Alfred J. Moo-Young

Effects of Androgen on Androgen Receptor Expression in Rat Testicular and Epididymal Cells: A Quantitative Immunohistochemical Study

Abstract Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7α-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0.01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0.01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.

Clinical trial with 3-keto-desogestrel subdermal implants.

The study was done to assess the clinical performance and in vivo steroid release rate of 3-keto-desogestrel subdermal implants designed to deliver 5 different doses of the progestin. Volunteers were healthy women of proven fertility who provided blood samples at scheduled intervals during treatment. No pregnancy occurred in 514 woman-months in users of implants delivering 30 and 40 micrograms per day of 3-keto-desogestrel. Three pregnancies, one ectopic, were observed in 109 woman-months recorded with implants delivering 20 micrograms per day or less. Ovulation was inhibited, as judged by depressed progesterone levels, in 57 of 59 (97%) blood samplings in women whose 3-keto-desogestrel plasma levels were greater than 0.28 nmol/L and in 39 of 75 (52%) of cases with lower levels. Users of 4 cm implants manufactured by The Population Council, New York, showed mean levels above 0.28 nmol/L until 18 months of use. Levels achieved with 4.4 cm implants manufactured by Organon, Oss, Holland, were less consistent. No changes were observed in the plasma lipoprotein pattern or clinical chemistry during treatment. The main complaint was the occurrence of bleeding irregularities, particularly with the lower doses. Ovarian cysts found during pelvic examination in 11 (22%) subjects disappeared spontaneously within 7-90 days. 3-keto-desogestrel implants releasing around 40 ug/day and providing plasma levels around 0.28 nmol/L afford efficient contraceptive protection.

Clinical trial with Nestorone subdermal contraceptive implants.

The clinical performance and the in vivo release rate of a single 4-cm Nestorone subdermal implant were investigated. Implants manufactured by two different procedures were compared. Volunteers were 70 healthy women of proven fertility. Forty women provided blood samples twice a week in the pretreatment cycle and for 5-6 weeks at 6-month intervals during treatment. Additional control cycles (n = 31) were studied in 19 Copper T users. No pregnancy occurred in 1570 woman-months. Nestorone plasma levels (x +/- S.E.) declined from 112 +/- 8 to 86 +/- 3 pmol/L (Implant A) and from 145 +/- 8 to 57 +/- 5 pmol/L (Implant B) from the first to the 24th month. Progesterone levels were < 9.5 nmol/L in 166 (93%) of 178 blood samplings taken during treatment. Progesterone levels > 16 nmol/L were found in only 7 sampling periods (3.9%) in treated women and in 70 (98.6%) out of 71 control cycles. No ovulation occurred with Nestorone plasma levels above 105 pmol/L. No abnormal changes were observed in plasma lipoproteins or other clinical chemistry parameters during treatment. The implants were well tolerated. The most frequent complaint was the occurrence of irregular bleeding. Enlarged follicles found during pelvic examination in 8 subjects (11.4%) disappeared spontaneously in 10 days to 6 weeks. Implants were removed because of medical (n = 10, 14.3%) or personal reasons (n = 6, 8.6%) or at the 24th month of treatment (n = 54, 77.1%). The estimated average daily in vivo release rate of Nestorone was 45-50 micrograms/day. A single Nestorone subdermal implant affords efficient contraceptive protection during two years.

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development

Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development.

Contraceptive efficacy and clinical performance of Nestorone implants in postpartum women.

The objective of this study was to evaluate the contraceptive efficacy and clinical performance of a Nestorone subdermal implant (NES) in the postpartum period. NES (n = 100) and Copper T intrauterine device (T-Cu; n = 100) acceptors initiated contraception at 8 weeks postpartum and were followed at monthly intervals during the first year and at 3-month intervals thereafter. Pregnancy rates, breastfeeding performance, infant growth, bleeding pattern, and side effects were assessed. Blood and milk NES concentration were measured. No pregnancy occurred in 2195 and 2145 woman-months of NES implant and T-Cu use, respectively. No effect of NES on lactation and infant growth and no serious adverse events were observed. Lactational amenorrhea was significantly longer in NES users (353 +/- 20 days) than in T-Cu users (201 +/- 11 days). More NES users (55.8%) experienced prolonged bleedings than did T-Cu users (36.2%). Concentrations of NES in breast milk ranged between 54-135 pmol/liter. The Nestorone implant is a highly effective contraceptive, safe for breastfed infants because the steroid is inactive by the oral route.

Contraception with subdermal implants releasing the progestin ST-1435: A dose-finding study

A new modified subdermal implant releasing the potent progestin ST-1435 was studied in eleven fertile-aged women. These implants have been developed for contraception and they have a life-time of two years. Three implant lengths of 4, 6 and 8 cm were tested to find the optimal steroid dose for inhibition of ovulation. Serum samples were collected twice per week during a six-week period every six months. The concentrations of serum ST-1435, estradiol and progesterone were determined by RIA. Ovulation was inhibited by all ST-1435 doses tested. The concentration of serum progesterone was below 6 nmol/l in all samples tested showing the absence of luteinization. The concentration of serum ST-1435 increased with increasing ST-1435 dose. Serum estradiol concentrations were quite variable, showing wide range and occasional high peak values typical of progestin treatment; the mean value of serum estradiol concentrations measured did not differ with different ST-1435 doses. The results of steroid determinations led to the conclusion that a single 4 cm subdermal implant is optimal for contraception. With this dosage level, ovulation is inhibited and side effects are minimized. Bleeding control was variable. No hormonal side effects due to the progestin ST-1435 were reported. This method, using a single 4 cm subcutaneous implant releasing the progestin ST-1435 with a life-time of two years, represents a promising alternative for inhibition of ovulation and contraception.

Effective long-term androgen suppression in men with prostate cancer using a hydrogel implant with the GnRH agonist histrelin ☆

OBJECTIVES To evaluate the effectiveness of a hydrogel implant containing the gonadotropin-releasing hormone (GnRH) agonist histrelin in suppressing testosterone production in men with prostate cancer and to determine the effective dose (one, two, or four implants). METHODS Forty-two men with prostate cancer and indications for androgen ablation were treated with one, two, or four implants. In two of the clinics, comprising 27 subjects, the treatment period was 12 months, with replacement with the same number of implants at 12-month intervals. In a third clinic, which treated 15 subjects, the implants were left in place for up to 30 months. The total experience was 605 treatment months. RESULTS The histrelin levels were detected in serum proportional to the number of implants placed. The response, however, was similar among all three dose levels, with testosterone and luteinizing hormone essentially completely suppressed. Serum testosterone levels decreased from 21.9 +/- 17.6 nmol/L to 0.93 +/- 1.57 nmol/L within 1 month and were maintained at 0.55 +/- 0.24 nmol/L at 6 months and 0.60 +/- 0.28 nmol/L after 12 months of treatment. Of the 38 assessable patients, 35 (92%) had castrate levels of testosterone within 4 weeks of the initial implant placement. All patients followed for up for 12 months after placement of the initial set of implants maintained suppression of testosterone production while the implant was in place. CONCLUSIONS The histrelin hydrogel implant provided adequate and reliable delivery of the potent GnRH agonist histrelin during at least 1 year using a single implant in men with prostate cancer. No apparent advantages were found in using more than one implant, and the question of the possible effectiveness of even lower doses remains open. This treatment modality appears to be both safe and effective.

Ovarian function during use of Nestorone® subdermal implants

Nestorone(R) progestin (NES) is a potent 19-nor-progesterone derivative which is biologically inactive when administered orally; however, it is an excellent option for implant contraception. The objective of this study was to evaluate ovarian function during use of either one 4-cm or two 3-cm NES implants for 24 months. A total of 60 volunteers were enrolled in each dose group. Vaginal ultrasound (VUS) and blood sampling for determinations of estradiol (E(2)), progesterone (P) and NES serum levels were carried out twice a week for 6 consecutive weeks, beginning in months 1, 6, 12, 18, and 24 of implant use. Serum levels of NES declined with time, with a more pronounced decrease during the first 18 months of implant use; thereafter, NES levels remained stable until the end of the study at 24 months. Luteal activity was very infrequent during the first year of use (<3%) but increased during the second year, occurring in 27% and 35% of the sampling periods in the 1-implant group, and 2% and 16% of the sampling periods in the 2-implant group, at months 18 and 24 of use, respectively. No luteal activity was observed with NES levels above 80 pmol/L. Serum P levels in periods of luteal activity were significantly lower than those of controls. Persistent anovulatory follicles were the most common VUS finding and this was associated with E(2) levels that remained within the normal range (101-1500 pmol/L) in the majority of the sampling periods studied. Considering that a single implant offers advantage for insertion and removal, a new single NES implant is being developed with a slightly higher release rate, to reduce effectively the incidence of ovulation and provide a greater margin of safety beyond 2 years.

Pharmacokinetics of 7α-methyl-19-nortestosterone (MENT™) delivery using subdermal implants in healthy men

We studied the pharmacokinetics of 7 alpha-methyl-19-nortestosterone (MENT), a potent synthetic androgen, administered by subdermal implants. The implants contained 112 +/- 4 mg of MENT acetate in a polyethylene vinyl acetate copolymer. MENT acetate released from the implants is rapidly hydrolyzed to MENT in vivo. Fifteen healthy Finnish men were randomized to have either one, two, or four implants inserted in the medial aspect of the upper arm. The implants remained in place for 4 weeks. Blood samples were obtained before implant insertion, 1, 2, 3, and 4 weeks after insertion, and 1 and 2 weeks after removal. Serum MENT concentrations were determined by gas chromatography with mass selective detection. The MENT levels attained in each implant group remained at a steady level during the 4 weeks of implant use. The mean steady state MENT concentrations in the one, two, and four implant groups were 0.6, 1.4, and 2.3 nmol/L, respectively. Serum MENT concentrations during implant use were clearly dose dependent; the between-subject effect of implants as well as the differences between each pair of groups were all statistically significant. The release rate of MENT from one, two, and four implants was calculated to be approximately 0.3, 0.8, and 1.3 mg/day, respectively. This study suggests that MENT acetate implants are a promising method for long-term androgen administration in hypogonadism and male contraception.

Influence of a levonorgestrel-containing contraceptive vaginal ring on plasma lipids and lipoproteins in cynomolgus monkeys

The effect of a contraceptive vaginal ring (CVR) containing levonorgestrel on plasma lipid and lipoprotein concentrations and characteristics was assessed in ten cynomolgus monkeys. The animals were fed a diet similar to the average American diet in fat (40% of calories) and cholesterol (0.2 mg/kcal) content. The objective of this study was to determine if changes in lipids and lipoproteins caused by progestogen administration parallel those seen in human females. A parallel pattern would recommend the cynomolgus monkey as a model for studying the effects of progestogens on the atherosclerotic process. Treatment with the CVR resulted in significant decreases in total plasma, VLDL + ILDL + LDL, and HDL cholesterol concentrations and a decrease in the percentage of HDL2 in total HDL. Plasma triglyceride concentrations were low throughout the study and consistent effects of the CVR were not seen. CVR treatment resulted in increases in TPC:HDL-C ratios and in the flotation rate of the LDL particle. The patterns of effects on HDL cholesterol, total plasma cholesterol, and HDL2 concentrations were similar to the progestogen-induced changes observed in human plasma lipids and lipoproteins. Based on these effects, the cynomolgus monkey appears to be a suitable model for the study of progestogen-induced changes in plasma lipids and lipoproteins and their consequent influences on coronary artery atherosclerosis.