GnRH agonist an- alogues and testosterone replacement have not produced consistent oligospermia [3], and GnRH antagonists may re- lease histamine from ...
BIOLOGY OF REPRODUCTION 55, 1377-1382 (1996)
Effects of Continuous Inhibin Administration on Gonadotropin Secretion and Subunit Gene Expression in Immature and Adult Male Rats' Stephen J. Winters, 2 ,37, Clifford R. Pohl,4 Adedayo Adedoyin,5 and Gary R. Marshall 4 ,6 Departments of Medicine, 3 Physiology and Cell Biology, 4 Pharmaceutical Sciences and the Center for Clinical Pharmacology,5 and Pediatrics,6 University of Pittsburgh, Pittsburgh, Pennsylvania 15213 School of Health Sciences, 7 Duquesne University, Pittsburgh, Pennsylvania 15219 Although most evidence has favored a preeminent role for intratesticular testosterone as a spermatogenic hormone [5], the emerging view is that FSH is also essential for sustaining spermatogenesis [6, 7]. For example, recombinant human FSH maintains spermatogenesis in adult rats treated with a GnRH antagonist [8], and passive immunization against FSH produces apoptosis in pachytene spermatocytes and spermatogonia [9]. Recombinant human inhibin administered i.v. in shortterm studies to intact and castrated male rats [10, 11] and rams [12, 13] preferentially suppressed serum FSH concentrations. Although at higher doses inhibin also reduced GnRH-stimulated LH release by rat pituitary cell cultures [14, 15], the applicability of these findings in vivo remains uncertain. To begin to explore the contraceptive potential of inhibin in the male, we examined the effects of recombinant human inhibin-A administered for 3 days via osmotic minipumps on gonadotropin secretion and levels of subunit mRNAs and GnRH receptor mRNA in male rats. Experiments were conducted in both immature and adult rats because of previous evidence for developmental differences in the effects of inhibin on FSH in this species [16, 17].
ABSTRACT To begin to examine inhibin as a male contraceptive, recombinant human (rh) inhibin-A was administered for 3 days via osmotic minipumps to male rats. Doses of inhibin of 0-150 mg/kg per day did not produce a concentration-dependent suppression of FSH secretion or pituitary FSHO mRNA levels in adult rats. Treatment of immature rats at a dose of 145 g/kg per day, which was without effect in adults, reduced plasma FSH levels by 49% (p < 0.01), and FSHP mRNA levels to 47 - 11% of control (p < 0.01). Inhibin also decreased levels of LHi mRNA (63 - 8% of control; p < 0.01), a-subunit mRNA (86 + 10% of control; p < 0.05), and GnRH-receptor mRNA (77 17% of control; p < 0.01) in immature rats. Rh inhibin-A was more effective in immature than in adult animals: plasma inhibin levels were increased (p = 0.03) by rh inhibin-A treatment only in immature rats. Pharmacokinetic studies revealed that the weight-adjusted clearance was greater (p < 0.01), and the elimination half-life of rh inhibin A was shorter (p < 0.01) in adult than in juvenile rats. These data indicate that partial suppression of FSHP mRNA by inhibin is associated with a decline in GnRH receptor gene expression, suggesting that the notion that inhibin can act as a male contraceptive through selective and complete inhibition of FSH production, without effect on LH and Leydig cell function, may be mistaken. In addition, increased inhibin clearance appears to contribute to the fall in plasma inhibin levels with maturation in the male rat.
MATERIALS AND METHODS Animals and Treatments
INTRODUCTION
Male Sprague-Dawley rats were purchased from ZivicMiller Laboratories Inc. (Allison Park, PA) and used according to a protocol approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh, PA. Rats were anesthetized with methoxyflurane (PittmanMoore Inc., Washington Crossing, NJ) and were implanted i.p. for 3 days with Alzet osmotic minipumps, model 1003D (Alza Corp., Palo Alto, CA), containing recombinant human (rh) inhibin-A (Genentech, Inc., South San Francisco, CA) in doses of 0-135 ig in 0.15 M NaCl, 0.05 M Tris, pH 7.5, containing 2% protease-free BSA, calculated to deliver 0-150 jig/kg per day. Previous studies have shown that maximum suppression of circulating FSH levels by an i.v. bolus of rh inhibin-A was observed at a dose of 10-12 ig/kg [10, 11]. Animals were killed by decapitation, and plasma was saved for measurement of LH, FSH, testosterone, and inhibin by RIA. Anterior pituitary glands were placed in 0.5 ml cold 4.0 M guanidinium thiocyanate solution (25 mM sodium citrate, pH 7.0, containing 0.5% sarcosyl, 0.1 M 2-mercaptoethanol), frozen in dry ice-acetone, and stored at -70C for measurement of gonadotropin subunit and GnRH receptor mRNA levels. Inhibin pharmacokinetics was studied in immature (age 23-25 days) and adult (age 7-8 wk) male rats. Animals were anesthetized with methoxyflurane, orchidectomized bilaterally, and implanted with right jugular catheters. Studies commenced in unrestrained conscious animals 72 h lat-
Current approaches to human hormonal male contraception use high doses of testosterone alone or GnRH analogues with testosterone replacement to suppress spermatogenesis by inhibiting FSH and LH secretion [1, 2]. Supraphysiological doses of testosterone produce unwanted sideeffects such as acne, polycythemia, weight gain, and the potential for increased risk for prostatic hypertrophy and cancer. Moreover, androgens have been abused, and are controlled substances in many regions of the United States. Therefore testosterone cannot be made available in an unrestricted fashion to the general public. GnRH agonist analogues and testosterone replacement have not produced consistent oligospermia [3], and GnRH antagonists may release histamine from mast cells and produce allergic side effects [4]. Thus other approaches are under investigation. Accepted August 8, 1996. Received May 13, 1996. 'This research was supported in part by the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, under a cooperative agreement (DPE2044-A-00-6063-00) with the United States Agency for International Development (A.I.D.), and by a grant from the NIH (R01 -HD19546). Presented inpart at the 77th Annual Meeting of the Endocrine Society, Washington, DC, 1995, Abstr P1-345. 2Correspondence: Stephen J. Winters, M.D., Department of Medicine, University of Pittsburgh Medical Center, Montefiore N919, 200 Lothrop Street, Pittsburgh, PA 15213-2582. FAX: (412) 692-4019, e-mail: wintersvaxa.muh.upmc.edu 1377
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TABLE 1. Effects of inhibin treatment on pituitary FSH[3 mRNA and plasma FSH levels in adult rats (mean +_SEM).* Dose (jig/kg/day)
n
FSHO mRNA (cpm)
0 5 20 50 100 150
6 3 3 3 3 3
1385 985 971 887 921 1279
+ 255 + 93 + 63 + 34 +148 + 40
plasma FSH (ng/ml) 9.3 8.1 8.5 6.5 6.2 8.1
+ 0.8 + 1.1 + 1.2 + 0.9' + 0.4t + 0.9
among means were compared by the Kruskal-Wallis test for FSH3 mRNA (p = 0.052) and by ANOVA for plasma FSH (p = 0.043); for FSHf mRNA levels, none of the values for rats treated with rh inhibin-A differed significantly from rats treated with vehicle. p < 0.05 vs. vehicle-treated controls. * Differences
er. After the collection of 0.5-ml blood samples at Time 0, animals received 40 Ixg/kg rh inhibin-A i.v. in 200 pLIsaline containing 2% protease-free BSA. Additional blood samples were drawn at 10, 20, 30, 45, 60, 90, 120, and 180 min; after each blood sample was drawn, an equal volume of saline containing 0.1% BSA and heparin (10 U/ml) was infused. Immunoassays Plasma LH and FSH levels were determined by RIA using reagents obtained from the National Hormone and Pituitary Program, NIDDK, through the University of Maryland School of Medicine, Baltimore, MD. Results are expressed in terms of LH and FSH RP-2 standards. The minimal detectable dose was 0.07 and 0.22 ng, and the within-assay coefficients of variation were 6% and 5% for LH and FSH, respectively. Plasma testosterone was assayed with a highly specific dextran-charcoal RIA following extraction of serum with diethyl ether. The within-assay coefficient of variation was 4%. Levels of rh inhibin-A in rat plasma were measured by the Monash heterologous RIA [ 18] using reagents provided by the Contraceptive Development Branch, NICHD (Bethesda, MD). The assay standard was rh inhibin-A. Binding of 25I-bovine inhibin to A/S 1989 ranged from 15% to 25%. The antibody used (No. 1989) cross-reacts 288% with bovine posttranslationally processed pro-aC subunit [19] and recognizes rh inhibin a-subunit [20]. The within- and between-assay coefficients of variation were 6.0% and 12.8%, respectively. RNA Extraction and Northern Blot Hybridization Total RNA was extracted by the guanidinium thiocyanate-phenol-chloroform procedure [21]. Pituitary glands were thawed, homogenized, and centrifuged; 2 M sodium acetate (pH 4.0), buffer-saturated phenol, and chloroform: isoamyl alcohol (25:1 v:v) were added to the supernatant. The RNA in the aqueous phase was precipitated with ethanol, redissolved in 4 M guanidinium thiocyanate solution, and reprecipitated with ethanol. The pellet was washed with 75% ethanol, desiccated, and dissolved in diethylpyrocarbonate-treated water. The concentration of total RNA was calculated by reading the optical density at 260 nm. The yield of RNA per pituitary was (mean + SEM) 33 + 3.0 pg for adult and 14 1.3 tLg for immature rats. Aliquots of pituitary RNA samples were electrophoresed in 1.2% agarose-formaldehyde gels. RNA was transferred to Nytran membranes (Schleicher & Schuell, Keene, NH), cross-linked to the membranes by baking for 2 h at 80-
90°C, and then irradiated with UV light for 2 min. Purified cDNAs (LH3, from Dr. James Roberts (Mount Sinai School of Medicine, New York, NY), FSHP3 from Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR), a-subunit from Dr. William Chin (Harvard Medical School, Boston, MA), and mouse GnRH receptor from Dr. Kevin Catt (National Institutes of Child Health and Human Development, Bethesda, MD) were labeled by the random primer method with [- 3 2P]dCTP (3000 Ci/mmol, New England Nuclear, Boston, MA) to 4.9-9.0 x 108 cpm/,g. Labeled probes were added to the hybridization solutions at a concentration of approximately 5 ng/ml, and incubated for 48-72 h. After autoradiography, slices corresponding to full-length mRNAs were excised and counted in a Packard 4530 scintillation counter (Packard, Downers Grove, IL). Membranes were rehybridized without stripping to cDNA for -actin or 18S ribosomal RNA for normalization of LHI3 and a-subunits, and to cyclophilin cDNA (from Dr. James Douglass, Oregon Health Sciences University, Portland, OR) for FSH[3 and GnRH receptor normalization. Data Analysis The means of two groups were compared using Student's t-test or the Mann-Whitney test. Dose-response effects were tested by ANOVA or the Kruskall-Wallis test followed by a comparison with values in vehicle-treated animals using Dunnett's test. Multiple comparisons were performed by ANOVA and Tukey's test. Pharmacokinetic parameters of inhibin disposition in adult and immature rats were estimated by standard noncompartmental methods. The area under the curve (AUC) was estimated by the log-trapezoidal method with extrapolation to infinity. The circulating half-life was calculated as 0.693/k, where k is the elimination rate constant estimated as the slope of the log-linear regression of the terminal elimination phase. The clearance was determined from the ratio of the DOSE administered to the total AUC. The volume of distribution at steady state, without the assumption of a specific compartmental model (Vdss), was calculated as the ratio of (DOSE*AUMC) to (AUC) 2 estimated by the log-trapezoidal method with extrapolation to infinity, and AUMC is the area under the first moment curve, which is the product of time and plasma concentration [22]. RESULTS Treatment of adult male rats with rh inhibin-A in doses of 0-150 uig/kg per day for three days produced no consistent dose-dependent effect on either plasma FSH or pituitary FSH3 mRNA concentrations (Table 1). Plasma FSH levels were reduced at doses of 50 and 100 ,ug/kg per day, but not at 150 jLg/kg per day. There was no significant effect of rh inhibin-A on pituitary FSH3 mRNA levels. Therefore, experiment 2 was conducted in immature as well as adult rats because of previous evidence for developmental differences in the effects of inhibin on FSH in this species [16, 17]. Table 2 summarizes plasma FSH, LH, and testosterone levels in 7-wk-old adult and 25-day-old immature male rats treated with rh inhibin-A at a dose of 145 lIg/kg per day or with vehicle for 3 days. Again no effect of inhibin was observed in adult rats. On the other hand, both plasma FSH and LH levels were suppressed (p < 0.05) by inhibin treatment of immature rats. FSH suppression was partial, however, in that FSH levels in plasma pools from hypophysec-
INHIBIN CONTROL OF GONADOTROPIN SECRETION TABLE 2. Effects of inhibin treatment on plasma FSH, LH, and testosterone concentrations in immature and adult rats (mean + SEM). Treatment FSH (ng/ml) vehicle inhibin LH (ng/ml) vehicle inhibin
Adult (n = 3/group)
Immature (n = 6/group)
9.44 ± 1.61 9.43 + 1.43
11.29 + 0.66 6.29 ± 1.80*
0.59 ± 0.14 0.66 ± 0.19
1.44 ± 0.34 0.54 + 0.10*
4.68 ± 1.26 6.98 ± 0.93
0.86 + 0.25 0.45 ± 0.05
Testosterone (ng/ml) vehicle inhibin
* p < 0.05, Mann-Whitney test; inhibin vs. vehicle.
tomized rats were < 0.19 ng/ml. Plasma testosterone levels were also slightly lower (p = 0.19; Mann-Whitney test) in inhibin-treated than in vehicle-treated immature rats. Figure 1 shows the percentage of suppression of the gonadotropin subunit mRNA levels in pituitaries from immature rats treated for 3 days with rh inhibin-A at a dose of 145 ,ug/kg per day compared to values in rats implanted with minipumps containing vehicle. As shown previously in vitro and in short-term studies in vivo [23-25], inhibin suppressed FSH3 mRNA levels. The observed decline was 53 11% (p < 0.01). In addition, pituitary at-subunit mRNA levels were reduced by 14 ± 10% (p < 0.05), and LHB mRNA levels were suppressed by 43 + 8% (p < 0.01). The results in Table 3 indicate that treatment of adult male rats with an equivalent dose of rh inhibin-A was without effect on any of the gonadotropin subunit mRNAs. Figure 2 shows the effect of inhibin on GnRH receptors examined by Northern blot hybridization of pituitary RNA to the GnRH receptor cDNA. As shown by others, the predominant mRNA in rat pituitary is in the range of 4.5 kilobases (kb) [26]. Three days of inhibin treatment suppressed (p < 0.01) GnRH receptor mRNA levels in immature rats to 41 11% of control, but was without effect in adult rat pituitaries. The concentration of GnRH receptor mRNA was higher (p < 0.05) in the pituitaries of vehicletreated immature rats than in adults when expressed per a-Subunit
LH-P
FSH-3
-10 -
-lO
_ -20
Z
-
-30-
I p
