Effects of exogenous melatonin prior to the breeding season on testis weight and epididymal androgen receptors in rams

DOMESTIC

ANIMAL

ENDOCRINOLOGY

Vol. 5(3):257-264,

1988

EFFECTS OF EXOGENOUS MELATONIN PRIOR TO THE BREEDING SEASON ON TESTIS WEIGHT AND EPIDIDYMAL ANDROGEN RECEPTORS IN RAMS’ F.R. Tekpetey

and R.P. Amann

Animal Reproduction Laboratory Colorado State University Fort Collins, Colorado 80523 Received

April

11, 1988

ABSTRACT Rams were

randomly

assigned to an experiment that evaluated effects of treatment for 45 d vs control; MEL vs CON) in mid-May through June on testis weight and concentration of epididymal androgen receptor in July or September (Sept). Mean testis weight of MEL and CON rams was not different in July (332 vs 283 g), but in Sept was less (P<.O5) for MEL rams than CON rams (268 vs 382 g). Testis weight of MEL rams was less (P<.O5) in Sept than in July. Caput, corpus and cauda epididymal tissues were used to prepare extracts which were analyzed for concentration of dihydrotestosterone (DHT) receptor using a new standard curve method. A standard extract was characterized using four independent &point Scatchard analyses and found to contain 6.0 fmol DHT receptor/mg wet tissue (Ka = 3.5 X lOaM’); this extract was used to establish standard curves for assays of unknown samples. Data on concentration of DHT receptors measured by Scatchard analysis and the standard curve method were highly correlated (r = 0.99; P<.Ol; n = 8). Concentrations of DHT receptor were not afTected by treatment, month of castration, or their interaction. However, for data pooled across treatment and month, concentration (fmol/mg protein) of DHT receptor was greater (P<.O5) in caput or corpus (125 or 122) than in cauda (92) epididymidis. The regional distribution of epididymal DHT receptors in this study conlirmed our previous findings. These results revealed that the melatonin treatment used in this study did not increase testis weight by early July, but caused premature decline in testis weight by Sept. However, the melatonin treatment did not inlluence concentration of DHT receptor in the epididymis.

(2.5 mg melatonin/d

INTRODUCTION

In most mammals, photoperiod is the principal cue that synchronizes endogenous circannual cycles in reproductive activity, so that in temperate latitudes there is a distinct breeding season. In short-day breeders such as sheep, reproductive activity is maximal in the fall when daylength is decreasing, but is low in late winter, spring and summer when daylength is increasing. The breeding season can be extended or the nonbreeding season abolished in ewes (1,2) or rams (3) by use of lighting regimes designed to simulate a short day (8 hr light: 16 hr dark) or sequences of short and long (16 hr light:8 hr dark) days. Although techniques of manipulating day length can increase spermatogenic activity and testicular growth in rams during the nonbreeding season (3), they are impractical for most breeders and are expensive. The pineal hormone, melatonin, has a major role in mediating effects of daylength on reproduction. Secretion of melatonin is highly correlated to the daily light-dark cycle in both ewes (4,5) and rams (6-8); there is a marked Copyright

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AND AMANN

increase in concentration of melatonin in blood occurring at night. Duration of elevated melatonin concentration provides an index for determination of night length (4,5,9) and under certain conditions “resets” or alters the annual cycle. Consequently, melatonin treatment of ewes by daily injection, feeding or via implants has been used to induce early onset of estrous cyclicity or extend the breeding season (lo- 12). Fewer studies with melatonin have been conducted with rams, although implanting melatonin in rams exposed to long days resulted in rapid onset of testicular redevelopment similar to that associated with exposure to short days (13). Effects of exogenous melatonin on epididymal function, which could intluence fertility, have not been reported. Epididymal function is androgen dependent and, in rams, the concentration of androgen receptors in the epididymis varies with region of the epididymis and season (14). Concentration of androgen receptor is higher in the caput and corpus than in the cauda epididymidis, and higher in the breeding than the nonbreeding season. Therefore, to determine if epididymal function might be altered in rams treated with melatonin, we compared the concentration of androgen receptor and testicular weight in rams treated with melatonin before the breeding season with values for control, untreated rams. MATJXIALS

AND METHODS

Experimental Treatment and Tissue Preparation. Tissues were obtained from a 2 X 2 random block design experiment, testing the effects of exogenous (or no) melatonin before the breeding season on reproductive capacity of rams at two periods (July and Sept). Adult western rams, previously exposed to the natural photoperiod, were assigned randomly to one of four groups (six rams/ group). Barns in two groups received a daily injection (at 1600 hr) of vehicle (1 ml satBower oil i.m.; CON) and rams in two groups received melatonin (2.5 mg in 1 ml oil i.m.; MEL) for 45 d starting in mid-May, when the natural photoperiod was about 15 hr light and 9 hr dark. This melatonin dosage and injection schedule were known to elevate serum concentration of melatonin for about 6 hr or until the onset of darkness on the longest day of the year (lo), simulating the melatonin secretory pattern on the shortest day of the year. Rams in one CON and one MEL group were castrated in early July (July), l-3 days after the last injection of oil or melatonin and the remaining rams were castrated in Sept (Sept), 77-80 days after terminating treatment. Castrations were performed under local anesthesia. Testes weights were recorded and epididymal tissue immediately was divided into the caput, corpus and cauda and immersed in ice-cold, low-salt TEDG buffer [lo mM Tris-HCI; 1.5 mM EDTA; 1.0 mM each of dithiothreital, phenylmethyl sulfonyl fluoride and sodium molybdate; 10% (v/v) glycerol; pH = 7.4; 141. High-salt buffer was identical, except that it contained 400 mM KCl. Epididymal tissues were processed for quantification of dihydrotestosterone (DHT) receptor (14). All steps were at 0-4C. Tissue was homogenized (1 g tissue in 4 ml buffer) to obtain low- and high-salt extracts which were snap frozen by immersing the 4-ml tubes in liquid nitrogen, and stored at -70C until assayed (14). Before quantification of DHT receptors, endogenous steroids were removed from low-salt extracts using dextran-coated charcoal (12.5 mg charcoal plus 1.25 mg dextran per 1 .O ml of extract); high-salt extracts were assumed not to contain endogenous free steroids.

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Receptor Binding Assay and Validation. A standard curve procedure (15) was adapted to quantification of DHT receptor and validated. Three standard extracts (1 g tissue in 5 ml TEDG buffer) were prepared by pooling low- and high-salt extracts from tissue representing the central caput through proximal corpus of six (SEl), two (SE2) or two (SE3) epididymides, respectively, aliquoted into 4-ml vials and stored at -70C. Aliquots of SE1 were thawed, stripped of endogenous steroid and characterized using four independent 8point Scatchard analyses (14). This extract contained 6.0 fmol DHT receptor per 1.0 mg wet tissue (affinity = 3.5 + 0.4 X lOaM.‘). Iater, additional aliquots of the same extract (SEl) were used to prepare IO-point standard curves for assays of test samples. Each standard curve was generated by incubating (in duplicate) 1.5 nM [SHJDHT (1,2,4,5,6,7[3H] dihydrotestosterone; 128 Ci/mmol) with increasing quantities of SE1 (25-400 ~1, equivalent to 5-80 mg wet tissue). Parallel tubes contained a loo-fold excess of nonradioactive DHT to determine nonspecific binding. After 30 hr at 4C (14), free steroid was separated from bound steroid by adding 500 pl of dextran-coated charcoal [0.6% (w/v) charcoal and 0.05% (w/v) dextran in TEDG buffer]. After incubation for 10 min at 4C, followed by centrifugation at 5,000 X g for 10 min, a 600+1 aliquot of supernatant was added to 4 ml of scintillation fluid and counted for 5 min. Counts for duplicate tubes were averaged and specific binding (primary value - non-specific binding) was calculated for each volume-of SE1. Using the concentration of DHT receptor in SE1 (from Scatchard analysis), number of receptors (fmol) for each volume of SE1 was calculated; these values were plotted to provide a standard curve (Figure 1). The amount of [3H]DHT specifically bound to a known volume of each sample extract, determined as described above for SE1, was compared to the standard curve and concentration of DHT receptor (fmol/mg wet tissue) in the sample extract was calculated. All sample extracts plus SE2 and SE3 were analyzed using duplicate tubes for two concentrations and invariably these points were parallel to the standard curve (Figure 1). Concentration of DHT receptor in a given tissue was computed as the sum of values for low- and high-salt extracts and expressed per milligram of total tissue protein (low-salt plus high-salt extracts). Protein concentration of each extract was measured, in duplicate, using the BioRad assay (BioRad Laboratories, Richmond, CA) with bovine serum albumin as the standard. To validate the standard curve technique, concentrations of DHT receptor in six extracts were determined concurrently using Scatchard analysis and the standard curve technique (Table 1); the difference (n = 8) averaged - 3.1% of the Scatchard value. The correlation between values obtained by the two methods was very high (r = 0.99; P
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concentration of DHT receptor used a model that included treatment, month of castration, region of epididymis (fixed), replicate and the interactions of treatment, month and region. Differences between means were tested using the Student-Newmann-Keuls multiple comparison.

0

IO

20

40

60 100

60

200

VOLUME EXTRACT (pill NUMBER DHT RECEPTORS

400

600

OR (fmol)

Fig. 1. A representative standard curve (o-0; y = - 11297 f 6660X when values designated 0 were excluded and X expressed as fmol DHT receptor and Y as cpm [‘H]DHT; rz=0.98) generated by plotting counts of [3H]DHT specifically bound to receptor vs fmol DHT receptor in standard extract SEl, as determined by Scatchard analysis (insert). Also shown are [sH]DHT counts specifically bound (0) to receptor in two other standard extracts (SE2, SE3) and a test-sample extract (S; caput high-salt) each assayed at two concentrations. TAEZU1.

Extract’ SE1 (n=4) SE2b SE2’ SE3b

SE3’ Sample 1’ Sample 24

COMPAIUSON

OP DATA ON

Ka(xlO’M-‘) 3.5 f 0.4 2.8 3.1 3.4 2.5 2.1 2.3

DHT

RECEPTORS MEASURED BY THE STANDARD CURVE METHOD k4TCHARD ANALYSES

DHT receptor (fmol/mg protein) Scatchard Standard curve --92.3 + 1.4

;::;

85.2 81.5 97.3 415.0 400.0 169.0

AND

Diff (%)

+ 75.9 75.9 108.0 400.0 383.0 144.0

6.9 2.1

-10.9

-

6.8

+11.0 I 2:; -14.8 *Extracts designated by superscripts b,c.d or e were analyzed in four separate assays. SEl, SE2 and SE3 are standard extracts; SE1 was used to prepare standard curves. Samples 1-4 were extracts of caput low-salt, caput high-salt, corpus high-salt and cauda high-salt, respectively. Values for Scatchard and standard curve methods are highly correlated; r = 0.99 (P<.Ol).

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ON OVINE

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RESULTS Effect of Treatment and Month of Castration on Testis Weight. Mean testis weight was affected (Pc.05) by the interaction of treatment and month of castration (Figure 2), but not by treatment or month. In July, there was no difference (P>.O5) in mean testis weight (SEM) between CON (281 f 3 1 g) and MEL (332 k 31 g) groups, but in Sept there was a difference. Mean testis weight of CON rams was greater (Pc.05) in Sept (382 -1- 20 g) compared to July, while mean testis weight of MEL rams was less (Pc.05) in Sept (268 + 20 g) than in July.

b ab

JULY SEPT MONTH OF CASTRATION Fig. 2. Effects of treatment and month on mean weight of the left and right testes (mean f SEM). Means with different superscript letters are different (P-C.05).

Effect of Treatment, of DHT Receptor.

Month

and Region

Treatment, month of treatment, month and region of epididymis of DHT receptor in the epididymis (Figure receptor differed among regions, and was than in cauda epididymidis (overall mean 92 + 7 fmol/mg protein, respectively).

of Epididymis

on Concentration

castration or interactions involving did not affect (P>.O5) concentration 3). However, concentration of DHT greater (P<.O5) in caput or corpus values were 125 f 7, 122 + 7 and

DISCUSSION

The affinity of the DHT binding site (Ka = 3.5 + 0.4 X 108.M’) in standard extract SE1 was similar to values for six other samples (mean Ka = 2.8 + 0.3 X 1 08.M-l ; Table 1) or reported previously (14,17). The androgen binding site measured using this Scatchard analysis has been shown to be highly specific for DHT and of an a.f&nity not influenced by season; androgen binding protein or testosterone-estradiol binding globulin that might be present in the tissue do not confound measurements (14). Thus, values reported herein are for DHT receptor. The standard curve procedure described herein does not permit determination of binding atlinity for each test sample. However, when analyzed at two

262

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AND AMANN

.

c II

SEPT

;’/ l-l

.

c M J-L iAPUT REGION

M

CORPUS OF

c M n :AUDA

EPIDIDYMIS

Fig. 3. Effects of treatment, month and region of the epididymis on concentration of DHT receptors (in low + high-salt extracts; mean * SEM) in rams castrated in July or Sept. Effects of treatment, month and interactions were not significant (P>.O5). ‘Mean for the cauda (pooled across treatments) diRered (P-C.05) from those for the caput or corpus.

concentrations, all test samples and two standard extracts (SE2 and SE3) showed parallelism with the standard curve. Thus, binding sites in the test samples had a6inities for DHT similar to those in the standard extract and, in confirmation of earlier data (14), the al%nity of DHT receptor did not change between July and Sept. Concentrations of DHT receptor in standard and sample extracts determined by both Scatchard analysis and the standard curve method were similar and highly correlated. Hence, the standard curve technique is a simple and valid method for quantifying DHT receptor in multiple samples or when relatively small amounts of tissue are available. Melatonin treatment for 45 d in late May and June caused no change in testicular weight in July, but induced low testicular weight in Sept (Figure 2). Under natural photoperiodic conditions, onset of testicular recrudescence in rams in early summer is perhaps the result of photorefractoriness to prevailing long days (18). The decline in testicular weight of MEL rams in Sept, may be due to an interruption of the process of photorefractoriness by the melatonin treatment in late May and June, thereby resensitizing those rams to the suppressive effects of long days. Premature decline in testicular function probably is an inevitable consequence of such treatment, unless circumvented by alternating 8-10 wk intervals of short and long days (3) or melatonin treatment with no treatment. A positive testicular response to treatment had been anticipated since Lincoln and Ebling (13) reported that constant administration of melatonin changed cyclicity of testicular activity in rams, and interfered with

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the normal photoperiodic regulation of reproduction. They implanted rams with melatonin during exposure to long days (16L:8D), and a 10% increase in testicular size (relative to controls) was apparent 14 wk later. However, when melatonin was implanted during short days (8L: 16D), there was a regression in testis size in both melatonin and control rams with no apparent difference between the two groups. Perhaps, failure to enhance testis development in July was because in most breeds of sheep, concentration of testosterone in blood begins to increase in mid-spring, followed by onset of the annual increase in testicular size in early summer (3). Thus, testicular growth might have been slightly enhanced, but “triggering” of development already had occurred. Administration of melatonin in mid-May in the present study also may have contributed to failure to detect a change in concentration of epididymal DHT receptor in either July or Sept. Although we did not measure plasma testosterone, it is likely that its concentration, and that of DHT receptor in epididymal tissue, had increased in early summer to approach maximal values by July which were maintained through Sept, but would decline by November. Indeed, when concentration of epididymal DHT receptor was measured in August to October, values were higher than in February to May (14). Regional differences in concentration of epididymal DHT receptor in both CON and MEL rams conIirm our previous findings (14). The higher concentration of DHT receptor in caput and corpus epididymidis provides additional support for the concept (14) that these regions are most dependent on androgenic stimulation which is important for sperm maturation. Based on data presented herein, we conclude that melatonin treatment of adult rams for 45 d starting in mid-May is of limited value. It is too late or inadequate to induce an increase in testicular weight by early July, and will cause a premature decline of testicular weight by Sept. However, melatonin treatment did not inlluence concentration of DHT receptor in the epididymis. ACKNOWLEDGMENTS

AND

FOOTNOTES

1 Supported by Cooperative Agreement CR 812725-01 from the US EPA and Grant HD-14,501. The tams used in this research were treated by C.F. Stokes and kindly made available by the Department of Animal Science, Colorado State University. Please address reprint requests and correspondence to R.P. Amann, 246 Physiology Building, Colorado State University, Fort Collins, Colorado 80523

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7. Almeida OFX, Lincoln GA. Photoperiodic regulation of reproductive activity in the 8. 9.

10. 11. 12. 13.

14.

ram: Evidence for the involvement of circadian rhythms in melatonin and prolactin secretion. Biol Reprod 27:1062-1075, 1982. Almeida OFX, Lincoln GA. Reproductive photorefractoriness in rams and accompanying changes in the patterns of melatonin and prolactin secretion. Biol Reprod 30:143-158, 1984. Karsch FJ, Bittman EL, Foster DL, Goodman RL, Legan SJ, Robinson JE. Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 40:185-232, 1984. Nett TM, Niswender GD. Intluence of exogenous melatonin on seasonality of reproduction in sheep. Theriogenology 17:645-653, 1982. Arendt J, Symons AM, Laud CA, Pryde SJ. Melatonin can induce early onset of the breeding season in ewes. J Endocrinol 97:395-400, 1983. Kennaway DJ, Dunstan EA,Staples LD. Photoperiodic control of the onset of breeding activity and fecundity in ewes. J Reprod Fertil, Suppl 34:187-199, 1987. Lincoln GA, Ebling FJP.Effect of constant-release implants of melatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams. J Reprod Fertil 73:241-253, 1985. Tekpetey FR, Amann RP. Regional and seasonal differences in concentrations of androgen and estrogen receptors in ram epididymal tissue. Biol Reprod 38: 105 l1060,

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15. Nett TM, Crowder ME, Moss GE, Duello TM. GnRH-receptor interaction. V. Downregulation of pituitary receptors for GnRH in ovariectomized ewes by infusion of homologous hormone. Biol Reprod 24:1145-1155, 1981. 16. Statistical Analysis Service @AS). Statistical Analysis System Institute, Inc., Gary, North Carolina, 1982. 17. Carreau S, Drosdowsky MA, Courot M. Androgen-binding proteins in sheep epididymis: Characterization of a cytoplasmic androgen receptor in the ram epididymis. J Endocrinol 103:273-279, 1984. 18. Almeida OFX, Lincoln GA. Reproductive photorefractoriness in rams and accompanying changes in the patterns of melatonin and prolactin secretion. Biol Reprod 30:143-158, 1984.