Functional characterization of the basal promoter of the murine LH receptor gene in immortalized mouse Leydig tumor cells.

The nuclear proteins of the LH receptor (LHR) expressing murine Leydig tumor cells (mLTC-1), binding to the LHR primary promoter, were studied by gel retardation assays. Nuclear extracts of HeLa cells, not expressing LHR, were used as control. Protein binding was characterized to the first 173 base pairs (bp) of the LHR 5'-untranslated region, comprising the basal transcriptional promoter activity in mLTC-1 cells, and accounting for the Leydig cell-specific LHR expression. The promoter fragment is GC-rich and contains several Sp1 sites, one activating protein 2 (AP-2) site, and a putative SF-1 binding site. Three subfragments of the 173 bp promoter, I (bases -1 to -55), II (-56 to -102) and III (-103 to -173), were separately analyzed. Fragments II and III formed several complexes with mLTC-1 and HeLa cell nuclear extracts. One complex with fragments II and III, using mLTC-1 and HeLa cell extracts, was similar to that formed with purified Sp1, and it could be removed by an Sp1 oligo and supershifted by an Sp1 antibody. Both fragments formed additional complexes with mLTC-1 cell extracts with no specificity for Sp1. Partly similar, though weaker, complexes were seen with HeLa cell extracts. The most clearcut differences between the protein/DNA complexes formed with LHR expressing mLTC-1 cells and non-expressing (HeLa, COS, HEK 293 and MSC-1) cells were found with fragment I. Extracts of the non-expressing cells formed one prominent protein/DNA complex which was missing in mLTC-1 cells. Purified Sp1 also bound to this fragment. The fragment containing the putative SF-1 binding site did not form any protein/DNA complexes with mLTC-1 cell proteins. In conclusion, the murine LHR primary promoter binds, in addition to the Sp1 and AP-2 transcription factors, several other proteins. The Sp1 protein can bind into at least three different sites in the basal promoter. The other binding proteins differ most clearly between LHR expressing and non-expressing cells in the promoter fragment closest to the translation start site, suggesting a key role for this part of the promoter in cell-specific LHR expression.

Inhibition of hCG-stimulated steroidogenesis in cultured mouse Leydig tumor cells by bisphenol A and octylphenols.

Some environmental chemicals exhibit estrogenic or antiandrogenic activity. Some of these, such as bisphenol A (bis A) and octylphenols, are used in large amounts in many applications. We have analyzed the effects of bis A and octylphenols on steroidogenesis in Leydig cells by measuring the LH receptor-mediated cAMP and progesterone (P) production in cultured mouse Leydig tumor cells (mLTC-1 cells). After preincubation of mLTC-1 cells for 48 h in the presence of bis A or one of the octylphenols in micromolar concentration, the hCG-stimulated cAMP and P formation in these cells was inhibited. Bis A or octylphenols could neither inhibit cAMP nor P formation stimulated by forskolin (Fk) or cholera toxin (CT) nor steroidogenesis stimulated by 8-Br-cAMP. The preincubation of mLTC-1 cells with estradiol or diethylstilbesterol (DES) at the concentration of 10(-8) mol/liter had no inhibitory effect on cAMP formation stimulated by hCG or Fk but P production was inhibited. Similarly, both estrogens inhibited P production stimulated by 8-Br-cAMP. Bis A or octylphenols had no effect on 125I-hCG binding to Leydig cell LH-receptors. Thus, these environmental chemicals appear to inhibit cAMP formation and steroidogenesis in mLTC-1 Leydig tumor cells by preventing the coupling between LH receptor and the adenylate cyclase. Since, estradiol did not inhibit hCG-stimulated cAMP production, the effects of bis A and octylphenols may not be estrogen related. This emphasizes the complexity of endocrine disruption: chemicals show multiple endocrine activities that may disturb several organs in distinct ways.

CD106 (VCAM-1) in testicular immunoregulation.

The expression of CD18, CD49d/CD29, CD44, CD54 and CD106 was studied in the testis of normal mice at various ages, in the cryptorchid testis, in the testis of estrogen-treated mice and in the testis of non-obese diabetic (NOD) mice, using immunocytochemistry to see which of these lymphocyte and endothelial adhesion proteins may be involved in lymphocyte regulation in the testis. CD18-, CD49d/CD29-, CD44- and CD54-expressing cells were not found in the normal > 10-week-old BALB/c mouse testis. Leydig cells expressed CD106 strongly at this age. In contrast to the > 10-week-old testis, only very few interstitial cells of the 2-week-old normal mice expressed CD106. The expression of CD106 increased gradually with age so that at 6 weeks of age the expression of CD106 was moderate in the interstitial tissue. In the experimentally abdominal testis, CD106 was expressed in the interstitial tissue as strongly as in the contralateral scrotal testis. CD44- and CD18-expressing cells were occasionally present in the interstitial tissue of the abdominal testis, but not in the contralateral scrotal testis. CD54 was present in the epithelium of the ductuli efferentes. In the testis of the estrogen-treated mice, CD106 was expressed in the interstitial tissue as strongly as in the normal mice. Occasional CD44- and CD18-expressing cells were found in the testicular capsule. In the testis of adult NOD mice, CD106 was present in the interstitial tissue, but none of the other studied proteins. Immunoblotting of CD106 from the adult testis under reducing conditions demonstrated a single broad band with a M(r) of 51-65 kDa. This is a novel isoform of CD106. In a modified Stamper-Woodruff assay, lymphocytes bound to the testicular interstitial tissue. In co-incubations of native Leydig cells and lymphocytes, anti-CD106 antibodies prevented formation of Leydig cell-lymphocyte rosettes more than isotype-matched irrelevant control antibodies, suggesting that Leydig cell lymphocyte binding occurs through CD106-CD49d interactions. In lymphocyte cultures in the presence of anti-CD3, anti-CD28, the M(r) > 5 K fraction of testis extract (containing CD106 as shown by immunoblotting) and anti-CD106 or control antibody, anti-CD106 did not consistently affect T cell 3H-TdR incorporation. The present results suggest that CD106 expressed by the Leydig cells may act as an adhesion-promoting molecule or a co-stimulatory factor for T cells migrating to the testis.

Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells.

The transcriptional activity of various lengths of the 5'-untranslated region (UTR) of the murine LH receptor (R) gene were studied using the luciferase reporter gene in transiently transfected mouse Leydig tumor cells (mLTC-1). Chinese hamster ovarian (CHO) and HeLa cells were used as controls. The basal transcriptional promoter activity in mLTC-1 cells resided within the first 173 base pairs (bp) of the 5'-UTR. Placing an LHR promoter fragment (bases -715/ -56) in front of the Herpes simplex virus thymidine kinase (TK) minimal promoter resulted in a 7-fold increase in luciferase activity. Deletion of bases -56 to -173 of the above construct totally abolished the increased luciferase activity, brought about by the LHR promoter sequences. Basically similar results on LHR promoter function were observed using CHO cells. In contrast, no LHR promoter activity was detected in HeLa cells, indicating a cell specific nature of its function. The first 173 bp promoter domain is GC-rich, with several SP-1 binding domains, and it bound specifically nuclear proteins isolated from mLTC-1 and HeLa cells. RNAse protection assays reconfirmed the presence of several transcription initiation sites within the first 310 bp of the 5'-UTR, also in the absence of the cognate LHR coding sequences. The most distal site at bp -310 did not function in the absence of the first 173 bp of the 5'-UTR. Other transcription initiation sites were identified closer to the translation initiation site. hCG (50 micrograms/l), 8-bromo (Br)-cAMP (100 mumol/l) and cholera toxin (100 microgram/l) displayed qualitatively similar negative effects on the LHR promoter activity in the transfected mLTC-1 cells when the constructs containing at least the first 565 bp of the LHR 5'-UTR were used, but the inhibitory effects were greatly decreased in constructs containing < or = 304 bp of the promoter region. Hence, the hCG/cAMP associated inhibitory effects interact with region(s) located mainly between bp -565 and -305 of the LHR promoter. The inhibitory role of cAMP on LHR gene expression was also confirmed at the level of hCO-binding and hCG stimulated cAMP production of mLTC-1 cells. In conclusion, the current results elucidate the cis- and trans-acting elements in the regulation of expression of the murine LHR gene in cultured mouse Leydig cells. The minimal basal promoter activity is within the first 173 nucleotides of the 5'-UTR and the structural elements of the negative LHR regulation by the cognate hormone and elevated cAMP levels are mainly located within nucleotides -305 to -565 of the 5'-UTR. The function of the murine LHR promoter is similar to, though not identical with that of the rat, but at variance with that of the human LHR gene.

Age-related variation of follicle-stimulating hormone-stimulated cAMP production, protein kinase C activity and their interactions in the rat testis.

To study further the ontogeny of hormonal regulatory mechanisms in the testis, we measured follicle-stimulating hormone (FSH)- and protein kinase C (PKC)-stimulated cAMP production, PKC activity, and messenger (m)RNA levels of the PKC isoenzymes alpha, beta and gamma in rat testes between day 19 of fetal life and day 90 postpartum. Human FSH (30 mg/l) stimulated slightly but significantly cAMP production of fetal testes (57%; p < 0.05). A higher response (3-fold; p < 0.01) was observed on the day of birth, and the maximum FSH effect on cAMP (23-fold) was observed on day 10 postpartum. Thereafter, a gradual decline of FSH response occurred towards adult age. Concerning testicular PKC, the soluble (inactive) form had its maximum at the age of 1 day and this PKC form declined gradually thereafter. The particulate (active) form was low at birth, increased 6-fold on days 8-11 of age, and declined thereafter. A significant age-dependent variation was also found in the mRNA level of the PKC alpha isoenzyme (maximum on day 10), whereas those of PKC gamma and PKC beta were undetectable at all ages in Northern blots. When the in vitro modulation of basal and FSH-dependent cAMP production by the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nmol/l) was studied, the substance alone was without effect at all ages studied. The TPA effect on FSH-stimulated cAMP production displayed age-dependent variation: a slight stimulation in fetal testes, no effect at birth, decrease between days 8 and 11, and no effect on day 30.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C and Gi-protein mediated modulation of cAMP production in different stages of the rat seminiferous epithelium.

The modulatory role of protein kinase C (PK-C)- and Gi-protein-mediated signal transduction systems was studied in the cyclic variation of follicle-stimulating hormone (FSH)-stimulated cAMP production of rat seminiferous tubules. FSH (Metrodin, Serono, 30 mg/l) stimulated cAMP production 10-fold (p less than 0.01) in a 3 h incubation of 5 mm segments of seminiferous tubules of stages II-VI of the epithelial cycle, but only 2-fold (p less than 0.01) in stages VII-VIII. The PK-C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nmol/l) suppressed the FSH effect on cAMP output by 50-70% (p less than 0.01) in stages II-VI, but had no effect in stages VII-VIII. If the tubular segments were preincubated for 3 h in the presence of pertussis toxin (PT, 100 micrograms/l), the FSH-stimulated cAMP production of stages VII-VIII increased by 100-200% (p less than 0.01), and now they also became responsive to the TPA suppression. Conversely, no effect of PT was observed in stages II-VI. Cholera toxin (CT, 100 micrograms/l) and forskolin (Fk, 100 mumol/l) nearly similarly stimulated the cAMP production in both stages studied (about 10-fold, p less than 0.01), and TPA and PT potentiated the effects in a non-additive fashion. In conclusion, both Gi-protein and PK-C-mediated mechanisms modulate cAMP production of rat seminiferous tubules. A clear cyclic variation can only be demonstrated in FSH-stimulated cAMP production, but not if the Gs-protein or adenylate cyclase are directly stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of protein kinase C activation on cyclic AMP and testosterone production of rat Leydig cells in vitro.

The role of protein kinase C in modulation of the endocrine function of rat Leydig cells was studied. Percoll-purified rat Leydig cells were stimulated with hCG, forskolin, cholera toxin, pertussis toxin and 8-bromo-cAMP in the presence and absence of two activators of protein kinase C, 12-0-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). The two activators had no effect on basal cAMP, but decreased hCG-stimulated, and increased cholera toxin- and forskolin-stimulated cAMP production. Cells pre-incubated with pertussis toxin showed enhanced rate of cAMP production in response to forskolin, but were no more responsive to TPA and OAG stimulation. These findings suggest that protein kinase C activation may on one hand inhibit the LH-receptor and Gs-protein coupling and on the other hand inhibit the Gi-protein mediated suppression of adenylyl cyclase activity. TPA and OAG effects on testosterone production were measured in the absence and presence of 8-bromo-cAMP stimulation. TPA enhanced basal testosterone production, but this effect was shifted to inhibition when steroidogenesis was stimulated by 8-bromo-cAMP. The OAG effect on testosterone production was inhibitory throughout the dose-response curve of 8-bromo-cAMP. The basal stimulation of testosterone production by TPA was probably due to a marginal increase of cAMP caused by inhibition of the Gi-protein, since a similar effect was observed by pertussis toxin, and thereafter TPA was without effect on testosterone. The inhibition of stimulated testosterone production by TPA and OAG indicates that protein kinase C activity also affects steroidogenesis at a step(s) beyound cAMP formation.

Pertussis toxin enhances follicle-stimulating hormone-stimulated cAMP production in rat seminiferous tubules in a stage-dependent manner.

Cyclic (c) AMP production was measured in vitro in dissected pieces of adult rat seminiferous tubules of defined stages of the seminiferous epithelial cycle (VII-VIII and XIV-IV) in the absence and presence of human follicle-stimulating hormone (FSH) (Metrodin, Serono). The basal rate of cAMP production was stage dependent, being about 2-fold higher in stages XIV-IV. FSH stimulated cAMP output in a time- and dose-dependent (stimulation at doses greater than or equal to 3 mg/l) fashion, and also the stimulability of stages XIV-IV (on average 2-fold) was greater than that of stages VII-VIII. When the tubular pieces were incubated in the presence of pertussis toxin (PT, 100 micrograms/l), the FSH-stimulated cAMP production of stages VII-VIII was enhanced by about 2-fold (P less than 0.01) whereas the basal rate was unaffected. In contrast, neither the basal nor the FSH-stimulated cAMP production of stages XIV-IV were affected by PT. Presence of the Gi-protein in both stages studied was demonstrated with PT-induced ADP ribosylation. However, no release of a putative activator of the Gi-protein could be demonstrated into spent media of the seminiferous tubules when incubated with freshly separated tubules. It is concluded that the poor FSH stimulability in cAMP production of certain spermatogenic stages of adult seminiferous tubules is at least partly due to endogenous inhibitors acting through the inhibitory Gi-protein. This inhibition could be demonstrated in a stage-dependent manner, and was present in stages with the lowest production and least stimulability of cAMP production by FSH.

Biological and immunological activity of human chorionic gonadotrophin in maternal serum during early pregnancy.

Serum chorionic gonadotrophin (hCG) levels were measured by three different assay methods in 20 women admitted to legal abortion and 21 patients having insulin-dependent diabetes, both during 8 to 14 weeks of pregnancy. Biological activity of hCG was determined with a mouse Leydig cell in-vitro bioassay, binding activity of the hormone to LH receptors by rat testis radioreceptorassay, and immunoreactivity by fluorimmunoassay. Bioassay and radioreceptorassay gave 1.4 and 1.7-fold higher hCG concentrations than fluoroimmunoassay using the same standard. Correlations between hCG levels measured by bioassay and fluoroimmunoassay (r = 0.81; P less than 0.01) and radioreceptorassay and fluoroimmunoassay (r = 0.95; P less than 0.01) were good. The results emphasize the heterogeneity of hCG in the pregnancy serum. Different domains of the molecules are recognized by assay methods based on antigenicity, receptor binding and biopotency of the hormone.

Sexual maturation of male rats treated postnatally with a gonadotrophin-releasing hormone antagonist.

Postnatal secretion of gonadotrophin by male rats was inhibited by a potent gonadotrophin-releasing hormone (GnRH) antagonist analogue (N-Ac-4-Cl-D-Phe1,4-Cl-D-Phe2,D-Trp3,D-Phe6,des-Gly10-GnRH-D-al anylamide; Org 30039; 2 mg/kg s.c. twice daily) on days 1-5, 6-10, 11-15 or 16-20 of life. The onset of puberty was determined by monitoring the separation of the preputium from the glans penis, i.e. balano-preputial separation (BPS). Rats treated on days 1-5 matured normally, whereas all treatments between days 6 and 20 delayed BPS (P less than 0.01). In adult rats (between 110 and 160 days of age), testis weights were reduced by 21-35% (P less than 0.01) in groups treated between days 1 and 15, although weights of the accessory sex glands were normal. Testicular FSH receptors were decreased by 31-47% (P less than 0.01) in all treatment groups, whereas the LH receptor content was decreased only in rats treated between days 1 and 5 (18%; P less than 0.05) and prolactin receptor content decreased only in rats treated up to day 10 (31-33%; P less than 0.01). Concentrations of serum testosterone, LH and FSH, and pituitary contents of LH and FSH were unaffected by neonatal treatment with Org 30039. Animals treated with Org 30039 had reduced fertility which was most pronounced (88%; P less than 0.01) in rats treated between days 1 and 5. However, motile sperm were detectable in the cauda epididymis of the infertile rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Histological and functional changes of the testis tissue during GnRH agonist treatment of prostatic cancer.

The purpose of this study was to examine long-term effects of GnRH agonists on human testicular histology and endocrine function. Patients with advanced prostate cancer (n = 7) were treated with the potent GnRH agonist analogue buserelin (Bu, Hoechst), 600 micrograms X 3/day intranasally. After 6 months, the patients were orchiectomized, and the testis tissue was used for histological studies and measurements of endocrine function in vitro. Fourteen other patients with matching ages and extent of the disease were castrated as the first form of therapy, and their testis tissue was used as controls (C). Severe atrophy of seminiferous tubules was seen in light microscopy in the testes of the Bu treated patients. Many tubules showed only Sertoli cells, and the seminiferous epithelium was frequently absent. In contrast, no clear changes were seen in the number of Leydig cells. Testicular content of testosterone (T) decreased greater than 95% by Bu treatment: C = 1.5 +/- 0.2 nmol/g wet wt (x +/- SE); Bu = 0.070 +/- 0.019 nmol/g. Likewise, a drop of 80% occurred in testicular high affinity receptors for FSH: C = 0.37 +/- 0.019 pmol/g; Bu = 0.067 +/- 0.009 pmol/g. In contrast, the number of LH receptors was unaffected by the treatment, C = 0.18 +/- 0.033; Bu = 0.18 +/- 0.032 pmol/g. When testis slices were incubated in the presence of maximally stimulating concentration of hCG (100 ng/ml), both groups of tissue responded similarly with a 50% increase in T production, albeit the absolute production rate was reduced by 95% in the Bu group. When several steroid precursors of T were analyzed in the incubation media, it appeared that decreased androgen synthesis was most clearly due to decreased 3 beta-hydroxysteroid dehydrogenase activity. It is concluded that long-term treatment with GnRH agonists in prostatic cancer patients brings about dramatic damage of seminiferous tubular function and reduces testicular androgen producing capacity, but has no effect on testicular capability of responding immediately to LH stimulation.

Gonadotropin-releasing hormone agonist activates protein kinase C in rat Leydig cells.

Gonadotropin-releasing hormone (GnRH) has specific receptor sites in rat Leydig cells and has direct effects on their steroidogenesis. The purpose of the present study was to examine whether activation of the calcium- and phospholipid-dependent protein kinase C (PK-C) is involved in GnRH effects on rat Leydig cells, as has been shown in pituitary gonadotrophs. Testosterone production of Percoll-purified Leydig cells was similarly stimulated (about 50-100%) by a GnRH agonist (buserelin, maximum effect at concentration of 10(-9) mol/l and above) and a tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, maximum effect at 10(-8) mol/l), which is known to activate PK-C. In contrast, a GnRH antagonist (10(-5) mol/l) and an inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate (10(-6) mol/l), were without effect on testosterone. None of these substances had clear effects on cAMP production. The maximum steroidogenic effects of GnRH agonist and TPA were the same whether used separately or together, suggesting that they share a common mechanism of action. TPA translocated the cytosolic proportion of Leydig cell PK-C activity to a membrane-associated form almost instantaneously, within 0.5-1 min. A similar translocation, though less complete, was observed in the presence of buserelin in 1-4 min. Inclusion of a 100-fold excess of a potent GnRH antagonist completely prevented the translocation of PK-C. These results provide evidence that GnRH agonist activates PK-C also in the testis tissue, and this may be the mechanism whereby it affects Leydig cell endocrine function.

Pituitary-testicular function of prostatic cancer patients during treatment with a gonadotropin-releasing hormone agonist analog. I. Circulating hormone levels.

Eight patients with advanced prostatic carcinoma (ages 59 to 78 years) were treated with a potent gonadotropin-releasing hormone (GnRH) agonist analog (buserelin, Hoechst; 600 micrograms intranasally, 3 times daily) and orchiectomized after 6 months of treatment. Endocrine responses were followed by serum hormone measurements during agonist treatment and for 3 months after orchiectomy. Six other patients (65 to 86 years) with advanced prostatic cancer had been orchiectomized as the first therapeutic measure and their blood samples were used as controls. In the GnRH agonist-treated patients, serum immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) decreased after initial stimulation by 70 to 80%, within 1 to 3 weeks (P less than 0.01). FSH partly recovered (P less than 0.05) after the first month of treatment. Serum prolactin (PRL) displayed a slight tendency to decline during buserelin treatment (P less than 0.05). Serum total and free testosterone (T) of the buserelin-treated patients decreased to the castrate range within 3 to 4 weeks after an initial 5-day increase (P less than 0.01). Serum progesterone and 17-hydroxyprogesterone (17-OHP-4) decreased to the castrate range (by 50 to 70%) in 1 week. Only minor changes were observed in sex hormone binding globulin (SHBG). Significant, acute elevations of LH, FSH, T, and 17-OHP-4 were observed only on day 1 after an injection of buserelin (500 microgram i.m.) and not when assessed between day 7 and month 6 of treatment. After 6 months of buserelin treatment, orchiectomy did not affect the serum steroids measured. After orchiectomy, immediate increases in serum LH, and somewhat later in FSH, were seen in the control patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary-testicular function of prostatic cancer patients during treatment with a gonadotropin-releasing hormone agonist analog. II. Endocrinology and histology of the testis.

Endogenous testosterone (T), LH and FSH receptors, and in vitro production of cyclic adenosine-3':5'-monophosphate (cAMP), T and some of its steroid precursors were measured in testicular tissue obtained at orchiectomy from seven prostatic cancer patients treated for 6 months with a potent gonadotropin-releasing hormone (GnRH) agonist analog (buserelin, Hoechst, 600 micrograms 3 times a day intranasally). In addition, histologic and morphometric studies were carried out on the testicular tissue. Testicular tissue from age-matched prostatic cancer patients (n = 14), whose first therapy was orchiectomy, served as controls. The peptide treatment decreased intratesticular T by 95% (P less than 0.01) and FSH receptors by 57% (P less than 0.01), but had no effect on LH receptors. The in vitro production of T decreased by 94% (P less than 0.01), but that of cAMP was unaffected. Besides T, the in vitro production of testicular 17-hydroxyprogesterone (17-OHP-4), androstenedione and 5 alpha-dihydrotestosterone (5 alpha-DHT) dropped by 71 to 90% (P less than 0.01 to 0.05) during buserelin treatment, but those of pregnenolone, progesterone and dehydroepiandrosterone (DHEA) were not affected. Histologic studies revealed considerable variation in the seminiferous epithelium of the control group, but spermatogenesis was highly suppressed in nearly all of the buserelin-treated group. The number of Sertoli cells was unaffected, but tubular diameters were reduced (P less than 0.05) by buserelin treatment. Leydig cells appeared dedifferentiated in this group, although their number per testis was not altered. These data indicate that gonadotropin suppression by GnRH agonist most likely affects testicular steroidogenesis by inhibiting 3 beta-hydroxysteroid dehydrogenase and a step(s) prior to pregnenolone formation. The treatment does not impair testicular LH binding or cAMP production, but clearly suppresses FSH receptors. Spermatogenesis in general is suppressed but with considerable variation.

Receptor binding properties and biologic action of deglycosylated human chorionic gonadotropin in human ovary and testis.

The receptor binding properties and biologic actions of chemically deglycosylated-asialo human choriogonadotropin (AHF-hCG) were studied in human ovary and testis. In corpus luteum and testis homogenates, the relative binding affinity of AHF-hCG was two- to fourfold higher in the ovary and five- to tenfold higher in the testis than that of native hCG. When assayed for luteinizing hormone (LH)-like activity in granulosa-luteal cells from in vitro fertilization patients and in testicular minces from patients undergoing orchiectomy for prostatic cancer, AHF-hCG did not stimulate cyclic adenosine monophosphate production. When added with hCG to granulosa-luteal cells or to testicular minces, AHF-hCG inhibited hCG-stimulated cyclic adenosine monophosphate production. These results indicate that the enhanced affinity to LH receptor caused by removal of the sugar moieties from hCG is associated with total inability to activate granulosa-luteal and Leydig cell adenylate cyclase, and that AHF-hCG is, in the human gonad, an hCG antagonist.

Blockade of rat testicular gonadotropin releasing hormone (GnRH) receptors by infusion of a GnRH antagonist has no major effects of Leydig cell function in vivo.

The purpose of this study was to examine the physiological functions of the gonadotropin releasing hormone (GnRH) receptors present in the rat testis. The receptors were blocked in situ by infusing one testis of adult rats for 7 days with 10-100 ng/h of a potent GnRH antagonist (N-Ac-Ala1, D-p-Cl-Phe2, D-Trp3,6-GnRH) using Alzet osmotic minipumps. The contents of the pump were delivered to the testis through a cannula perforating, and fixed, to the tunica albuginea. A plastic cannula alone was attached to the contralateral testis, to act as a control. Infusion of the antagonist resulted in a dose-dependent decrease of testicular GnRH receptors, up to 90%. Some of the antagonist also occupied GnRH receptors in the contralateral testis and pituitary, but these effects were always clearly less than in the infused testis. None of the doses used affected circulating levels of gonadotropins, prolactin (Prl) or testosterone. However, when the endocrine parameters of the two testes were compared, the 100 ng/h dose of the antagonist resulted in a significant (P less than 0.01-0.05) 16-32% decrease in the testicular content of testosterone, and LH, FSH and lactogen receptors. Similar effects (inhibition of the same parameters by 22-42%) were observed when immature (30-day-old) male rats were treated for 1 week with intratesticular infusions of the antagonist. It is inferred from these observations that, in physiological circumstances, testicular GnRH receptors may mediate stimulatory effects of Leydig cell LH and lactogen receptors, and testosterone synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and activation of protein kinase C in the rat testis tissue.

The distribution and role of the calcium-activated, phospholipid-dependent protein kinase C (PK-C) was studied in rat testis. When testis tissue was homogenized in the presence of 2 mmol/l EDTA and EGTA, the majority (greater than 70%) of the PK-C activity was soluble, the rest was released from the particulate fraction by solubilization with 0.3% Triton X-100. Without chelating agents the soluble PK-C activity was undetectable, and only partially recovered from solubilized membranes. Preincubation of the tissue with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7) mol/l) translocated PK-C to the membranes, and the majority of this activity was recovered by solubilization. Mobility of testicular soluble PK-C activity in HPLC-DEAE cellulose chromatography was similar to that of the brain enzyme. This single step purified testicular PK-C activity 140-fold. The specific activity and subcellular distribution of PK-C was similar in whole testis tissue and separated seminiferous tubules (160-210 pmol 32P X mg protein-1 X min-1 in the soluble and particulate fractions), but 2- to 3-fold higher in purified Leydig cells. However, the majority of total testicular PK-C activity appeared to be of tubular origin. Unilateral cryptorchidism for 1 week reduced PK-C of the abdominal testis by 50%, and the activity of dissected seminiferous tubules varied according to the epithelial wave. Both findings suggest that the bulk of the activity resides in the seminiferous epithelium. Involvement of PK-C in Leydig cell function was demonstrated using the TPA, which at 10(-7) mol/l inhibited basal cAMP production by 50% (P less than 0.01) but increased that of testosterone by 2- to 3-fold (P less than 0.01). On the other hand, when incubated with hCG, TPA inhibited both cAMP and testosterone production; the ED50s of hCG stimulation increased 4- to 10-fold with both parameters. It is concluded that PK-C activity is present in both the seminiferous tubules and Leydig cells, and is involved in the regulation of these testicular compartments. Its total activity and subcellular distribution are at variance according to the functional state and endocrine milieu of the testis.

Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists.

Clinical and experimental studies are described on the effects of a gonadotropin-releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions.