What is the effect of MRI with targeted biopsies on the rate of patients discontinuing active surveillance? A reflection of the use of MRI in the PRIAS study.

BACKGROUND: The reduction of overtreatment by active surveillance (AS) is limited in patients with low-risk prostate cancer (PCa) due to high rates of patients switching to radical treatment. MRI improves biopsy accuracy and could therewith affect inclusion in or continuation of AS. We aim to assess the effect of MRI with target biopsies on the total rate of patients discontinuing AS, and in particular discontinuation due to Grade Group (GG) reclassification. METHODS: Three subpopulations included in the prospective PRIAS study with GG 1 were studied. Group A consists of patients diagnosed before 2009 without MRI before or during AS. Group B consists of patients diagnosed without MRI, but all patients underwent MRI within 6 months after diagnosis. Group C consists of patients who underwent MRI before diagnosis and during follow-up. We used cumulative incidence curves to estimate the rates of discontinuation. RESULTS: In Group A (n = 500), the cumulative probability of discontinuing AS at 2 years is 27.5%; GG reclassification solely accounted for 6.9% of the discontinuation. In Group B (n = 351) these numbers are 30.9 and 22.8%, and for Group C (n = 435) 24.2 and 13.4%. The three groups were not randomized, however, baseline characteristics are highly comparable. CONCLUSIONS: Performing an MRI before starting AS reduces the cumulative probability of discontinuing AS at 2 years. Performing an MRI after already being on AS increases the cumulative probability of discontinuing AS in comparison to not performing an MRI, especially because of an increase in GG reclassification. These results suggest that the use of MRI could lead to more patients being considered unsuitable for AS. Considering the excellent long-term cancer-specific survival of AS before the MRI era, the increased diagnostic accuracy of MRI could potentially lead to more overtreatment if definitions and treatment options of significant PCa are not adapted.

SPCG-15: a prospective randomized study comparing primary radical prostatectomy and primary radiotherapy plus androgen deprivation therapy for locally advanced prostate cancer.

OBJECTIVE: To describe study design and procedures for a prospective randomized trial investigating whether radical prostatectomy (RP) ± radiation improves cause-specific survival in comparison with primary radiation treatment (RT) and androgen deprivation treatment (ADT) in patients with locally advanced prostate cancer (LAPC). MATERIALS AND METHODS: SPCG-15 is a prospective, multi-centre, open randomized phase III trial. Patients are randomized to either standard (RT + ADT) or experimental (RP with extended pelvic lymph-node dissection and with addition of adjuvant or salvage RT and/or ADT if deemed necessary) treatment. Each centre follows guidelines regarding the timing and dosing of postoperative RT and adjuvant treatment such as ADT The primary endpoint is cause-specific survival. Secondary endpoints include metastasis-free and overall survival, quality-of-life, functional outcomes and health-services requirements. Each subject will be followed up for a minimum of 10 years. RESULTS: Twenty-three centres in Denmark, Finland, Norway and Sweden, well established in performing RP and RT for prostate cancer participated. Each country's sites were coordinated by national coordinating investigators and sub-investigators for urology and oncology. Almost 400 men have been randomized of the stipulated 1200, with an increasing rate of accrual. CONCLUSIONS: The SPCG-15 trial aims to compare the two curatively intended techniques supplying new knowledge to support future decisions in treatment strategies for patients with LAPC The Scandinavian healthcare context is well suited for performing multi-centre long-term prospective randomized clinical trials. Similar care protocols and a history of entirely tax-funded healthcare facilitate joint trials.

KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine.

Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.

Increase of prostate biopsy-related bacteremic complications in southern Finland, 2005-2013: a population-based analysis.

BACKGROUND: The most severe manifestations of prostate biopsy complications are bacteremic infections. These complications are increasing alarmingly. METHODS: A retrospective cohort study of 17 183 transrectal prostate biopsies performed at the Helsinki and Uusimaa hospital district in southern Finland during 2005-2013. Biopsies were linked to a database of positive blood cultures, yielding 111 bacteremic cases, and yearly bacteremia rates were determined. By multiple regression analysis, demographic risk factors of the whole biopsy cohort for developing bacteremia or fluoroquinolone (FQ)-resistant bacteremia were studied. Clinical risk factors for bacteremia caused by an FQ-resistant organism and for serious bacteremic outcomes were studied by univariate and multivariate analyzes. RESULTS: The average bacteremia rate was 0.7% (111 of 17 183 biopsies) and an increase was observed from 0.5% in 2005 (95% confidence interval (CI): 0.3-0.9) to 1.2% in 2012 (95% CI 0.8-1.8); 53.2% were caused by an FQ-resistant organism. In univariate regression analysis, previous biopsy sessions and increasing calendar year of biopsy associated with the risk of developing bacteremia (odds ratio (OR) 1.232, 95% CI: 1.020-1.488, P=0.030 and OR 1.164, 95% CI: 1.079-1.255, P<0.001, respectively), but only increasing calendar year of biopsy remained statistically significant (OR 1.155, 95% CI: 1.070-1.247, P<0.001) in multivariate analysis. Foreign travel within 3 months was associated with FQ resistance in multivariate analysis (OR 7.158, 95% CI: 1.042 to infinite, P=0.045). The study failed to show any significant clinical risk factors for serious bacteremic outcomes (requiring intensive care, developing deep infection foci or death). CONCLUSIONS: The postbiopsy bacteremia rate doubled during the study period and half of the cases were caused by FQ-resistant organisms. Recent foreign travel increased the risk for FQ resistance. Future research efforts should be aimed at better identifying risk factors, targeted prophylaxis and reducing the need for biopsies.

Systematic knockdown of epigenetic enzymes identifies a novel histone demethylase PHF8 overexpressed in prostate cancer with an impact on cell proliferation, migration and invasion.

Our understanding of key epigenetic regulators involved in specific biological processes and cancers is still incomplete, despite great progress in genome-wide studies of the epigenome. Here, we carried out a systematic, genome-wide analysis of the functional significance of 615 epigenetic proteins in prostate cancer (PrCa) cells. We used the high-content cell-spot microarray technology and siRNA silencing of PrCa cell lines for functional screening of cell proliferation, survival, androgen receptor (AR) expression, histone methylation and acetylation. Our study highlights subsets of epigenetic enzymes influencing different cancer cell phenotypes. Plant homeo domain (PHD) finger proteins have a key role in cell survival and histone methylation, whereas histone deacetylases were primarily involved in regulating AR expression. In contrast, JumonjiC-domain (JmjC) containing histone lysine demethylases (KDMs) mainly had an impact on cell proliferation. Our results show that the KDMs JARID1B, PHF8, KDM3A, KDM3B and KDM4A were highly expressed in clinical PrCa samples. The PHD-finger protein 8 (PHF8), a transcriptional coactivator with both PHD- and JmjC-domains, was moderately to strongly expressed in 80% of clinical PrCa samples, whereas 76% of normal and benign samples were negative or only showed weak PHF8 expression. Strong PHF8 expression correlated significantly with high Gleason grade and was borderline significant for poor prognosis. The results of functional PHF8 knockdown implicate a role in cell migration and invasion, as shown by cell motility and 3-D invasion assays. Our study suggests that various cellular phenotypes are regulated by distinct subsets of epigenetic enzymes. Proteins interpreting and modifying histone methylation, such as JmjC-domain and particularly PHD-finger proteins like PHF8, are activated in subsets of PrCa's and promote cancer relevant phenotypes.

A high pretreatment plasma oestradiol level is associated with a low risk of acute myocardial infarction in parenteral oestrogen therapy for locally advanced prostate cancer.

OBJECTIVE: To evaluate the relationship of pretreatment plasma oestradiol (ppE2) and testosterone (ppT) level to acute myocardial infarction (AMI) in patients with locally advanced prostate cancer primarily treated with parenteral polyoestradiol phosphate (PEP) or orchidectomy, considering the effect of age, performance status, pretreatment diseases and medication, and tumour stage and grade. PATIENTS AND METHODS: The present Finnprostate 6 study comprised 234 patients randomized to oestradiol or intramuscularly administered PEP (240 mg/month) therapy. Each patient was followed until the end of the primary therapy (up to 10 years) or until the first AMI (lethal or not). RESULTS: The risk of AMI, when the PEP and orchidectomy groups were analysed together, was lower in patients with a high ppE2 level, and this risk was independent of the ppT level, pretreatment diseases, medication, age, performance status, disease stage or grade. In the PEP therapy group the risk of AMI was statistically significantly lower in patients with a high ppE2 level (>or=93 pmol/L) than in those with a low ppE2 level (<93 pmol/L; risk ratio 0.28, 95% confidence interval 0.10-0.84, P = 0.022). There was no such difference in the orchidectomy group. The ppT level had no association with the risk of AMI. CONCLUSIONS: A high ppE2 level is associated with a low risk of AMI in patients with locally advanced prostate cancer treated with PEP; there was no such association for ppT level. In the orchidectomy group the ppE2 or ppT level was not statistically significantly associated with the risk of AMI.

Cavoatrial extension of renal cell cancer: results of operative treatment in Helsinki University Hospital between 1990 and 2000.

BACKGROUND AND AIMS: To evaluate the technical procedures and the post-operative survival of patients having been operated for renal cell cancer with cavoatrial tumour thrombus (RCC-T). MATERIAL AND METHODS: Between 1990 and 2000 the cardiac unit at Helsinki University Central Hospital operated on seven patients for RCC-T. A cardiac surgeon along with a urologist, performed all seven operations using sternolaparotomy (either midline or Chevron incision) with cardiopulmonary bypass. RESULTS: The average duration of the operations was eight hours (range 6-11 hours) and the average perfusion time was 118 minutes (range 35-206). Hypothermic circulatory arrest was used for one patient with an arrest time of 31 minutes. Only with one patient could the cavotomy be closed directly. In four patients a cava resection was performed and closed either with a pericardium patch or a Gore-Tex prosthesis. In two patients the cava was ligated below the renal veins. During the post-operative intensive care, there were two deaths. Of the remaining patients, five were alive after six months, four after 12 months, three after six years and one patient is still alive after 12 years of follow-up. CONCLUSIONS: In agreement with previously published results, although peri-operative mortality is relatively high with RCC-T patients, long-term post-operative survival is possible.

Quantification of prostate specific antigen mRNA levels in circulation after prostatic surgery and endocrine treatment by quantitative reverse transcription-polymerase chain reaction.

Various methods to detect prostatic cells in circulation have given conflicting results. This is probably because qualitative rather than quantitative methods have been used to detect mRNA from prostatic cells. A quantitative method has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) for detection of prostate specific antigen (PSA) mRNA in peripheral blood. A competitive internal mRNA standard was used for quantification of absolute amounts of PSA mRNA. The detection limit of the assay was 7 copies of mRNA, and the highest level of circulating PSA mRNA in 88 control subjects was 25 copies per milliliter of blood. This method was used to study the influence of prostatic surgery and endocrine treatment on prostatic cells in the circulation of 56 patients undergoing biopsy, radical prostatectomy, transurethral resection of the prostate (TURP), orchiectomy, or androgen blockade. Blood samples were drawn before, during and up to 26 weeks after these procedures had been carried out. The highest level of PSA mRNA in controls was 25 copies per milliliter of blood. After RP, TURP or orchiectomy, PSA mRNA levels increased above this level in 27%, 29%, and 25% of the samples, respectively. After prostate biopsy, two out of 15 patients became positive. PSA mRNA levels that were elevated by surgery became undetectable within 1-3 days. No significant correlation was found between PCR positivity and the clinical characteristics of the patients. It is concluded that the level of PSA mRNA in peripheral blood increases after prostatic surgery, indicating temporary dissemination of prostatic cells. However, preoperative levels do not correlate with serum PSA, stage or grade.

The pattern of inhibin/activin alpha- and betaB-subunit messenger ribonucleic acid expression in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

To further investigate the regulatory mechanisms responsible for the control of testicular inhibin/activin subunit gene expression, inhibin-alpha, -betaA, and -betaB messenger RNA (mRNA) levels were assessed after ethylene dimethane sulfonate (EDS)-induced destruction of Leydig cells (LC) in different animal models: the intact rat, the rat treated with high doses of testosterone, and the unilaterally cryptorchid rat. In intact rats, EDS selectively eliminates the mature adult-type LCs, activating the proliferation and differentiation of preexisting LC precursors into a new population of functionally active LCs. In this model, a single dose of EDS (75 mg/kg BW, ip) induced a significant increase in testicular inhibin-alpha and -betaB mRNA levels 5 days after treatment (5.0- and 5.5-fold increases, respectively), whereas inhibin-betaA mRNA remained undetectable upon Northern hybridization in control and EDS-treated testes. Moreover, in situ hybridization analysis demonstrated that the increased expression of inhibin-alpha and -betaB mRNAs observed 5 days after EDS takes place mainly in Sertoli cells. Along with LC repopulation, the expression level of inhibin-alpha and -betaB messages declined, and inhibin-alpha mRNA returned to control values on day 40 after EDS. This treatment, however, failed to alter the pattern of testicular expression of FSH receptor and androgen-binding protein mRNAs, thus suggesting selectivity for the above effects. In EDS-treated rats supplemented with high doses of testosterone, the preexisting mature LCs are destroyed, but, due to elevated testosterone concentrations, disruption of spermatogenesis is attenuated, and the post-EDS rise in serum gonadotropins is blocked; the latter prevents LC regeneration. In this model, a 5.0-fold increase in inhibin-alpha mRNA levels, similar to that found in intact animals, was detected 5 days after EDS administration, but the rise in inhibin-betaB levels was partially delayed. In addition, the blockade of LC repopulation resulted in permanent elevation of inhibin-alpha and -betaB messages throughout the study period. In unilaterally cryptorchid rats, the abdominal testis shows disrupted spermatogenesis and altered paracrine environment that expedites LC repopulation after EDS treatment. In this model, the abdominal testes showed a significant 2.5-fold increase in inhibin-alpha mRNA levels 5 days after EDS, but no effect was found in those of inhibin-betaB. Further, the faster rate of LC repopulation resulted in precocious decline of inhibin-alpha mRNA levels. Finally, the expression of inhibin/activin subunit mRNAs was monitored during postnatal testicular development, specifically at the time of regression of fetal-type LCs and appearance of those of the adult type. High levels of expression of inhibin-alpha and -betaB mRNAs were detected in neonatal and infantile testes. A sharp decline in both messages took place between days 15-20, i.e. at the time when fetal-type Leydig cells are replaced by adult-type cells. From this time point onward, inhibin-alpha and -betaB mRNA levels remained low, ranging between 15-30% of the maximum. In conclusion, our results suggest that the adult-type LCs differentially modulate the expression of inhibin/activin subunit genes and point to a major inhibitory role in this cell type on expression of the inhibin-alpha gene.

Experimental cryptorchidism induces a change in the pattern of expression of LH receptor mRNA in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

In the rat, the cytotoxic drug ethylene dimethane sulfonate (EDS) selectively eliminates mature Leydig cells (LCs) from testicular interstitium, activating a complex process of proliferation and differentiation of pre-existing LC precursors. We observed previously that after EDS treatment, the early LC precursors persistently express a truncated 1.8 kb form of LH receptor (LHR) mRNA. This prompted us to study whether experimental cryptorchidism, known to alter the process of LC repopulation, can influence the pattern of testicular LHR mRNA expression after EDS administration. EDS treatment completely eliminated mature LCs both in control and unilaterally cryptorchid (UC) rats. This response was followed by gradual reappearance of newly formed, functionally active LCs, as evidenced by the recovery in testicular LHR content and plasma testosterone levels in both experimental groups. Noteworthy, the rate of LC repopulation was higher in the abdominal testes of UC rats, in keeping with previous findings. Interestingly, the 1.8 kb LHR transcript was persistently expressed in scrotal testes at all time-points, but undetectable upon Northern hybridization in abdominal testes at early stages after EDS administration, when low levels of expression of truncated LHR transcripts could only be detected by semi-quantitative RT-PCR analysis. In addition, the faster LC repopulation in cryptorchid testes was associated with precocious recovery of the complete array of LHR mRNA transcripts, including the 1.8 kb species. These changes appeared acutely and irreversibly, as unilateral positioning of scrotal testes into the abdomen resulted in a rapid loss of expression of the 1.8 kb LHR transcript, whereas scrotal relocation of the UC testes failed to alter the pattern of LHR gene expression. In conclusion, experimental cryptorchidism changes the pattern of LHR mRNA expression in rat testis after selective LC destruction by EDS. This change, i.e. repression of the 1.8 kb LHR transcript after EDS administration, is acute and irreversible, and likely related to the impairment of testicular microenvironment following cryptorchidism. However, even though at low levels, the expression of truncated forms of LHR mRNA appears to be a universal feature of proliferating LC precursors. The UC testis may represent a good model for analysis of the regulatory signals involved in the control of LHR gene expression.

Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo.

We have previously described the preparation, purification and partial characterization of recombinant (rec) forms of rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the present study, the special functional features of these hormones were studied further, in vitro and in vivo, and compared with human recLH and recFSH, as well as with human urinary choriongonadotropin (hCG) and rat pituitary LH (NIDDK-RP3). In radioreceptor assay, the affinity of hCG binding to rat testis membranes was 5-fold higher than that of human recLH and 100-fold higher than that of rat recLH. In in vitro bioassay, using dispersed adult mouse interstitial cells or a mouse Leydig tumor cell line (BLT-1), hCG and human recLH were 10- to 20-fold more potent than rat recLH. Correspondingly, rat pituitary LH was about 10-fold less potent than rat recLH, and evoked a maximum testosterone response that was about half of that elicited by the other LH/CG preparations. Rat recFSH was about 10-fold less potent than human recFSH in stimulating cAMP production of a mouse Sertoli cell line (MSC-1) expressing the recombinant rat FSH receptor. The circulating half-times (T1/2) of rat and human rec hormones were assessed after i.v. injections into adult male rats rendered gonadotropin-deficient by treatment with a gonadotropin-releasing hormone antagonist. A novel immunometric assay was used for the rat FSH measurements. In the one-component model the T1/2 values of rat and human recLH were 18.2 +/- 1.9 min (n = 7) and 44.6 +/- 3.1 min (n = 7) respectively and those of rat and human recFSH were 88.4 +/- 10.7 min (n = 6) and 55.0 +/- 4.2 min (n = 6) respectively; the two-component models revealed similar differences between the rec hormone preparations. Collectively, rat recLH was eliminated significantly faster from the circulation than human recLH (P < 0.0001). In contrast, the elimination of rat recFSH was significantly slower than that of human recFSH (P = 0.02). In conclusion, rat recFSH and rat recLH display lower biopotencies per unit mass than the respective human hormones in vitro, and also in vivo for LH. This is paralleled by shorter T1/2 of rat recLH than the respective human hormone in the circulation, whereas human recFSH has a shorter T1/2 than human FSH. The special functional features of the rat rec gonadotropins emphasize the use of these preparations on studies of gonadotropin function in the rat, an important animal model for reproductive physiology.

Stable transfection of the rat follicle-stimulating hormone receptor complementary DNA into an immortalized murine Sertoli cell line.

A plasmid expressing the rat FSH receptor (R) cDNA under the Simian virus (SV) 40 promoter/enhancer was stably transfected into a mouse Sertoli cell (SC) line (MSC-1) established from transgenic mice carrying a fusion gene of the human anti-Müllerian hormone (AMH) promoter sequences linked to the SV40 T-antigen gene (Peschon et al., 1992). The original cell line has numerous SC characteristics, but it was reported not to express the inhibin-alpha and follicle-stimulating hormone (FSH)R genes. The new FSHR expressing cell line possessed approximately 2000 per cell with equilibrium association constant (Ka) of 1.5 x 10(9) l/mol. In Northern blots, an FSHR mRNA species of 2.6 kb was found. The cells responded to recombinant human FSH (recFSH) and pertussis toxin (PT) with stimulated cAMP production. Moreover, PT enhanced the FSH-stimulated cAMP production in these cells, indicating the presence of a functional Gi protein. 12-O-tetradecanoylphorbol-13-acetate (TPA) suppressed the FSH-stimulated cAMP production of the cells, which effect was similar to that observed previously upon protein kinase C (PKC) activation in rat seminiferous tubules in vitro. Hence, the FSHR signalling, and its modulatory pathways, were intact in the FSHR expressing MSC-1 cell line. RT-PCR with inhibin-alpha specific oligonucleotide primers. followed by Southern hybridization, indicated that, unlike previously shown, the original and the FSHR expressing MSC-1 cells do express the inhibin alpha gene. FSH stimulation of the cells decreased their proliferation and, unexpectedly, the inhibin-alpha mRNA levels. The cells have functional features both from neonatal and mature SC. A feature of the former cells is the lack of FSH-stimulated up-regulation of inhibin-alpha expression; in fact FSH decreased this message. The antiproliferative, and apparently differentiating, effect of FSH on these cells resembled mature SC functions. Since adult SC do not proliferate in vitro, the new FSHR expressing and proliferating cell line provides a useful in vitro model for studying some facets of SC functions, though keeping in mind that these transformed cells do not behave identically with adult SC in vivo. The constitutive expression of FSHR in these cells allows the study of posttranscriptional events in the FSHR regulation.

Gonadotropin-independent regulation of steroidogenesis in the fetal rat testis.

UNLABELLED: The goal of the present study was to determine whether the onset of fetal Leydig cell steroidogenesis is dependent on gonadotropic stimulation. The relationships between the onset of pituitary LH synthesis and secretion, and the response of testicular steroidogenesis to LH and various putative paracrine factors were examined. We found by reverse transcription-polymerase chain reaction (RT-PCR) that the LHbeta-subunit gene expression in the fetal pituitary gland starts on embryonic day (E) 16.5. Plasma LH was very low (< 5.0 ng/L) until E19.5 and increased significantly thereafter. In contrast, the greatest increase in the testicular testosterone had already occurred between E18.5 and E19.5. Hence, fetal testicular steroidogenesis must start independent of LH stimulation. Basal testosterone production in incubations of fetal testis (E16.5-19.5) was high, 50-80% of the hCG-stimulated level. In contrast, in dispersed fetal Leydig cells, basal steroidogenesis was consistently low. This suggests the presence of paracrine factors in the intact testes that stimulate their steroidogenesis. Effects of various putative paracrine factors were thereafter tested on the fetal testis. We found for the first time that both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP-27) markedly stimulated fetal, but not adult, Leydig cells. IN CONCLUSION: 1) Pituitary LH cannot be the initial stimulus for fetal testicular steroidogenesis. 2) Some paracrine factor(s) probably turn on and maintain early fetal testicular steroidogenesis before the later onset of LH secretion, although a constitutive component in the onset of steroidogenesis is also possible. 3) VIP and PACAP-27 are likely candidates for a paracrine stimulus of the fetal testis.

Mechanisms of desensitization of follicle-stimulating hormone (FSH) action in a murine granulosa cell line stably transfected with the human FSH receptor complementary deoxyribonucleic acid.

The desensitization of follicle-stimulating hormone (FSH)-evoked cAMP synthesis occurs upon continuous or repeated hormonal stimulation, and it involves the hormone-receptor interaction and post-receptor events. These mechanisms were studied in a murine granulosa cell line (KK-1) stably transfected with the human FSH receptor (hFSHR) complementary deoxyribonucleic acid (cDNA) under a powerful viral promoter. Hence, the FSHR transcriptional regulation was eliminated from the experimental model. Stimulation of the cells with recombinant human FSH (rhFSH) or a phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), resulted in clear desensitization, i.e. subsequent rhFSH-stimulated cAMP formation was 73.4 +/-2.2%, (P < 0.001) and 66.3 +/-3.4%, (P < 0.0001), respectively, of that of cells preincubated in medium. TPA prestimulation evoked also clear inhibition (65-74% of control) of rhFSH or forskolin (a non-specific activator of adenylate cyclase) induced progesterone production. The suppression by TPA preincubation of the rhFSH-induced cAMP synthesis was completely abolished by the protein kinase C (PKC) inhibitor staurosporine (STR). Preincubation with STR exhibited a significant (P < 0.0001) increasing effect on the rhFSH-stimulated cAMP accumulation. The specific involvement of PKC was further evidenced by other inhibitors, all of them exerted significant elevation of cAMP synthesis following rhFSH restimulation. Furthermore, only the PKC beta isoform appeared to be constitutively expressed in these cells during desensitization. Prestimulation of the G-protein activity by sodium fluoride (NaF) or cholera toxin (CT), followed by rhFSH challenge, accounted for a decrease in the cAMP-mediated responsiveness, down to 69.4 +/- 2.8 or 74.2 +/- 1.9%, of control (P < 0.001), respectively, indicating that the post-receptor events are critical for desensitization. [125I]iodo-rhFSH binding to the cells did not change significantly during desensitization and the different stimulations. In contrast, approximately 50% increase (P < 0.001) occurred in the steady-state levels of FSHR mRNA in the cells stimulated with FSH. This was apparently due to prolonged half-time of mRNA, and not to altered transcription, since the FSHR cDNA was driven by a powerful viral promoter. In accordance, the cells transfected with Simian Virus (SV40) promoter-driven luciferase gene did not display alterations in luciferase activity following stimulatory treatments. The effects of the post-receptor stimulations (NaF or CT) on [125I]iodo-rhFSH binding were minor (8-12% reduction). Taken together, these data provide evidence that the agonist-responsive hFSHR desensitization appears through a PKC-beta isoform-mediated modulation of cAMP production. The desensitization of FSH action involves modifications of functional properties of the existing components of the FSH signal transduction complex, and does not require concomitant suppression of transcription or translation of the FSHR gene.

Molecular mechanisms of reappearance of luteinizing hormone receptor expression and function in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

Considering the major role of LH in the control of Leydig cell (LC) development and function, we aimed to characterize further the pattern of LH receptor (LHR) expression in two experimental paradigms: the rat treated with ethylene dimethane sulfonate (EDS), in which the selective destruction of preexisting mature LCs induces the proliferation and differentiation of newly formed LCs, a process that takes place in the presence of high levels of gonadotropins; and the EDS-rat treated with a high dose of testosterone (EDS + T), in which the LH secretion is suppressed, and consequently LC development after EDS arrested. In EDS rats, serum T was suppressed and testicular LHR binding became undetectable on days 5 and 15 after treatment. The pattern of LHR messenger RNA (mRNA) expression was profoundly modified: only one of the splice variants [1.8-kilobase (kb)] persisted, whereas the others disappeared. On days 20 and 45 after EDS, along with LC repopulation, serum T and LHR binding recovered, and the pattern of LHR mRNA expression gradually returned to that resembling controls. In EDS + T rats, a similar drop in testicular LHR binding and change in the pattern of LHR mRNA expression was detected on days 5 and 15 after treatment. However, on days 20 and 45, no recovery either in LHR binding or in expression of the longer LHR mRNA splice variants was observed, showing that LH is needed to induce LHR expression in repopulating LCs, at least to a quantitatively significant level. To gain further insight into the mechanism(s) by which LH acts on LC precursors, the translational status of the 1.8-kb LHR transcript, persistently expressed after EDS, was analyzed and compared with that of the 6.8-kb message. In polysome distribution analysis of total testicular RNA, the 6.8-kb LHR message was highly associated with polysomes, whereas the 1.8-kb variant was mainly localized to prepolysomal fractions, both in control and EDS testes, thus predicting lower translational efficiency. In addition, considering that only LCs express LHRs in the testis, the time course of the reappearance of functional receptors was mapped by evaluating testicular responsiveness to human recombinant LH in vitro. No response to LH stimulation was detected 5 days after EDS. However, cAMP response to LH was observed on days 15 and 20, regardless of the presence of high (EDS) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of functional LHRs, qualitatively, can take place in the absence of measurable LH levels. In EDS-treated rats, the appearance of the cAMP response coincided with those of pregnenolone, progesterone, and T. In contrast, no LH-induced steroid release was observed in EDS + T rats, indicating that steroidogenic response in developing LC requires LH priming. In conclusion, the appearance of functional LHRs, at a low level of expression, in LC precursors is an LH-independent developmental event, essential for the subsequent LH-dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results cast doubt on a major functional role of the truncated (1.8-kb) form of LHR mRNA, which persists after EDS at a high level of expression, in the early Leydig cell precursors.

Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species.

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testicular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) containing the complete amino acid-coding sequence (676 amino acids). Northern hybridization analysis of monkey testicular RNA, using a complementary RNA probe corresponding to the full-length cDNA, demonstrated major transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, and 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a striking feature, i.e. the absence of an 81-bp nucleotide sequence corresponding to exon 10, present in the LHR cDNAs of all other species studied to date. The monkey LHR cDNA displayed 83-94% overall sequence homology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridization of monkey genomic DNA using a human exon 10 probe demonstrated its presence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA showed similar high affinity (Kd = 0.25 nmol/liter) of [125I]iodo-hCG binding as those transfected with human LHR cDNA (Kd = 0.20 nmol/liter). The cells expressing the recombinant monkey and human LHR displayed similar responses of extracellular cAMP and inositol trisphosphate to hCG. In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.

Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium.

Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII-I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P < 0.01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I-IV were threefold higher than in stages VI-VII (P < 0.01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.

Developmental expression of the prolactin receptor gene in rat gonads.

The prolactin receptor (PRLR) is a member of the cytokine/prolactin/GH receptor family, and it is widely expressed in various mammalian tissues. Expression of the two different forms of PRLR, differing in the length of their cytoplasmic domains, was studied in rat gonads during fetal and postnatal development. The two forms of PRLR mRNA were analyzed by reverse transcription (RT)-PCR using primer pairs specific for the different forms. The specificity of the cDNA species generated by RT-PCR was verified by Southern hybridization using nested 32P-labeled oligonucleotides. The results indicated that both forms of PRLR mRNA are expressed in the rat testis and ovary, which is in agreement with previous reports. The onset of expression of the two PRLR forms occurs on day 14.5 of fetal life in rat testis. In the ovary, the long form of PRLR mRNA appears 1 day before the short form, i.e. these forms begin to be expressed on fetal days 14.5 and 15.5 respectively. In situ hybridization with antisense cRNA probes specific to each form of the PRLR mRNAs demonstrated specific hybridization of both forms, localized in Leydig cells from day 18.5 of fetal life and at the postnatal ages studied. Compared with our previous findings concerning the ontogeny of LH receptor gene expression, PRLR gene expression starts earlier in development and exhibits no sexual dimorphism. The presence of two forms of PRLR mRNA in the fetal gonads suggest that they might play differential roles in gonadal development and function.

Isolation and characterization of testis-specific cDNAs for luteinizing hormone beta-subunit in the rat.

To study further the unexpected expression of the luteinizing hormone (LH) beta-subunit in the rat testis, we identified in a rat testicular cDNA library three LH beta clones with lengths of 3.2, 2.4 and 0.86 kb (TLH beta 1, TLH beta 2 and TLH beta 3). The clones were identified using a 32P-labeled cDNA probe complimentary to the known rat pituitary LH beta mRNA. Clone TLH beta 2 corresponds in size to the main LH beta mRNA species (2.7 kb) detected by Northern hybridization in the rat testis. Sequence analysis indicated that the different sizes of the three clones are due to alternative RNA splicing and differences at the 5' ends of transcripts. The sequence of one open reading frame deduced from TLH beta 1 is almost identical with the pituitary LH beta peptide, differing only in three amino acids in the putative signal peptide. It might encode a functional testis-specific LH beta peptide. Shorter transcripts from clones TLH beta 2 and TLH beta 3 may correspond to short testicular LH beta peptides. The present findings provide further evidence in the rat for expression of testis-specific mRNA variants of the LH beta gene. Their translation products may form a novel class of testicular para/autocrine factors.