Effect of vitamin C on androgen independent prostate cancer cells (PC3 and Mat-Ly-Lu) in vitro: involvement of reactive oxygen species-effect on cell number, viability and DNA synthesis.

Studies have described the protective role of vitamin C (ascorbic acid) in certain types of cancer. In this study, we report the effects of vitamin C treatment of two androgen independent prostate cancer cell lines from human (PC3) and rat (Mat-Ly-Lu or MLL) sources. In vitro treatment of PC3 and MLL with sodium ascorbate acid (0-10 mM) resulted in a decrease in cell viability and thymidine incorporation into DNA. These effects of vit. C were dose and time dependent. Ascorbate induced these changes through the production of hydrogen peroxide since addition of catalase (100-300 units/ml), an enzyme that degrades hydrogen peroxide, inhibited the effects of ascorbate on these cell lines. In contrast, superoxide dismutase, an enzyme that dismutates superoxide and generates hydrogen peroxide did not prevent ascorbate-induced changes emphasizing the involvement of reactive oxygen species (ROS) in cellular damage. That singlet oxygen scavengers such as sodium azide and hydroquinone, hydroxyl radical scavengers such as D-mannitol and DL-alpha-tocopherol did not counteract the effects of ascorbate on thymidine incorporation suggests that these free radicals are not involved in cellular damage. In conclusion, these results suggest that vitamin C inhibits tumor growth by virtue of producing reactive oxygen species. These results suggest that ascorbate is a potent anticancer agent for prostate cancer cells.

Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations.

BACKGROUND: Neurotensin (NT), a neuroendocrine peptide, exerts trophic effects in vivo and stimulates growth of some tumor cells in vitro. Androgen-sensitive prostate cells derived from lymph node carcinoma of the prostate (LNCaP) secrete NT and exhibit growth responses to NT. This study examines NT secretion, NT receptor and NT-growth responses in androgen-independent prostatic carcinoma (PC3) cells derived from prostate adenocarcinoma metastatic to bone. METHODS: Binding of 125I-NT to PC3 membranes was studied by filtration. NT was measured by RIA. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for NT and NT receptor mRNA. Growth was measured as 3H-thymidine incorporation into DNA. RESULTS: Scatchard analyses gave two binding components (Kd1 = 40 pM and Kd2 = 300 pM) in equal amounts (15-30 x 10(3) sites/cell). The bioactive region of NT was essential and the specific, non-peptide NT antagonist, SR48692, inhibited (IC50 = 3 nM). GTP analogs, sodium ion and SH-directed alkylating agents also inhibited. Glutaraldehyde crosslinking labeled two substances (M(r) of 23 and 46 kDa). RT-PCR indicated robust expression of authentic NT receptor but little for NT precursor. NT was stable in PC3 cultures but it was not found in cells or conditioned media. Incubated with PC3 cells, NT exhibited a mitogenic effect with bell-shaped dose-response and maximum at 100 pM NT. CONCLUSIONS: PC3 cells expressed genuine NT receptors and generated growth responses to physiologic levels of NT which were blocked by SR48692. If NT contributes to the survival of prostate tumor cells upon androgen deprivation therapy, NT antagonists might be useful agents in further treatment.

17 beta-estradiol induced prostatitis in the rat is an autoimmune disease.

PURPOSE: Studies suggest that alteration in steroid hormone levels may be one of the factors causing nonbacterial prostatitis (NBP) in rats. We hypothesized that hormonally induced prostatitis in the rat may be an autoimmune disease. Studies were carried out to prove this hypothesis. MATERIALS AND METHODS: We injected 17 beta-estradiol (E2; 250 micrograms/kg. subcutaneously) or vehicle into 1-year-old male rats for 30 days, and isolated and cultured the splenocytes in the presence of con-A (Experiment 1). Approximately 10(7) splenocytes were adoptively transferred into young naive syngeneic rats. To find out whether or not the inflammation is mediated by T-lymphocytes, a pure population of T-lymphocytes from E2-treated 3-month-old rats was injected into young naive syngeneic rats (Experiment 2). To rule out the possibility that the inflammation was due to con-A itself, we cross-linked some T-cells with anti-CD3 antibody before adoptive transfer (Experiment 2). RESULTS: The recipients of splenocytes from E2-treated animals presented evidence of inflammation in terms of lymphocytic infiltration and presence of degranulated mast cells. Furthermore, we observed in these animals an increase in histamine-releasing peptide (HRP) levels, an indication of plasma extravasation. The T-cells stimulated by anti-CD3 antibody produced a similar degree of inflammation, thereby ruling out the possibility that the inflammation was due to con-A. The results also indicated that the immune response to antigen(s) is not dependent on the age of the animal but is dependent on a population of CD3+ T-cells. CONCLUSION: Our results demonstrate that hormonal imbalance and autoimmunity in male rats produce the symptoms of nonbacterial prostatitis.

Effects of sialoadenectomy and epidermal growth factor on testicular function of sexually mature male mice.

The effect of sialoadenectomy (Sx) and epidermal growth factor (EGF) administration on testicular function was investigated in 8-week old C3H mice. Animals were divided initially into three groups: sham operated controls, Sx, and Sx + EGF treated (100 micrograms./kg./day subcutaneously for 28 days). Sialoadenectomy completely depleted the circulating levels of EGF and reduced body weight and reproductive organ weights. However, kidney weight was not affected. Quantitative analysis of spermatogenesis showed a decrease in preleptotene and pachytene spermatocytes and round spermatids, which resulted in a decrease in sperm counts. Sperm motility and fertility were also significantly decreased. Endocrinologic studies showed a 2- and 6-fold elevation in intratesticular and serum levels of testosterone and a decrease in luteinizing hormone (LH) levels. Follicle stimulating hormone levels were not altered. Administration of EGF to the Sx animals maintained reproductive organ weights, spermatogenesis and levels of LH and testosterone closer to control values; however, sperm motility was not maintained at control value. That sialoadenectomy resulted in a decline in androgen-dependent parameters, in spite of an elevation in testosterone levels, and EGF maintained them closer to the control value suggested that EGF may modulate androgen action. A comparison was therefore carried out between the effects of Sx and administration of flutamide (F), an androgen receptor blocker. Animals were subjected to Sx, F treatment (100 mg./kg./day subcutaneously for 28 days), Sx + F, or Sx + F + EGF. The effects of Sx and F treatment on organ weights, sperm counts and sperm motility were more or less similar. As expected, flutamide treatment increased LH and FSH levels, and testosterone levels were normal. The Sx + F animals showed no further decrease in organ weights, sperm count and motility. Treatment with Sx + F increased intratesticular and serum levels of testosterone by 2- and 10-fold. Circulating levels of LH and FSH were the same as in the flutamide-treated group. Administration of EGF to Sx + F maintained all these parameters, except sperm motility, closer to the control value. These results suggest that EGF either bypasses flutamide effects and acts directly or that EGF modulates androgen action at one or more steps in the signal transduction pathway in the male reproductive organs.

Epidermal growth factor: receptor binding and effects on the sex accessory organs of sexually mature male mice.

The role of epidermal growth factor (EGF) in maintaining the integrity of the male sex accessory glands was investigated in the mouse. In the sexually mature male C3H mouse, EGF levels were highest in the submandibular gland, followed by the seminal vesicles and the prostate. Twenty eight days after sialoadenectomy (Sx), EGF fell below detectable limits in the serum and in the seminal vesicles. However, the prostate still retained 22% of its immunoreactive EGF. There was a seven-fold increase in serum testosterone after sialoadenectomy. Despite this drastic rise in testosterone, both prostatic and seminal vesicular weights were reduced, serum levels of LH were suppressed only by 37% and FSH levels were not altered. All these changes were abolished by the simultaneous administration of exogenous EGF at 100 micrograms./kg./day for 28 days. Both prostatic and seminal vesicular membranes contained binding sites for 125I-EGF. Binding was maximal after one hour of incubation at room temperature. Two classes of binding sites were shown for either organ (Kd = 1.2 nM, n = 56 fmol/mg. and Kd = 74 nM, n = 540 fmol/mg. for prostate; Kd = 0.9 nM, n = 29 fmol/mg. and Kd = 93 nM, n = 150 fmol/mg. for seminal vesicle). The binding of 125I-EGF was displaced by excess EGF and TGF-alpha but not by insulin, ILGF-2 and PDGF. These data suggest that EGF may have an important role in maintaining the integrity of the seminal vesicle and the prostate in the mouse. While the seminal vesicle appears to acquire EGF by uptake from the environment, the prostate may have the ability to synthesize EGF locally.

The mechanism of cyclosporine's action in the inhibition of testosterone biosynthesis by rat Leydig cells in vitro.

We have previously demonstrated that cyclosporine inhibits testosterone (T) biosynthesis in vivo. To better understand the mechanism by which CsA inhibits T synthesis, interstitial cells were isolated from rat testes and incubated in the standard medium 199 with or without CsA (0-10 micrograms/ml) in the presence or absence of human chorionic gonadotropin (hCG, 10(-7) M) and 8-bromo cyclic AMP (cAMP, 0.5 mM) for 3 hr at 32 degrees C. The levels of cAMP and T were determined by RIA. CsA did not inhibit the basal secretion of T, but inhibited hCG-stimulated T production in a dose-dependent manner (4 ng/10(6) cells vs. 10 ng/10(6) cells at a CsA dose of 5 micrograms/ml, P less than 0.05). Radioligand binding of 125I-hLH to testicular membranes was not affected by CsA, as CsA did not compete with hCG/LH for binding sites (25-28% binding with or without CsA). Similarly, the MIX-stimulated cAMP production was not affected by CsA (24.03 +/- 1.09 vs. 20.60 +/- 0.38 pmol/10(6) cells), suggesting that CsA does not inhibit the accumulation of the second messenger. However, when interstitial cells were incubated with CsA in the presence of cAMP, a significant dose-dependent decline in T secretion was observed (7 ng/10(6) cells vs. 20 ng/10(6) cells at a CsA dose of 5 micrograms/ml). To determine whether CsA inhibits the steps beyond cAMP stimulation of T secretion, the kinetic parameters (Km and Vmax) of steroidogenic enzymes, delta 4-3 keto-17 alpha hydroxylase (17 alpha-hydroxylase), and delta 4-3 keto-17 beta hydroxy steroid dehydrogenase (17B-HSD) were determined by using Michaelis Menten analysis. Results are shown in the presence of CsA vs. no CsA: Km and Vmax values for 17 alpha-hydroxylase were (2.32 vs. 7.98 microM) and (27.96 vs. 100.97 pmol/mg protein/min), respectively. For 17B-HSD the Km and Vmax were (2.14 vs. 1.52 microM) and (15 vs. 15 pmol/mg protein/min), respectively. These results indicate that CsA inhibits the activity of 17 alpha-hydroxylase uncompetitively and 17B-HSD activity competitively. In conclusion the primary site for CsA inhibition is the cAMP stimulation and, CsA inhibits T synthesis at multiple sites.

The effects of an LHRH agonist on testicular function in the cryptorchid rat.

To assess the effects of an LHRH analog (LHRH-ethylamide des gly10 D-Leu6) on the function of the cryptorchid testis, an animal model was created, using prepubertal male Sprague-Dawley rats (21 days old). The animals were divided into six groups: 1) sham operated; 2) sham + LHRH treated; 3) cryptorchid; 4) cryptorchid + LHRH treated; 5) cryptorchid and orchidopexed; 6) cryptorchid, LHRH treated and orchidopexed. Two weeks post cryptorchidism, the animals were treated with either saline or LHRH-analog (one microgram./rat/day) subcutaneously for a total of twenty five days. Orchidopexy was performed following hormonal treatment. The effects of treatment were assessed in terms of body and reproductive organ weights, serum testosterone and LH levels, sperm counts and motility and fertility. The results demonstrated that experimental cryptorchidism impaired reproductive function, which was restored by orchidopexy, alone. Treatment of cryptorchid rats (groups 4 and 6) with LHRH did not produce any lasting improvement in spermatogenesis or fertility.

Effects of subchronic treatment with cis-platinum on testicular function, fertility, pregnancy outcome, and progeny.

Cis-platinum-based chemotherapy is known to impair spermatogenesis, but the effects of paternal cis-platinum treatment on the progeny are unknown. To study this effect, sexually mature male Sprague-Dawley rats were administered intraperitoneal injections of saline or cis-platinum (0.5 mg/kg per day) for 9 weeks. Every week, one set of control and treated animals was mated with females in proestrus. Nineteen days later, the females were subjected to laparotomy, and the numbers of corpora lutea, resorptions, and normal and abnormal fetuses were noted. In conjunction, the effects of treatment on the hypothalamo-pituitary-gonadal axis of the treated males were evaluated. Cis-platinum-treated animals failed to grow; the weights of the reproductive organs and the sperm counts declined from week 2 onward, and sperm motility was reduced throughout the testing period. Circulating and intratesticular levels of testosterone declined from week 3 of treatment and follicle-stimulating hormone levels were not affected. Serum levels of luteinizing hormone declined from week 3 and were not detectable from week 6 onward. However, the pituitary response to gonadotropin-releasing hormone was intact in all treated groups. There was no significant decrease in fertility, but a prominent increase in pre- and postimplantation losses of fetuses after cis-platinum treatment was observed. There was also a decrease in the male-to-female ratio of the offspring. A small but significant number of malformed and growth-retarded fetuses was also found among the offspring of cis-platinum-treated males. These results suggest that subchronic treatment with low doses of cis-platinum may affect progeny; such effects are seen in addition to the apparent alteration in a number of measures of reproductive function of treated males.

Short term effects of cis-platinum on male reproduction, fertility and pregnancy outcome.

In order to find out the short term effects of cis-platinum treatment on reproductive function of the treated male rats and their progeny, sexually mature male Sprague-Dawley rats were given a single intra-peritoneal injection of either saline or cis-platinum (2, 4, 8 mg./kg.body wt.). One week following the treatment, the animals were mated with proestrus females of proven fertility. The females were scored positive or negative depending upon whether or not spermatozoa were seen in the vaginal smear after mating. However, all the females were watched until day 18, and on day 19, the females that became pregnant were subjected to laparotomy, and the number of corpora lutea, implantation sites and fetuses were counted. The fetuses were weighted and observed for the presence of any morphological abnormalities. Significant pre-implantation loss was seen in the treated groups. The weights of the fetuses were also significantly lower than those from the control group. Analysis of the effects of cis-platinum on the reproductive system of treated males revealed that cis-platinum reduced the reproductive organ weights, sperm counts, sperm motility, fertility and the levels of testosterone, LH and FSH. These results suggest that cis-platinum has a profound deleterious effect on the reproductive system and on the fertility potential of the treated male rats.

Reversal of the toxic effects of cyclosporine on male reproduction and kidney function of rats by simultaneous administration of hCG + FSH.

We have shown earlier that the administration of cyclosporine impairs testicular function and causes a decrease in sperm counts, sperm motility and fertility. In order to determine whether or not the deleterious effects of CsA could be reversed by hormonal therapy, we injected sexually mature male Sprague Dawley rats with cremaphor + saline or CsA (40 mg./kg./d) alone or in combination with human chorionic gonadotropin (hCG; five micrograms./d/rat) and follicle stimulating hormone (FSH; five micrograms./d/rat). The injections were given subcutaneously for 14 days. As expected, CsA administration decreased the body and reproductive organ weights, testicular and epididymal sperm counts, sperm motility and fertilizing ability. Serum levels of LH were elevated and testosterone was decreased. The administration of FSH + hCG to the CsA treated rats restored the body and reproductive organ weights, sperm counts and motility. Seventy five percent of gonadotropin treated males were fertile as compared to 25% in the CsA treated group. In the hormone treated group, the blood levels of CsA were 50% of that of CsA treated group. In order to verify whether or not the decline in the blood levels of CsA was the cause for the amelioration of CsA-induced changes in the reproductive function, we compared the CsA + hormone treated group with another group treated with five mg./kg./d CsA which had blood levels of CsA comparable to the former group. In the five mg./kg./d group the reproductive functions were significantly lower than the CsA + hormone treated group suggesting, therefore, that the restoration of reproductive functions in the CsA + hormone treated group is a result of hormonal treatment. Administration of CsA (40 mg./kg./d) reduced the kidney weight and increased the levels of serum creatinine: these changes were also ameliorated by the administration of hCG + FSH.

Quantitative maintenance of spermatogenesis in cyclosporine-treated rats by exogenous administration of testosterone propionate.

The authors had previously shown that the subcutaneous administration of cyclosporine (CsA) resulted in an impairment of spermatogenesis. Testosterone levels declined and gonadotropin levels increased, suggesting that CsA primarily affects the synthesis and secretion of testosterone. In this study, the authors attempted to determine whether the exogenous administration of testosterone would maintain spermatogenesis in animals treated with a very high dose of CsA. Sexually mature, male Sprague-Dawley rats were treated subcutaneously with CsA (40 mg/kg per day) alone, or in combination with testosterone propionate (TP; 2 and 5 mg/d per rat), for 14 days. As expected, CsA reduced the body and reproductive organ weights and the levels of serum testosterone, while elevating the levels of follicles-stimulating hormone (FSH) and luteinizing hormone (LH). Quantitative analysis of spermatogenesis revealed a decline in all the different types of germ cells in tubules at stage VII of the cycle of the seminiferous epithelium. Administration of TP in 2 and 5 mg/d per rat doses restored the body and reproductive organ weights and the circulating levels of FSH. The serum levels of LH were below the assay's minimum level of detectability. Analysis of spermatogenesis revealed a dose-dependent increase in the germ cell counts after the administration of 2 and 5 mg of TP. The circulating levels of CsA were also significantly reduced after TP administration. These results revealed that CsA-induced alteration in spermatogenesis can be prevented by the exogenous administration of testosterone.

Evaluation of the effect of experimental cyclosporine toxicity on male reproduction and renal function. Reversal by concomitant human chorionic gonadotropin administration.

Administration of cyclosporine to rats has been shown to impair testicular function, resulting in a decrease in sperm counts and fertility. In order to determine whether or not the deleterious effects of CsA could be reversed by hormonal therapy, mature male Sprague Dawley rats were treated with CsA (40 mg/kg/day, s.c.) alone or in combination with human chorionic gonadotropin (hCG) (5 micrograms/day/r; s.c.) for 14 days. Cyclosporine administration decreased the body weight (290 +/- 5.30 vs. 339 +/- 8.7 g; P less than 0.05) and reproductive organ weights (testis 1.49 +/- 0.42 vs. 1.60 +/- 0.03 g; epididymis 0.41 +/- 0.02 vs. 0.49 +/- 0.002 g; seminal vesicle 0.61 +/- 0.09 vs. 1.60 +/- 0.05 g; prostate 0.28 +/- 0.04 vs. 0.60 +/- 0.06 g; P less than 0.05) testicular sperm counts (5.80 +/- 0.42 vs. 8.49 +/- 0.48 x 10(7)/100 mg tissue; P less than 0.05) and epididymal sperm counts, (28.2 +/- 0.95 vs. 51 51.62 +/- 2.17 x 10(7)/100 mg tissue; P less than 0.05) and fertility (25% vs. 100%). Serum levels of LH were elevated (101.98 +/- 21.48 vs. 25.6 +/- 5.18 ng/ml; P less than 0.05) and testosterone was decreased (0.48 +/- 0.07 vs. 2.06 +/- 0.56 ng/ml; P less than 0.05). The administration of hCG to the CsA-treated rats restored the reproductive organ weights (testis 1.56 +/- 0.043 g; seminal vesicle 1.04 +/- 0.05 g; prostate 0.70 +/- 0.06 g) and sperm counts (testicular 7.88 +/- 1.0 x 10(7)/100 mg tissue; epididymal 59.86 +/- 4.16 x 10(7)/100 mg tissue; P less than 0.05) Serum levels of testosterone (18.63 +/- 4.45 ng/ml) and LH (431.65 +/- 31.41 ng/ml) were significantly elevated, as compared with control and CsA-treated groups (P less than 0.05). All the rats in the gonadotropin-treated group were fertile, as compared with 25% in the CsA-treated group. CsA reduced the kidney weight (1.17 +/- 0.02 vs. 1.27 +/- 0.03 g; P less than 0.05) and increased the levels of serum creatinine (0.97 +/- 0.07 vs. 0.59 +/- 0.03 mg/dl; P less than 0.05): these changes were ameliorated by the administration of hCG (kidney weight 1.35 +/- 0.03 g; creatinine 0.76 +/- 0.09 mg/dl).

Cyclosporine: its effects on testicular function and fertility in the prepubertal rat.

The authors examined the effects of the immunosuppressive drug cyclosporine (CsA) on the male reproductive system in prepubertal rats. Twenty-one-day-old rats were subcutaneously injected with either cremaphorsaline vehicle or CsA (1 and 2 mg/kg/d). The animals were treated until they were 66 days old. Cyclosporine did not affect the weights of the body or testis but decreased the weights of all sex accessory organs. Quantitative analysis of the tubules in stage VII of spermatogenesis revealed a decline in the cell counts of pachytene spermatocytes and step VII spermatids. Testicular and epididymal sperm counts and motility were decreased by 50% and fertility by 60%. Cyclosporine lowered serum testosterone despite an elevation of LH, indicating that the drug directly inhibited testosterone synthesis. Serum creatinine levels were normal in the treated animals, precluding renal failure as the cause for this impairment. Intratesticular concentrations of pregnenolone and 17-hydroxy progesterone were significantly elevated, while those of progesterone, androstenedione, and testosterone were markedly reduced. Determination of steroidogenic enzyme activities indicated that the administration of CsA inhibited the activity of delta 5-3B-hydroxy steroid dehydrogenase-delta 5-4 isomerase (3 beta-HSD). These results clearly indicate that CsA in the doses used is harmful to the male reproductive function in prepubertal rats.

Effect of cyclosporine A on male reproduction in rats.

We examined the effects of the immunosuppressive drug cyclosporine A on male reproduction in sexually mature rats. The drug was administered subcutaneously in 3 doses (10, 20 and 40 mg. per kg. daily) for 14 days. Cyclosporine A administration resulted in a dose-dependent decline in body and reproductive organ weights, histology of the testis showed definite degenerative changes, and sperm counts and motility decreased. Spermatozoa from treated rats retained the cytoplasmic droplet and they were decapitated. Consequently, sterility occurred in rats treated with high doses of cyclosporine A. A reduction in circulating testosterone and an increase in gonadotropins were noted. The possibility existed that the cyclosporine A-induced alterations in the male reproductive system were secondary to the hepatic or nephrotoxic effects of the drug. Therefore, we assessed liver and kidney function by assay of the serum levels of bilirubin, alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and creatinine. Except for an increase in bilirubin, none of the other liver function studies was altered, thereby eliminating hepatocellular damage as the etiology of impaired reproductive function in cyclosporine A-treated rats. We observed an increase in creatinine levels in animals treated with higher doses of cyclosporine A, consistent with mild renal failure. However, the fact that a decline in reproductive function was seen even in the group treated with 10 mg. per kg. cyclosporine A daily, which had normal serum creatinine levels, suggests that the alterations in male reproduction seen with this therapy are not entirely the result of nephrotoxicity.

The effect of streptozotocin-induced diabetes on the neuroendocrine-male reproductive tract axis of the adult rat.

Studies in the streptozotocin rat model for diabetes mellitus suggest that sexual dysfunction in these animals may result from diabetes-induced alterations of the neuroendocrine-reproductive tract axis. Our investigation was performed to better define the effects of diabetes on the neuroendocrine sex accessory organ axis in the male rat. Rats were rendered diabetic, and were either left untreated or treated with insulin, testosterone or both. Diabetes resulted in decreased body and reproductive organ weights, as well as diminished sperm counts and motility and prostatic acid phosphatase. Seminal fructose was increased. A significant decrease in serum LH, FSH and testosterone was noted. Insulin treatment of the diabetic rats restored serum gonadotropins, reproductive organ weight, sperm counts and motility, and seminal fructose to control levels. Prostatic weight and prostatic acid phosphatase levels remained abnormal. Testosterone restored the above mentioned parameters to control levels, with the exception of LH. Treatment with insulin and testosterone had a synergistic effect on spermatogenesis. A GnRH stimulation test demonstrated that pituitaries of diabetic animals had a blunted response, with diminished LH and FSH secretion. In the diabetic animal, there are both pituitary and testicular abnormalities which may be responsible for reproductive dysfunction.

Effects of vitamin C deficiency on physiology of male reproductive organs of guinea pigs.

Vitamin C dietary deficiency in guinea pigs markedly affected the androgen-sensitive parameters of the reproductive tissues--testis, epididymis, vas deferens, and accessory sex glands--and caused an "androgen-deprived effect" in these target organs. This in turn altered their internal milieu and caused changes in their metal ion profile and the morphology, motility, and density of spermatozoa in the cauda epididymis and vas deferens. The sensitivity of the vas deferens to adrenaline was also reduced in scorbutic guinea pigs, thus decreasing their fertility rate. The primary action seems to be at the level of the testis, and androgen deprivation and partial antifertility effects are probably secondary manifestations consequent to the primary effect. The present study demonstrated that ascorbic acid is essential for maintaining the physiological integrity of the androgen target reproductive organs in guinea pigs.

Pituitary binding of 3H-labeled Sertoli cell factor in vitro: a potential radioreceptor assay for inhibin.

We have previously demonstrated that binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The present study further characterized the binding of [3H]SCF to rat anterior pituitary in vitro and explored its potential use as a radioreceptor assay for SCF and other inhibin preparations. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 mu Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of approximately 20,000 dpm/micrograms protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing one pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37 C; 30 min) or heat (100 C; 10 min). Storage of the pituitary glands at -20 C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high mol wt fraction of ram testis fluid (mol wt, greater than 10,000), low mol wt fraction of ram testis fluid (mol wt, less than 5,000), ovine testicular lymph, and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low mol wt fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). In contrast, various noninhibin substances tested [rat androgen-binding protein, bovine LH, BSA, native GnRH, or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-EA and D-Ala6-des-Gly10-GnRH-N-EA] did not significantly compete for the [3H]SCF binding. The binding ability correlated well with inhibin biological activity in vitro. These results provide additional evidence for the presence of SCF-binding sites in the rat anterior pituitary which interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. Our results also indicate that the pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations.