Influence of the vitamin D-binding protein on the serum concentration of 1,25-dihydroxyvitamin D3. Significance of the free 1,25-dihydroxyvitamin D3 concentration.

The influence of the serum binding protein (DBP) for vitamin D and its metabolites on the concentration of its main ligands, 25-hydroxyvitamin D(3) (25-OHD(3)) and 1,25-dihydroxyvitamin D(3) (1,25-[OH](2)D(3)) was studied. The concentration of both 1,25-(OH)(2)D(3) and DBP in normal female subjects (45+/-14 ng/liter and 333+/-58 mg/liter, mean+/-SD, respectively; n = 58) increased during the intake of estro-progestogens (69+/-27 ng/liter and 488+/-90 mg/liter, respectively; n = 29), whereas the 25-OHD(3) concentration remained unchanged. A positive correlation was found between the concentrations of 1,25-(OH)(2)D(3) and DBP in these women. At the end of pregnancy, the total concentrations of 1,25-(OH)(2)D(3) (97+/-26 ng/liter, n = 40) and DBP (616+/-84 mg/liter) are both significantly higher than in nonpregnant females and paired cord serum samples (48+/-11 ng/liter and 266+/-41 mg/liter, respectively). A marked seasonal variation of 25-OHD(3) was observed in pregnant females and their infants, whereas in the same samples the concentrations of both DBP and 1,25-(OH)(2)D(3) remained constant throughout the year. The free 1,25-(OH)(2)D(3) index, calculated as the molar ratio of this steroid and DBP, remains normal in women taking estro-progestogens, however, and this might explain their normal intestinal calcium absorption despite a high total 1,25-(OH)(2)D(3) concentration. In pregnancy the free 1,25-(OH)(2)D(3) index remains normal up to 35 wk of gestation, but during the last weeks of gestation, the free 1,25-(OH)(2)D(3) index increases in both circulations. A highly significant correlation exists between the (total and free) 25-OHD(3) and 1,25-(OH)(2)D(3) concentrations in maternal and cord serum both at 35 and 40 wk of gestation.

Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP.

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.

Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway.

Increased expression of fatty acid synthase (FAS) is observed in a clinically aggressive subset of various common cancers and interference with FAS offers promising opportunities for selective chemotherapeutic intervention. The mechanisms by which FAS expression is (up)-regulated in these tumors remain, however, largely unknown. Recently we demonstrated that in LNCaP prostate cancer cells FAS expression is markedly elevated by androgens via an indirect pathway involving sterol regulatory element-binding proteins (SREBPs). Here, we also show that growth factors such as EGF are able to stimulate FAS mRNA, protein and activity. Several observations also indicate that the effects of EGF on FAS expression are ultimately mediated by SREBPs. EGF stimulates SREBP-1c mRNA expression and induces an increase in mature nuclear SREBP-1. Moreover, in transient transfection studies EGF stimulates the transcriptional activity of a 178 bp FAS promoter fragment harboring a complex SREBP-binding site. Deletion or mutation of this binding site abolishes these effects and ectopic expression of dominant negative SREBP-1 inhibits FAS expression and induction in intact LNCaP cells. Given the frequent dysregulation of growth factor signaling in cancer and the key role of SREBP-1 in lipid homeostasis, growth factor-induced activation of the SREBP pathway is proposed as one of the mechanisms responsible for up-regulation of lipogenic gene expression in a subset of cancer cells.

Identification of a functional androgen-response element in the exon 1-coding sequence of the cystatin-related protein gene crp2.

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Androgens stimulate lipogenic gene expression in prostate cancer cells by activation of the sterol regulatory element-binding protein cleavage activating protein/sterol regulatory element-binding protein pathway.

Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin.

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.

Morphological and functional characteristics of isolated acini used for in vitro studies of prostatic secretion.

Acini of the rat ventral prostate were isolated by interstitial injection of a collagenase-containing medium, subsequent incubation in the same medium and repeated aspiration through pipette tips with decreasing gauge of the tip opening. Functional integrity of the isolated acini was assessed by morphological studies, including transmission and scanning electron microscopy, freeze-fracturing, and immunocytochemistry. Incubation studies with different incubation media were performed monitoring O2-consumption as a parameter of functional activity, in addition to the incorporation rate of radioactively labeled amino acids into newly synthesized proteins. Optimal incubation conditions (shaking water bath, 20 strokes/min, 37 degrees C, gassing with carbogen at 15 min intervals) were found with M 199 medium supplemented with dihydrotestosterone. Stimulation of prostatic secretion was maximal with 10(-7) M of pilocarpin, which was more effective than carbamylcholine. Incorporation of precursors into prostatic proteins proceeded for about 2 h at a linear rate. Thereafter a rapid loss of functional and morphological integrity of the isolated acini was observed including disintegration, vacuolation and lysis of individual cells. The system of isolated prostatic epithelium developed is a useful tool in the study of prostatic secretion in vitro in short term experiments.

Secretion of prostatic binding protein by rat ventral prostate: influence of age and androgen.

Rat ventral prostate of adult male rats contain a large amount of prostatic binding protein (PBP). Immunological evidence indicates that this protein is a specific secretion product of this gland. The amount and concentration of PBP in ventral prostate show marked changes as a function of age. PBP is low but detectable (0.009 and 0.002 U/mg prostate) in 5- and 10-day-old rats and increases thereafter in a biphasic way to adult levels (0.619 U/mg prostate). After castration of PBP drops to 0.054 U/mg prostate after 10 days and 0.030 U/mg prostate after 21 days. The concentration of PBP returns to precastration levels after 2 weeks of androgen treatment. Estradiol and progesterone are ineffective in this respect. The antiandrogen, cyproterone acetate, counteracts the stimulatory effect of testosterone propionate.

Prostatic stromal cells and testicular peritubular cells produce similar paracrine mediators of androgen action.

The role of mesenchymal-epithelial interactions in androgen action was explored using Sertoli cells as the epithelial cells and testicular peritubular cells or prostatic stromal cells as mesenchymal cells. Footsole fibroblasts served as a control. The secretion of transferrin was used as an androgen-regulated parameter of Sertoli cell function. It is demonstrated that coculture of peritubular or stromal cells with Sertoli cells markedly increases the production of transferrin. This effect requires a 4-day latent period and is maximal with low concentrations (10%) of mesenchymal cells. Stimulatory effects of androgens can only be demonstrated at suboptimal concentrations of the latter cells. Fibroblasts are inactive. At least two mechanisms contribute to these stimulatory effects. Peritubular cells and stromal cells share the ability to promote the deposition of an extracellular matrix when cocultured with Sertoli cells. When Sertoli cells are seeded on this matrix, the production of transferrin is increased. This effect requires no latent period and is independent of the presence of androgens during the period of matrix deposition. In addition, peritubular cells and stromal cells produce diffusible mediators which increase transferrin production by Sertoli cells. In both cell types, the production of these mediators is controlled by androgens, and their action is preceded by a 4-day latency period. The mediators have a comparable mol wt (45,000) and resemble P Mod-S, known to be secreted by peritubular cells. These data suggest that mesenchymal-epithelial interactions play a role in androgen-supported maintenance of adult function and that mesenchymal tissue from different androgen target tissues produces similar or identical mediators of androgen action.

An effect of androgens on the length of the poly(A)-tail and alternative splicing cause size heterogeneity of the messenger ribonucleic acids encoding cystatin-related protein.

The 22-kilodalton glycoprotein, expressed in the rat ventral prostate under the influence of androgens, is structurally a cystatin-related protein (CRP), as has been shown by copy DNA sequencing. In fact, two slightly different forms (CRP-1 and CRP-2) are expressed in the prostate; one of them (CRP-1) is also expressed in the exorbital lacrymal gland. In both glands, the CRP-1 messenger RNA (mRNA)s are androgen regulated. Moreover, androgens also influence the size of these mRNAs, which show marked heterogeneity (from 760-950 nucleotides). Indeed, the smaller forms are predominant in castrated animals, whereas the large forms are observed immediately after androgen induction. Hybridization with oligo(dT) followed by ribonuclease H treatment revealed that differences in length of the poly(A)-tail are responsible for this effect of androgens. Indeed, two well defined forms of CRP mRNA subsisted after removal of the poly(A)-tail by this treatment. In the less abundant shorter form (CRP-1 delta), 123 nucleotides are deleted by alternative splicing at the junction between the third and the fourth exon. The variant mRNA encodes a truncated protein, wherein the last 27 amino acids are replaced by a hydrophobic stretch of 8 amino acids. No alternative splicing was observed for the CRP-2 mRNA.

Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells.

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-alpha (Her-alpha) and Her-beta stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-alpha and NDF-beta is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-beta on intact Sertoli cells and reveals parallel displacement curves for Her-beta, Her-alpha, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 microg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP.

Microscopic evaluation of LNCaP cells stained with the lipophilic dye Oil red O revealed that androgens induce a marked stimulation of lipid droplet accumulation. As determined by quantitative analysis of the Oil red O extracted from the stained cells, stimulatory effects of the synthetic androgen R1881 became apparent at concentrations as low as 10(-11) M. Maximal induction (15-fold) was reached at 10(-8) M. Increases were observed 2 days after hormone addition and were maximal 1 day later. Accumulation of lipid droplets was also induced by mibolerone (another synthetic androgen) and by the natural androgens testosterone and dihydrotestosterone. In agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, stimulation of lipid accumulation was also apparent after treatment with progesterone and estradiol. Cortisol and the synthetic glucocorticoid dexamethasone were ineffective. The androgen antagonist Casodex (bicalutamide) abolished the stimulatory effect of R1881, further supporting the involvement of the androgen receptor. In agreement with this conclusion, no changes in lipid accumulation were observed after androgen treatment of the androgen receptor-negative prostate tumor lines PC-3 and DU-145. To investigate the nature of the lipids affected by androgens, lipid extracts were analyzed by TLC, complemented with enzymatic lipid analyses. Androgens were shown to have major effects on the content of triglycerides and cholesterol esters (33- and 7-fold stimulation, respectively), the two main classes of lipids stained by Oil red O. Phospholipid and cholesterol contents were increased by a factor of 2. Incorporation studies with [2-14C]acetate revealed that androgens caused a major stimulation of 2-14C incorporation into triglycerides and cholesterol esters (11- and 13-fold, respectively), suggesting that androgens act at least in part at the level of lipid synthesis. Taken together, these findings indicate that androgens, besides affecting proliferation and protein secretion, also markedly stimulate the production and accumulation of neutral lipids, revealing a novel interesting aspect of androgen regulation of LNCaP cells.

Androgens transcriptionally regulate the expression of cystatin-related protein and the C3 component of prostatic binding protein in rat ventral prostate and lacrimal gland.

In this report, it is demonstrated that the C3 component of prostatic binding protein (PBP) is also expressed and androgen regulated in the exorbital lacrimal gland, as shown previously for cystatin-related protein (CRP), another abundant secretory protein from the ventral prostate. The presence of C3 messenger RNA (mRNA) could be demonstrated by both Northern blot hybridization and PCR amplification and sequencing. The mRNAs encoding the C1 and C2 components of PBP, however, were undetectable. At the protein level, the C3 component in the lacrimal gland is glycosylated and linked by disulfide bridges to a new 10-kDa component not reacting with the PBP antiserum. As shown previously for CRP, the expression of C3 in the lacrimal gland requires the simultaneous presence of androgens and a functional androgen receptor. The effects of castration and androgen treatment on CRP and C3 mRNA concentrations were studied by Northern blot and dot blot hybridization; effects on transcription rates were determined by nuclear run-on assay. Two days after castration, the relative abundance of CRP mRNA had declined significantly (P < 0.01) to 10.5 +/- 1.5% (+/-SEM) of precastration levels in the prostate and to 14.5 +/- 8.0% in the lacrimal gland; the transcription rates declined to 14.3% and 10.0%, respectively. The C3 mRNA level and transcription rate in the prostate showed a more moderate decrease (P < 0.05) to 40.6 +/- 8.5% and 41.7%, but were hardly measurable in the lacrimal gland. Androgen administration resulted in a rapid increase in the transcription rates, which reached or exceeded control levels after 6-9 h of treatment and clearly preceded the increase in mRNA levels. It is concluded that the lacrimal gland, which can be studied conveniently in female and long term androgen-depleted animals offers a suitable model for the study of androgen-regulated gene expression.

Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions.

Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.

Marked elevation and cyclic variation of corticosteroid-binding globulin: an inherited abnormality?

Unexplained high serum corticosteroid-binding globulin (CBG) concentrations [mean values, 78.8 and 55.7 mg/L; normal women, 38.8 +/- 3.8 (+/- SD) mg/L] were found repeatedly in two apparently healthy sisters who were not pregnant or taking exogenous estrogens. One had substantial variations in serum CBG and sex hormone-binding globulin concentrations during the menstrual cycle, which paralleled the normal cyclic changes in serum estradiol. The other woman was postmenopausal and had a high serum CBG concentration despite of low serum estradiol levels. We conclude those women have an inherited abnormality in CBG production.

Genetic variation of human sex hormone-binding globulin: evidence for a worldwide bi-allelic gene.

Genetic variation of human sex hormone-binding globulin (SHBG) has been investigated on 1690 unrelated neuraminidase-treated serum samples using isoelectric focusing followed by transfer to nitrocellulose membranes and immunostaining. Three clearly distinct isoelectric focusing patterns, consistent with the expression of an autosomal genetic system, were identified. Using allele frequencies, calculated on the basis of a bi-allelic gene, an excellent agreement between observed and expected phenotype numbers was obtained in every examined population sample. Family data along with the observed distribution of the three SHBG phenotypes among racially different groups and sexes indicate that SHBG is worldwide encoded by two autosomal codominant alleles. Compared with healthy Belgian blood donors no statistically significant differences were noted for the allele frequencies among 399 patients and 70 hirsute women of Belgian origin. Evidence is also presented that the subunit produced by the variant allele (SHBG2) has a higher molecular mass than the one produced by the regular allele (SHBG1) and that the three SHBG genotypes have identical binding characteristics for 5 alpha-dihydrotestosterone.

Secretion rates of LH and FSH during infusion of LH-FSH/RH in normal women and in patients with secondary amenorrhea: suggestive evidence for two pools of LH and FSH.

Normal women in the early follicular phase and in the luteal phase of the cycle, and patients with secondary amenorrhea received on consecutive days a rapid intravenous injection (50 mug) and a two or four-hour infusion (25 mug/h) of synthetic LH-FSH/RH. The responses of LH and FSH were evaluated by the measured plasma concentrations, as well as by the calculated pituitary secretion rates and by the amounts of hormone released. To estimate these pituitary secretion rates of LH and FSH, a simplified mathematical model is proposed. During an infusion of LH-FSH/RH the secretion rates of both LH and FSH increased in the three groups of women in a biphasic way with a dip after 1 to 2h of infusion, suggesting that besides the pool mobilized by a rapid intravenous injection of LH-FSH/RH there is a second pool of (stored) gonadotropins. For LH the increase above baseline concentrations was higher in group II (luteal phase) than in group I (follicular phase) or in group III (amenorrhea) and this both after a bolus injection and during infusion of LH-FSH/RH. For FSH a similar pattern of response prevailed during an infusion of LH-FSH/RH. After a bolus administration, however, the FSH release was relatively higher in group III (amenorrhea) than in both groups of normal women in which the increases were about the same. The latter finding suggests that the first pool of FSH is released by a different mechanism than the second pool.

Biochemical characterization of peritoneal fluid in women during the menstrual cycle.

Peritoneal fluid was collected at laparoscopy in women during the menstrual cycle and was assayed for protein and steroid hormone content. The total protein concentration in peritoneal fluid and the concentrations of the steroid hormone-binding proteins, transcortin and sex hormone-binding globulin, the polypeptide hormones, LH, FSH, and PRL, correlated with the plasma concentration but were lower; they were, respectively, 68%, 71%, 68%, 42%, and 34% of the plasma concentration. The concentrations of steroid hormones secreted by the ovary, i.e. 17 beta-estradiol, progesterone, androstenedione, and testosterone, were always equal or higher in peritoneal fluid than in plasma. In contrast, the concentrations of cortisol, a nonovarian steroid hormone, was 40% lower in peritoneal fluid than in plasma. No cyclic variations were observed in the peritoneal fluid concentrations of androstenedione and testosterone, two steroid hormones secreted by the stromal component of the ovary. On the contrary, the concentrations of 17 beta-estradiol and progesterone secreted by the follicular apparatus of the ovary increased sharply in peritoneal fluid after ovulation, reaching values of 44000 pg/ml and 3000 ng/ml, respectively. They declined progressively, whereas in plasma, peak concentrations were achieved only in the midluteal phase. In conclusion, the concentrations of 17 beta-estradiol and progesterone are much higher in peritoneal fluid than in plasma for at least 1 week after ovulation. We suggest that the secretion of the early, not yet vascularized, corpus luteum is directed preferentially toward the peritoneal cavity, creating a specific hormonal environment for the released oocyte and the oviduct.

Structure of rat genes encoding androgen-regulated cystatin-related proteins (CRPs): a new member of the cystatin superfamily.

Cystatin-related proteins (CRPs) are abundant androgen-regulated secretory glycoproteins that are specifically synthesized in the ventral prostate and lachrymal gland of the rat. Two complete 6-kb genes, Crp1 and Crp2, have been cloned and characterized. They are differentially expressed and encode slightly different proteins. The genes each contain four exons which are interrupted by large introns. An alignment of their sequences demonstrates an overall homology of 90%. The 3' end of a third gene, Crp3, from which only a 1.5-kb fragment was isolated, displays a sequence identity of 84%. These data indicate the existence of a Crp multigene family. The 5' flanking regions of Crp1 and Crp2 are highly homologous and contain a GATAAA sequence 29 nt upstream from the transcription start point. This TATA-box-like element is also found in the promoters of the genes encoding cystatin type-2 proteins. No other recognizable transcription control elements can be detected. Potential binding sites (ARE) for the androgen receptor are scattered throughout the entire genes. The exon/intron organization of the genes encoding CRPs, the size of the exons and their encoding amino acid sequences exhibiting a characteristic spacing of the Cys residues are structural elements displaying a remarkable similarity with the corresponding elements in the genes encoding cystatin type-2 proteins. CRPs must therefore belong to the cystatin superfamily. However, due to their additional domain encoded in an extra exon 2, CRPs must be classified as a new family, type 5.

The differentiation-related gene 1, Drg1, is markedly upregulated by androgens in LNCaP prostatic adenocarcinoma cells.

A differential display technique was used to identify androgen-regulated genes in LNCaP prostatic adenocarcinoma cells. One of the genes markedly upregulated by androgens proved to be identical to differentiation-related gene 1 (Drg1; also described as RTP, Cap43 and rit42), a gene whose expression has recently been shown to be diminished in colon, breast and prostate tumors. We show that Drg1 is abundantly expressed in the (androgen-exposed) human prostate and that its expression is stimulated some 14-fold in androgen-treated LNCaP cells. The ligand specificity of the induction reflects the altered specificity of the mutated androgen receptor in LNCaP. In androgen receptor negative tumor lines basal expression is slightly higher than in LNCaP but inducibility is absent. These data suggest that Drg1 is a novel marker of androgen-induced differentiation in the human prostate.