in androgen biosynthesis and action led to the observation of a significant association between a susceptibility to prostate cancer and common genetic variants in some of those genes.

Effect of long-term treatment with the antiestrogen EM-652.HCl on pituitary estrogen receptor alpha and prolactin mRNA expression in intact, ovariectomized and gonadotropin-releasing hormone-treated female rats.

Estrogen receptor alpha (ERalpha) is the predominant estrogen receptor subtype in the anterior pituitary gland. In order to assess the influence of the pure antiestrogen EM-652.HCl on ERalpha gene transcription, we have studied the effect of long-term administration of the antiestrogen in ovariectomized rats as well as in intact female rats treated or not with the GnRH-agonist D-trp(6), des-Gly-NH2(10) GnRH ethylamide (GnRH-A), a treatment which induces pharmacological castration. To evaluate the degree of pituitary responsiveness to changes in estrogen exposure, prolactin (PRL) mRNA levels were also measured. ERalpha and PRL mRNA levels were evaluated by quantitative in situ hybridization. It was found that, 49 weeks after ovariectomy (OVX), pituitary ERalpha mRNA levels were decreased by 55%. Long-term administration (49 weeks) of EM-652.HCl to OVX animals resulted in a further 41% decrease in ERalpha mRNA. On the other hand, ovariectomy induced an 82% decrease in PRL mRNA levels while the administration of EM-652.HCl to OVX animals did not further decrease PRL mRNA. The administration of EM.652.HCl or GnRH-A alone to intact rats during 52 weeks did not significantly modify pituitary ERalpha mRNA levels. Concomitant administration of both GnRH-A and EM-652.HCl induced 41 and 47% decreases in ERalpha mRNA levels, when compared to the levels measured in vehicle-treated and GnRH-treated animals respectively. Combined administration of EM.652.HCl and GnRH-A induced 56 and 65% decreases in PRL mRNA, respectively. When EM-652.HCl was administered concomitantly with GnRH-A, the inhibitory effect on PRL mRNA levels was more marked than that observed in GnRH-A-treated animals. The present data demonstrate that when circulating estrogens are absent or maintained at very low levels by GnRH administration, EM-652.HCl can still depress ERalpha gene transcription. It is suggested that estrogens can positively regulate pituitary ERalpha gene transcription and that the antiestrogen EM-652.HCl can downregulate by itself pituitary ERalpha gene transcription.

Expression of the hamster sperm protein P26h during spermatogenesis.

P26h is a hamster sperm protein of 26 kDa that has been previously characterized as a surface protein covering the acrosome acquired during epididymal transit. P26h is involved in sperm-egg interactions. Recently, it has been shown that the P26h transcript is highly expressed in the testis, and the P26h cDNA has been cloned from a hamster testicular cDNA library. Herein we report the production of a fusion protein (maltose binding protein-P26h) with the whole P26h cDNA encoding sequence and the production of a polyclonal antiserum against it. In Western blots, this antiserum recognized both the P26h extracted from cauda epididymal spermatozoa and the MBP-P26h. We also determined the age of appearance of P26h and which germ cell types express P26h mRNA and its translational product. Northern blots and in situ hybridization analysis showed that P26h transcripts appear at 3 wk of age, within the first round of spermatogenesis in the golden hamster. In situ hybridization showed that P26h transcripts are expressed in spermatocytes and round spermatids, whereas immunostaining revealed the presence of P26h in the cytoplasm of round spermatids and elongated spermatids. P26h was undetectable in testicular spermatozoa. Both in situ hybridization and immunostaining showed P26h expression to be dependent of the testicular cell type and the epithelium cycle. The implications for P26h in sperm-egg interaction and the testicular origin of P26h are discussed.

The androgen-conjugating uridine diphosphoglucuronosyltransferase-2B enzymes are differentially expressed temporally and spatially in the monkey follicle throughout the menstrual cycle.

UDP-glucuronosyltransferase (UGT) enzymes enhance the polarity of steroid hormones by catalyzing their conjugation with the sugar group from UDP-glucuronic acid. Previous results have shown that the monkey is a suitable animal model to study steroid glucuronidation in steroid target tissues. In humans, as in the monkey, the main androgen metabolites found in the circulation are 5alpha-androstane-3alpha,17beta-diol-glucuronide and androsterone glucuronide, and high levels of androsterone glucuronide were also measured in human follicular fluid. Ovarian androgens play a significant role as precursors for estrogens and may modulate the recruitment and growth of follicles. To analyze the expression pattern of UGT2B enzymes involved in androgen metabolism throughout the menstrual cycle, cynomolgus monkey ovaries were collected during the mid and late follicular and luteal phases. Microsomal proteins and total RNA were analyzed for UGT2B expression in the whole ovary. Western blot and specific RT-PCR analyses demonstrated no significant changes in the expression of UGT2B protein or transcripts during the menstrual cycle. Immunocytochemistry analysis showed that UGT2B proteins are expressed in the cytoplasm of thecal and granulosa cells of growing follicles. Interestingly, the thecal cells of secondary follicles and of corpus luteum were extensively stained, whereas luteal granulosa cells were not labeled. These results suggest an important regulation of cell type-specific UGT2B expression during follicular development. Previous results demonstrated similar changes in the expression of the androgen receptor. The colocalization of the androgen receptor and UGT2B enzymes in the same cell types of the ovary provide evidence for a potential role of glucuronidation as a modulator of the intracellular androgen response during follicular development.

Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis.

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.

Immunoelectron microscopic localization of 3beta-hydroxysteroid dehydrogenase and type 5 17beta-hydroxysteroid dehydrogenase in the human prostate and mammary gland.

The subcellular distribution of steroidogenic enzymes has so far been studied mostly in classical endocrine glands and in the placenta. In the peripheral intracrine organs which synthesize sex steroids there is no indication about the organelles which contain the enzymes involved in steroid biosynthesis. We have thus investigated the subcellular localization of two enzymes involved in the production of sex steroids, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Using specific antibodies to these enzymes, we conducted immunoelectron microscopic studies in two peripheral tissues, namely the human prostate and mammary gland. In the prostate, immunolabelling for both 3beta-HSD and type 5 17beta-HSD was detected in the basal cells of the tube-alveoli as well as in fibroblasts and endothelial cells lining the blood vessels. In all the labelled cell types, the gold particles were distributed throughout the cytoplasm. No obvious association with any specific organelle could be observed, although some concentration of gold particles was occasionally found over bundles of microfilaments. In mammary gland sections immunolabelled for 3beta-HSD or type 5 17beta-HSD localization, labelling was observed in the cytoplasm of the secretory epithelial cells in both the acini and terminal ducts. Immunolabelling was also found in the endothelial cells as well as in fibroblasts in stroma and blood vessels. The gold particles were not detected over any organelles, except with the occasional accumulation of gold particles over microfilaments. The present data on the localization of two steroidogenic enzymes leading to the synthesis of testosterone indicate that these enzymes are located not only in epithelial cells but also in stromal and endothelial cells in both tissues studied. The absence of any association of the enzymes with membrane-bound organelles appears as a common finding in the reactive cell types of two peripheral tissues.

Targeted disruption of the mouse prosaposin gene affects the development of the prostate gland and other male reproductive organs.

The prosaposin gene encodes a 65-70 kilodalton (kd) protein, which is secreted or targeted to lysosomes. In lysosomes, prosaposin is the precursor of 4 activator proteins, designated saposins A, B, C, and D, which promote by acidic hydrolases, the degradation of glycosphingolipids with short oligosaccharide chains. Mutations of the prosaposin gene have been linked to several lysosomal storage disorders. An animal model was recently developed by creating a null allele in embryonic stem cells through gene targeting in order to investigate the phenotypic diversity of prosaposin mutations, the involvement of this protein in lysosomal storage diseases, and to develop potential therapeutic approaches. Mutant homozygous mice die at 35-40 days of age and neurological disorders contribute to their early death. Secreted prosaposin is present in milk and in cerebrospinal and seminal fluids. In the nervous system, prosaposin exhibits a trophic activity. Examination of reproduc-tive organs in homozygous mutant males shows several abnormalities such as a decrease in testis size with reduced spermiogenesis, and an involution of the prostate, seminal vesicle, and epididymis, although levels of testosterone in blood remain normal. In the prostate of homozygous mutants, only basal cells appear to be present, whereas secretory cells are absent. The epithelia in efferent ducts is formed by ciliated cells, whereas heterozygotes exhibit a majority of nonciliated cells. Our data indicate that prosaposin is involved in the development and maintenance of male reproductive organs. In prostatic epithelium, targeted disruption of the prosaposin gene appears to inactivate the mitogen-activated protein kinase pathway and to interfere with differentiation of secretory cells.

Frequency of inhibitor development in severe haemophilia A children treated with cryoprecipitate and low-dose immune tolerance induction.

The frequency of factor VIII inhibitor development was evaluated in a hundred severe haemophilia A patients < 18 years of age (mean 10.4 +/- 5.1 years); 25 were previously untreated patients (PUPs), with a mean age of 11.2 +/- 2.9 months. All were followed up for 3 years from December 1996. Immune tolerance (IT) was induced with low-dose factor VIII (FVIII); 25-50 IU kg(-1) every other day for the 10 haemophiliacs who developed persistent inhibitors. The incidence of inhibitors for PUPs was 3/25 (12%; 95% confidence interval [CI], 0. 7-24.7%) and were detected after 4, 15 and 20 exposure days (mean 13 +/- 8.2 days; 95% CI, 3.7-22.2%). Children with maximum inhibitor levels of > 40 Bethesda units (BU) per mL (n=4) received IT therapy as 25 U kg(-1) FVIII in the form of cryoprecipitate every other day for 1-4 months (mean 2.4 +/- 1.6 months; 95% CI, 0.8-3.9%), which was successful in all of them. FVIII (50 U kg(-1)) was given every other day for six patients with maximum inhibitor level > 40 BU mL(-1) for 3-9 months (mean 5.4 +/- 3.2 months; 95% CI, 2.9 -7.9%) with success in 4/6 (66.6%; 95% CI, 28.8-104.3%). Patients who showed a good IT response had an inhibitor level < or = 30 BU mL(-1), were < or = 9 years of age at inhibitor development with few exposure days to FVIII and had an early immune tolerance. In conclusion, inhibitor development in severe haemophilia A children exclusively treated with cryoprecipitate is low. Early low-dose IT induction for high responders may be achieved successfully if inhibitor level is < or = 50 BU mL(-1).

Unique features of the basal cells of human prostate epithelium.

The prostate gland is globally composed of epithelium and stroma. The epithelium plays an important role in the development of both benign and malignant disorders while the stroma is involved in benign prostatic hyperplasia. While the prostatic epithelium of the majority of laboratory animals is well recognized as a pseudostratified columnar, the classification of the human prostatic epithelium is controversial. Moreover, the role of the basal cells of the human prostatic epithelium is still uncertain. These cells have been described as undifferentiated cells, precursors of luminal cells, reserve and myoepithelial cells. The objective of the present study was to assess the similarities and/or differences between the epithelium of the human prostate and that of other laboratory animals and thus derive information about the potential functions of basal cells in the human prostate. In the human, basal cells form a continuous layer of cells resting on the basement membrane and upon which rests a layer of luminal cells. This results in a stratified columnar epithelium of two layers of cells, unlike the sporadic appearance of basal cells observed in other species where it results in a pseudostratified epithelium. In addition, the ratio of basal to luminal cells in the human is about 1:1, while the average ratio in the other animal species examined is about 1:7. Furthermore, the gap junctional proteins connexin 26 and 43, are present between basal and luminal cells in the human, thus suggesting that these cells communicate directly with each other. In addition, the ultrastructure of the human basal cells shows morphological evidence of differentiated but not of undifferentiated cells. Moreover, the presence of junction-like structures between adjacent basal cells suggests that these cells form a blood-prostate barrier. In this way, basal cells could prevent substances derived from the blood from directly coming in contact with the luminal cells. Human basal cells could thus regulate functions of the luminal cells by being part of a two-cell mechanism somewhat analogous to thecal and granulosa cells in the ovary.

Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease.

In women and men, an important proportion of estrogens and androgens are synthesized locally at their site of action in peripheral target tissues. This new field of endocrinology has been called intracrinology. In postmenopausal women, 100% of active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors while, in adult men, approximately 50% of androgens are made locally in intracrine target tissues. The last and key step in the formation of all estrogens and androgens is catalyzed by members of the family of 17beta-hydroxysteroid dehydrogenases (17 beta-HSDs) while different 17 beta-HSDs inactivate these steroids in the same cell where synthesis takes place. To date, seven human 17 beta-HSDs have been cloned, sequenced and characterized. The 17 beta-HSDs provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs.

Cellular localization of uridine diphosphoglucuronosyltransferase 2B enzymes in the human prostate by in situ hybridization and immunohistochemistry.

UDP-glucuronosyltransferase (UGT) enzymes catalyze the transfer of the glucuronide group from UDP-glucuronic acid to several exogenous or endogenous compounds, including steroid hormones. Although it is widely recognized that the liver is a major site of steroid glucuronidation, RT-PCR analysis has shown the expression of UGT2B transcripts in extrahepatic steroid target tissues such as the prostate. Measurement of androgen metabolites in human prostate revealed high levels of C(19) steroid glucuronides such as androsterone glucuronide and 3alpha-diol glucuronide, thus suggesting an important role of UGT2B enzymes in androgen metabolism. To investigate the cellular localization of UGT2B expression in the human prostate, the present in situ hybridization studies demonstrated the presence of UGT2B transcripts in epithelial cells lining the acinii. All basal cells were intensively labeled, whereas the luminal secretory cells were moderately labeled. To confirm these results, an immunohistological analysis was performed using a specific anti-UGT2B antibody. The presence of UGT2B proteins was observed in both basal and luminal cells of prostate epithelium, in fibrocytes of stroma and blood vessels, and in endothelial cells of blood vessels. Using a specific anti-UGT2B17 antibody, the expression of this androsterone-conjugating UGT enzyme was found exclusively in basal cells of the epithelium. These results demonstrate the expression of androgen-conjugating UGT2B enzymes in human prostatic epithelium. Moreover, they show for the first time a cell type-specific expression of an UGT2B isoform.

Immunocytochemical localization of estrogen receptors alpha and beta in the human reproductive organs.

To better define the role of estrogens in reproductive functions, we have proceeded to the immunocytochemical localization of the estrogen receptor (ER) subtypes, ERalpha and the recently discovered ERbeta+, in human reproductive tissues. In the ovary, ERbeta+ immunoreactivity was found in nuclei of granulosa cells of growing follicles at all stages from primary to mature follicles, interstitial gland, and germinal epithelium cells. Nuclear staining for ERalpha occurred in thecal, interstitial gland, and germinal epithelium cells. In the uterus, strong ERalpha immunoreactivity was detected in nuclei of epithelial, stromal, and muscle cells. Similar localization was obtained for ERbeta+, although the staining was much weaker. In the vagina, only ERalpha could be detected; a nuclear reaction was observed in deep layers of the stratified epithelium as well as in stromal and muscle cells. In the mammary gland, both ER subtypes were observed in epithelial and stromal cells. In the testis, ERbeta+ was detected in nuclei of Sertoli and Leydig cells, whereas ERalpha immunoreactivity was only observed in Leydig cells, with no tubular labeling. In the efferent ducts, only ERbeta+ could be detected, whereas neither ERbeta+ nor ERalpha could be found in the epididymis. In the prostate, ERbeta+ nuclear immunolabeling was observed in both basal and secretory cells in alveoli as well as in stromal cells, whereas ERalpha could not be detected. The present results demonstrate that there is a cell-specific localization for each of the ER subtypes in the majority of the reproductive organs studied. Moreover, they contribute to establish the exact sites of action of estrogens in male and female human reproductive systems.

Intracrinology and the skin.

The skin, the largest organ in the human body, is composed of a series of androgen-sensitive components that all express the steroidogenic enzymes required to transform dehydroepiandrosterone (DHEA) into dihydrotestosterone (DHT). In fact, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors, especially DHEA. Secretion of this precursor steroid by the adrenals decreases progressively from the age of 30 years to less than 50% of its maximal value at the age of 60 years. DHEA applied topically or by the oral route stimulates sebaceous gland activity, the changes observed being completely blocked in the rat by a pure antiandrogen while a pure antiestrogen has no significant effect, thus indicating a predominant or almost exclusive androgenic effect. In human skin, the enzyme that transforms DHEA into androstenedione is type 1 3beta-hydroxysteroid dehydrogenase (type 1 3beta-HSD) as revealed by RNase protection and immunocytochemistry. The conversion of androstenedione into testosterone is then catalyzed in the human skin by type 5 17beta-HSD. All the epidermal cells and cells of the sebaceous glands are labelled by type 5 17beta-HSD. This enzyme is also present at a high level in the hair follicles. Type 1 is the 5alpha-reductase isoform responsible in human skin for the conversion of testosterone into DHT. In the vagina, on the other hand, DHEA exerts mainly an estrogenic effect, this effect having been demonstrated in the rat as well as in post-menopausal women. On the other hand, in experimental animals as well as in post-menopausal women, DHEA, at physiological doses, does not affect the endometrial epithelium, thus indicating the absence of DHEA-converting enzymes in this tissue, and avoiding the need for progestins when DHEA is used as hormone replacement therapy.

Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis.

During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermatozoa. During this process there occurs remodeling of cell structure and loss of most of the cytoplasm and a large fraction of cellular proteins. To evaluate the role of the ubiquitin proteolytic system in this protein loss, we measured levels of ubiquitinated proteins and rates of ubiquitin conjugation in extracts of testes from rats of different ages. Endogenous ubiquitin-protein conjugates increased till day 30 and then reached a plateau. In parallel, there was a progressive increase in the rate of conjugation of ubiquitin to proteins in testis extracts from these animals. To test the importance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extracts was carried out and the remaining conjugation activity in supernatants was assayed. Depletion of either enzyme family resulted in decreased conjugation. However, most of the conjugation activity and, more importantly, the increased conjugation during development were UBC4-dependent. Immunocytochemistry demonstrated a marked increase in expression of UBC4 in spermatids, consistent with the UBC4-dependent activation of conjugation seen in vitro. In situ hybridization studies evaluated the contribution of various UBC4 isoforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 mRNA was restricted to germ cells with high levels of expression in round and elongated spermatids. UBC4-testis had previously been shown to be expressed only in spermatids. Our data suggest that induction of various UBC4 isoforms activates overall conjugation and plays an important role in the cellular remodeling and protein loss occurring during spermatogenesis.