Structure and expression of a new complementary DNA encoding the almost exclusive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human adrenals and gonads.

The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) enzyme catalyzes the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3 beta HSD catalyzes the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. Clinical observations in patients with 3 beta HSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3 beta HSD cDNA (type I) as probe suggested the existence of multiple related 3 beta HSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type II) encoding 3 beta HSD after screening of a human adrenal lambda gt22A library. The nucleotide sequence of 1676 basepairs of human 3 beta HSD type II cDNA predicts a protein of 371 amino acids with a calculated molecular mass of 41,921 daltons, which displays 93.5% and 96.2% homology with human placental type I and rhesus macaque ovary 3 beta HSD deduced proteins, respectively. To characterize and compare the kinetic properties of the two isoenzymes, plasmids derived from pCMV and containing type I or type II 3 beta HSD full-length cDNA inserts were transiently expressed in HeLa human cervical carcinoma cells. In vitro incubation with NAD+ and 3H-labeled pregnenolone or dehydroepiandrosterone shows that the type I protein possesses a 3 beta HSD/delta 5-delta 4 isomerase activity higher than type II, with respective Km values of 0.24 vs. 1.2 microM for pregnenolone and 0.18 vs. 1.6 microM for dihydroepiandrosterone, while the specific activity of both types is equivalent. Moreover, incubation in the presence of NADH of homogenates from cells transfected with type I or type II 3 beta HSD indicates that dihydrotestosterone is converted into 5 alpha-androstane-3 beta, 17 beta-diol, with Km values of 0.26 and 2.7 microM, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3 beta HSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3 beta HSD mRNA species in the placenta and skin and represent the predominantly expressed species in mammary gland tissue. The present data show for the first time that adrenals and gonads express a type of 3 beta HSD isoenzyme that is distinct from the type expressed in the placenta.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of adrenal 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase expression and activity by adrenocorticotropin and corticosterone in the rat.

This study describes the regulation of adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) expression and activity by ACTH and corticosterone, alone or in combination, in intact male and female rats as well as the effect of ACTH on 3 beta HSD expression and activity in the adrenals of hypophysectomized female animals. The effect of treatment on total 3 beta HSD mRNA levels was measured by dot blot hybridization using rat 3 beta HSD cDNA, while the specific regulation of type I and type II 3 beta HSD mRNAs was analyzed by ribonuclease protection assay. The concentration of 3 beta HSD protein was measured by Western blot, using cross-reacting antibodies raised against purified human placental 3 beta HSD, while 3 beta HSD enzymatic activity was measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione. The present data show that the trophic effect of ACTH on male and female rat adrenals is accompanied by increases in total 3 beta HSD mRNA, enzymatic activity, and protein content. Hypophysectomy, on the other hand, causes a marked decrease in 3 beta HSD mRNA levels and enzymatic activity, which is completely reversed by administration of ACTH. On the other hand, corticosterone treatment results in a marked inhibition of 3 beta HSD mRNA levels, enzymatic activity, and protein content in intact animals; this effect is probably mediated by a decrease in ACTH secretion. The present data show that ACTH and corticosterone, via its inhibitory action on ACTH secretion, have potent and opposite effects on the expression of two 3 beta HSD genes in the rat adrenal; a parallel effect is observed on both type I and II 3 beta HSD. Such data suggest that 3 beta HSD could well play a major role in the regulation of steroid formation in the adrenal cortex.

Structure and sexual dimorphic expression of a liver-specific rat 3 beta-hydroxysteroid dehydrogenase/isomerase.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes the obligatory oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-3-ketosteroids which lead to the formation of all classes of steroid hormones. We report the molecular cloning of a third type of cDNA clone encoding rat 3 beta- HSD isolated from a rat liver lambda gt11 cDNA library. The nucleotide sequence of 1955 bp determined from overlapping cDNA clones predicts a protein of 372 amino acids which displays 80% similarity with that of rat type I and type II 3 beta-HSD proteins. RNA blot analysis reveals the presence of mRNA transcripts of 2.1 kb in male liver in contrast to the 1.7 kb mRNA species detected in adrenal and gonad poly(A)+ RNA. Ribonuclease protection assays using type I, type II and type III specific cRNA probes demonstrate the liver-specific expression of type III mRNA while the two others are expressed in adrenals and gonads. The type III mRNA species was below the detection limit in intact female liver while in hypophysectomized females, its accumulation was restored to 55% of the levels measured in intact or hypophysectomized male rats. The present data describe the presence of a third type of 3 beta-HSD mRNA species and its marked sexual dimorphic gene expression in the liver which apparently results from pituitary hormone-induced gene repression in female rat liver tissue.

Regulation of 3-ketosteroid reductase messenger ribonucleic acid levels and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase activity in rat liver by sex steroids and pituitary hormones.

We have recently characterized three types of complementary DNA clones encoding predicted isoenzymes of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family. Transient expression in nonsteroidogenic cells reveals that the type III isoenzyme specific for male liver does not display oxidative activity for classical substrates of 3 beta-HSD, in contrast to the two other 3 beta-HSD isoenzymes, thus showing exclusively 3-ketosteroid reductase (3-KSR) activity. In order to better understand the sex-specific control of 3 beta-HSD activity and type III 3-KSR gene expression in rat liver, we have studied in adult animals of both sexes the effect of sex steroids and hypophysectomy, pituitary implants, PRL, and GH on type III 3-KSR messenger RNA (mRNA) levels and 3 beta-HSD/delta 5-delta 4 isomerase activity as measured by the conversion of [14C]dehydroepiandrosterone into [14C] delta 4-androstenedione. Ribonuclease protection assay using types I-, II-, and III-specific complementary RNA probes reveals that type III transcripts are the only species detectable in liver RNA extracted from intact males, whereas no hybridization signal was detectable with any of the three probes in intact female liver RNA. In males, 15 days after castration, liver type III 3-KSR mRNA levels decreased by 80% compared to intact controls, whereas 3 beta-HSD activity was reduced by 48%. Administration of dihydrotestosterone (DHT) increased by 8.25-fold type III 3-KSR mRNA concentration and completely reversed the inhibitory effect of orchiectomy on 3 beta-HSD activity. In ovariectomized animals, treatment with DHT markedly increased type III 3-KSR mRNA accumulation and 3 beta-HSD activity, thus leading to values similar to those measured in intact males. Simultaneous treatment with 17 beta-estradiol almost completely abolished the stimulatory effect of DHT in female rats, whereas no significant effect was seen in males. Twenty-four days after hypophysectomy, type III 3-KSR mRNA levels were decreased by 50-55% in males, whereas in females these transcripts markedly increased from undetectable to 28-36% of the value measured in intact male rats. Treatment with DHT or 17 beta-estradiol for a period of 9 days starting 15 days after hypophysectomy had no effect in male and female rats. On the other hand, treatment with ovine PRL (1 mg, twice daily) had no effect in males but completely blocked the elevation of type III 3-KSR mRNA levels and 3 beta-HSD activity observed after hypophysectomy in females.(ABSTRACT TRUNCATED AT 400 WORDS)

Combination of an antiandrogen and a 5 alpha-reductase inhibitor: a further step towards total androgen blockade?

While the elimination of androgens of testicular origin can be easily achieved by orchiectomy or medical castration with LHRH agonists, the action of adrenal androgen precursors which are converted into the active androgen 5 alpha-dihydrotestosterone (DHT) in the prostatic tissue itself can be partially neutralized by antiandrogens which compete with DHT for binding to the androgen receptor. In order to increase the efficiency of androgen blockade, we have used 4-MA, an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone into DHT, to reduce intracellular DHT concentrations and thus facilitate the action of the antiandrogen Flutamide. The present data show that the inhibitory effects of 4-MA (17 beta, N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) and of the antiandrogen Flutamide are additive on prostatic growth and on androgen-sensitive prostatic binding protein mRNA levels in the rat, thus clearly suggesting that such a combination could provide the basis for a further improvement in the therapy of prostate cancer.

Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase expression and activity in the hypophysectomized rat ovary: interactions between the stimulatory effect of human chorionic gonadotropin and the luteolytic effect of prolactin.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes an obligatory step in the conversion of pregnenolone and other 5-ene-3 beta-hydroxysteroids into progesterone as well as precursors of all androgens and estrogens in the ovary. Since 3 beta-HSD is likely to be an important target for regulation by pituitary hormones, we have studied the effect of chronic treatment with LH (hCG), FSH, and PRL on ovarian 3 beta-HSD expression and activity in hypophysectomized adult female rats. Human CG (hCG) [10 IU, twice a day (bid)], ovine FSH (0.5 microgram, bid), and ovine PRL (1 mg, bid) were administered, singly or in combination, for a period of 10 days starting 15 days after hypophysectomy. In hypophysectomized rats, PRL exerted a potent inhibitory effect on all the parameters studied. In fact, PRL caused a 81% decrease in ovarian 3 beta-HSD mRNA content accompanied by a similar decrease in 3 beta-HSD activity and protein levels. In addition, ovarian weight decreased by 40% whereas serum progesterone fell dramatically from 1.92 nmol/liter to undetectable levels after treatment with PRL. Whereas hCG alone had only slight stimulatory effects on 3 beta-HSD mRNA, protein content and activity levels, treatment with the gonadotropin partially or completely reversed the potent inhibitory effects of oPRL on all the parameters measured. FSH, on the other hand, had no significant effect on 3 beta-HSD expression and activity. In situ hybridization experiments using the 35S-labeled rat ovary 3 beta-HSD cDNA probe show that the inhibitory effect of PRL is exerted primarily on luteal cell 3 beta-HSD expression and activity. On the other hand, it can be seen that hCG stimulates 3 beta-HSD mRNA accumulation in interstitial cells. The present data show that hCG and PRL exert potent and opposite cell-specific effects on ovarian 3 beta-HSD expression, activity, and content in the rat ovary. Moreover, the present study could suggest that female infertility associated with hyperprolactinemia in women could well be related, at least in part, to the potent inhibitory effect of PRL on ovarian 3 beta-HSD expression and activity.

Blockade of androstenedione-induced stimulation of androgen-sensitive parameters in the rat prostate by combination of Flutamide and 4-MA.

In order to mimic the human situation in which adrenal steroid precursors are converted to the active androgen dihydrotestosterone (DHT) in prostatic tissue, we have used castrated rats supplemented with the precursor steroid androstenedione (delta 4-dione) released from Silastic implants. While it is well known that the action of DHT can be partially neutralized by antiandrogens which compete for binding to the androgen receptor, we have used 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA), an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone into DHT, in order to decrease intraprostatic DHT levels and thus facilitate the action of the antiandrogen. Animals were treated for 7 days with Flutamide (FLU, 2 mg) or 4-MA (4 mg) injected subcutaneously, twice daily, alone or in combination. 4-MA administered alone caused a 54% inhibition of delta 4-dione-stimulated ventral prostate weight while FLU exerted a 74% inhibitory effect and 4-MA+FLU further improved inhibition to 81%. We then measured, by in situ hybridization, the levels of prostatic mRNAs encoding the C1 and C3 components of the prostatic binding protein (PBP) which are highly specific and sensitive markers of androgen action. PBP-C3 mRNA levels fell by 95% following castration while treatment with delta 4-dione completely reversed the effect of castration. Administration of FLU or 4-MA independently caused 33% and 10% decreases, respectively, of PBP-C3 mRNA levels stimulated by delta 4-dione while the combination of both compounds further inhibited PBP-C3 mRNA levels to reach a 55% inhibition. Similar effects were observed on PBP-C1 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of human chorionic gonadotropin (hCG) and prolactin (PRL) on 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) expression and activity in the rat ovary.

Using a recently cloned rat ovary 3 beta-HSD cDNA and antibodies raised against purified human placental 3 beta-HSD, we have studied the effects of treatment with human chorionic gonadotropin (hCG) and hyperprolactinemia achieved by pituitary implants, alone or in combination, on the expression and activity of ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) in intact adult rats. 32P- and 35S-labeled cDNA probes were used to evaluate the effects of treatments on 3 beta-HSD mRNA levels by dot blot and in situ hybridization, respectively, while enzymatic activity was measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione. The present data show that hCG exerts a marked trophic effect on rat corpora lutea with an increase in total ovarian 3 beta-HSD mRNA levels, 3 beta-HSD protein content as well as enzymatic activity, resulting in an increase in serum progesterone levels. Prolactin-secreting pituitary implants alone, on the other hand, while exerting small effects on 3 beta-HSD expression and activity, led to a marked potentiation of the stimulatory effect of hCG on all parameters. The present data show that hCG and PRL act synergistically to stimulate ovarian progesterone secretion via an increase in 3 beta-HSD mRNA levels, protein content and enzymatic activity.

Distribution of 17 beta-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues.

The interconversion of estrone (E1) and 17 beta-estradiol (E2), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol is catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). The enzyme 17 beta-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17 beta-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17 beta-HSD mRNA in some human tissues. Enzymatic activity was measured using 14C-labeled E1, E2, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highest level of 17 beta-HSD activity, low but significant levels of E2 as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E2----E1, T----4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17 beta-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.

Reassessment of nonplanimetric transrectal ultrasound prostate volume estimates.

OBJECTIVES: To assess the accuracy and reproducibility of nonplanimetric transrectal ultrasound (TRUS) volume estimates because inaccurate volume estimates may potentially undermine the value of serum prostate-specific antigen density (PSAD) in early prostate cancer detection. METHODS: We prospectively evaluated 535 consecutive male patients with two consecutive volume determinations performed by the same ultrasonographer at the time of the same visit. RESULTS: Pearson correlation coefficients between two consecutive gland volume estimates ranged from 0.82 to 0.85 depending on the formula used; however, these correlation coefficients corresponded to an average 25% difference between the first and second gland volume estimates. CONCLUSIONS: Although two consecutive nonplanimetric TRUS volume estimates show statistically good correlation, clinically up to a 25% volume difference should be expected between two such volume estimates. In consequence, nonplanimetric TRUS volume estimates should be interpreted with caution, especially when used for PSAD calculation, in the early detection of prostate cancer.

Role of prostate-specific antigen density after applying age-specific prostate-specific antigen reference ranges.

OBJECTIVES: To determine the potential role of prostate-specific antigen (PSA) density (PSAD) in the early detection of prostate carcinoma if we apply age-specific PSA reference ranges (2.5 ng/mL or less for ages 40 to 49 years, 3.5 or less for ages 50 to 59, 4.5 or less for ages 60 to 69, and 6.5 or less for ages 70 to 79. METHODS: We retrospectively reviewed 3234 cases referred to us by urologists for transrectal ultrasound (TRUS) between January 1, 1991, and September 28, 1993. We included 2429 patients in the study, ages 40 to 79 years, with Hybritech or Abbott IMx serum PSA determinations and without previously diagnosed prostate cancer. We performed digital rectal examination (DRE) and TRUS in all cases, and TRUS-guided biopsies when indicated. We used stringent criteria to define 736 cases without clinical evidence of malignancy that were designated as a "benign group." RESULTS: In the benign group, we found serum PSA to increase with age in parallel with the increase in prostate volume with age (r = 0.25 and r = 0.26, respectively). The association between serum PSA and prostate volume was stronger (r = 0.46). Using multiple regression analysis, prostate volume accounted for 18% of the variation in serum PSA, whereas age accounted for only an additional 2%. PSAD, which directly relates serum PSA to prostate volume, showed a weak association with age (r = 0.1). In the entire study population of 2429 cases, 555 patients had negative DRE and TRUS results and a serum PSA level between the age-specific upper limit of normal and 10.0 ng/mL. According to the proposed age-specific algorithm, these patients would have required automatic biopsies. Of these, 315 cases (56.8%) still had a PSAD of less than 0.15. We performed biopsies in 108 of these 315 and detected only two cancers, for a positive biopsy rate (PBR) of 1.9%. The remaining 240 cases had a PSAD of 0.15 or higher, and we performed biopsies in 217 of these cases and detected 59 cancers, for a PBR of 27.2%. CONCLUSIONS: The use of age-specific PSA reference ranges does not totally account for the effect of prostate volume on serum PSA. Therefore PSAD can still be used to reduce safely the number of biopsies performed in patients with negative DRE and TRUS results and a serum PSA level 10.0 ng/mL or less and above the age-specific upper limit of normal.

Prospective evaluation of prostate-specific antigen density and systematic biopsies for early detection of prostatic carcinoma.

Significant controversies persist in regard to the need for systematic biopsies in patients with serum prostate-specific antigen (PSA) levels above 4 ng/mL (Hybritech assay), especially if they show no signs of prostatic cancer on digital rectal examination (DRE) or transrectal ultrasonography (TRUS). We evaluated 565 consecutive patients referred to us for prostatism, suspicious lesions on DRE, or an elevated serum PSA level. These patients do not represent a purely screened population. A detection rate of 38.4 percent was achieved by performing directed biopsies of suspicious lesions on DRE and/or TRUS, and systematic biopsies of all patients with serum PSA levels above 4 ng/mL. Among 142 patients with serum PSA between 4.1 and 10 ng/mL, but without suspicion for cancer on DRE and TRUS (DRE- TRUS-), a large number of patients (6.2) were subjected to systematic biopsies to detect one cancer. A receiver-operating characteristics curve for PSA density (PSAD) applied to this population confirmed that the best cut-off point for biopsies was a PSAD of 0.15, below which only two of twenty-three cancers would have been missed, sparing biopsies in 77 of 142 patients. A similar approach was applied to DRE- TRUS- patients with serum PSA levels above 10 ng/mL. The number of cancers in those with serum PSA between 10.1 and 14 ng/mL was too low to establish a PSAD cut-off point. In patients with serum PSA above 14 ng/mL, the best PSAD cut-off point for biopsies was 0.3, below which two of thirteen cancers would have been missed, sparing biopsies in 19 of 39 patients. We conclude that PSAD can safely reduce the number of patients subjected to systematic biopsies without significantly compromising cancer detection.

Outcome of sextant biopsy according to gland volume.

OBJECTIVES: To reassess positive rate of sextant biopsy according to gland size. METHODS: We evaluated 1974 consecutive men with systematic sextant biopsy, among whom we examined biopsy yield according to gland-volume intervals of 10 cc. RESULTS: Decreasing yield of sextant biopsy is strongly associated with increasing gland volume (P < 0.001). Highest biopsy rate (39.6%) was recorded among men with prostates smaller than 20 cc. The lowest biopsy rate (10.1%) was recorded among men with prostates between 80 and 89.9 cc. Among men with biopsy-proven cancer, age, serum prostate-specific antigen, and Gleason grade were comparable (P > 0.05) throughout the range of gland-volume intervals. CONCLUSIONS: Our findings suggest that gland size represents an important determinant contributing to the yield of sextant biopsy in men at risk of harboring a nonpalpable, isoechoic cancer. Consequently, an individualized sector biopsy approach, based on prostate volume, may warrant consideration because it may ensure superior detection of clinically significant disease among all men at risk, regardless of prostate size.

Complications of ultrasound-guided prostate biopsy peripheral zone only versus combined peripheral and transition zone biopsy.

The main objectives of this study were to reassess complications associated with transrectal ultrasound-guided biopsies (TRUSBx) of the peripheral zone (PZ) and to compare morbidity of exclusive PZ biopsy to morbidity associated with two additional transition zone (TZ) biopsies. We distributed a self-administered questionnaire assessing TRUSBx complications to 883 consecutive patients who underwent two systematic TZ TRUSBx in addition to systematic sextant PZ TRUSBx, and to 383 consecutive patients who underwent exclusive PZ TRUSBx. Of 316 (35.8%) patients subjects to TZ and PZ TRUSBx, 71% experienced hematuria, 63% hematospermia, 39% rectal bleeding, and 2.2% temperature elevation greater than 38°C. Of 137 (35.8%) patients who exclusively underwent PZ TRUSBx, 57%, 62%, 36%, and 1.4% reported these complications, respectively. Symptom duration and severity were similar in both groups. Although we report a substantially higher incidence of biopsy complications than previously published, these complications are self limited and require no intervention. Furthermore there appears to be no significant difference between complication rates associated with exclusive PZ biopsy compared with those associated with two additional biopsies of the TZ.

Normal gonadal functions and fertility after 23 months of treatment of prepubertal male and female dogs with the GnRh agonist [D-Trp6, des-Gly-NH2(10)]GnRH ethylamide.

The GnRH agonist [D-Trp6, des-Gly-NH2(10)]GnRH ethylamide was administered to prepubertal male and female dogs daily for 23 months by subcutaneous injection. During GnRH agonist treatment, plasma steroid levels, namely dehydroepiandrosterone, androst-5-ene-3 beta-17 beta-diol, androstenedione, testosterone, dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane, 3 beta, 17 beta-diol were markedly inhibited in male animals, whereas in female animals, the plasma concentration of DHEA and delta 5-diol were decreased. Within 2 months following cessation of therapy, all steroids increased to normal adult levels. Morphological studies reveal that treatment of male animals with the GnRH agonist is accompanied by a small volume of seminiferous tubules, Leydig cells, and prostate gland, whereas in the ovaries of female animals, there is a large number of primordial follicles, a few primary follicles, but no secondary follicles. In the pituitary gland of animals of both sexes, LH-secreting cells have high levels of glycogen particles in their cytoplasm and tend to be either of normal appearance with dilated rough endoplasmic reticulum (RER) or strongly atrophied with a dark-stained cytoplasm, a contraction of RER, and a decrease in the number of secretory granules. Reticular cells of the connective tissue also show high levels of glycogen particles. After the 14 month recovery period, spermatogenesis has a normal adult appearance, the prostate gland shows a normal secretory epithelium, and secondary follicles are easily observed in the ovary. Gonadotrophs are free of glycogen accumulation, but reticular cells continue to show an accumulation of glycogen particles in their cytoplasm. Two male and two female animals were mated after the recovery period and produced normal offspring with normal fertility. The present results indicate that GnRH agonist treatment achieves a blockade of sexual maturation and that following cessation of treatment, normal pituitary-gonadal functions resume, with apparently normal fertility and normal offspring.

Anal reeducation for postoperative fecal incontinence in congenital diseases of the rectum and anus.

From October 1993 to March 1996, 14 patients with anorectal disease were referred to an anal reeducation clinic. Initial evaluation allowed the authors to identify three classes of defects: lack of proprioception in the sphincters, use of synergistic muscles (gluteal) to compensate for anal dysfunction, and inversion of command by contraction, rather than relaxation, of abdominal muscles. Patients were treated by electrostimulation through an anal probe as well as biofeedback therapy coupled with home exercises. This therapy resulted in rapid correction of the abnormal motor commands and erroneous use of accessory muscles. All patients became able to isolate their continence muscles with success, with documented increase in strength, rapidity of response, and duration of contraction. The mean Kelly score went from 1.46 (range, 0 to 4.5) to 3.07 (range, 0.5 to 5.5). This physiological improvement also increased patient motivation and discipline toward continence and subsequently their quality of life.

[Perineal retraining in urinary stress incontinence]. / La rééducation périnéale dans l'incontinence urinaire d'effort.

Twenty patients suffering from urinary stress incontinence were treated by perineal reeducation. The assessment included a medical and urological questionnaire, a physical examination, a urine analysis and culture, a cystoscopy, urinary flow and cystometry, a urethral pressure profile and a subjective evaluation of the perineal musculature. The 20 patients selected had documented stress incontinence, had never been operated on for incontinence and had a stable bladder at urodynamic assessment. Treatment was identical for all patients and included 12 biofeedback and electrostimulation sessions over a 4 to 6 week period. The questionnaire, urodynamic and perineal assessment were repeated at the end of treatment. No complication occurred. Micturition frequency decreased in all patients. Clinical correction of incontinence was observed in ten patients, improvement in nine and no change in one for an overall cure or improvement rate of 95%. The urethrocystocele evaluation did not change. Perineal evaluation and urodynamic parameters were only slightly improved. At follow-up evaluation 6 to 9 months post treatment, a 75% cure or improvement rate was still present. Perineal reeducation is a non morbid and effective modality to correct urinary stress incontinence. Its long term efficacy and its use for other types of incontinence has to be demonstrated.

Translocation of FGF2 to the cell surface without release into conditioned media.

Like most cells in culture, stably transfected COS-1 cells (CF18) that constitutively overexpress basic fibroblast growth factor (FGF2) do not release the growth factor into conditioned media. Yet, when cells were biotinylated, 30% of the total cell-associated immunoreactive FGF2 was detected on the cell surface. Under similar conditions, up to 70% of the total immunoreactive FGF2 in transfected endothelial cells (MAE ZIP) or untransfected rat (C6) and human (U87MG) glioblastoma cell lines was detected on their cell surface. When peripheral plasma membrane proteins were removed from the cell surface with 0.1 M sodium carbonate, the amount of exported FGF2 was significantly reduced, whereas cell viability was unaffected. FGF2 then reappeared on the cell surface in a time-dependent manner. Ouabain, a cardenolide previously shown to inhibit the export of FGF2 from transiently transfected COS-1 cells, blocked the appearance of FGF2 onto the surface of transfected CF18 cells and MAE ZIP cells but had no detectable effect on C6 and U87MG cells. The observation that the translocation of FGF2 onto the cell surface is dissociated from its release into conditioned medium is consistent with FGF2's being rarely found in biological fluids but always cell associated and in the extracellular matrix. The findings point to a role played by the protein export pathway in controlling FGF2 activity and the normal physiological function that this growth factor plays in cell growth and differentiation. The widely accepted presumption that the absence of FGF2 in conditioned media reflects its inability to exit the cell needs to be reevaluated.