| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis |
-Reductase Isoenzymes 1 and 2 in the Rat Testis During Postnatal Development1
Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5-reductase (5R) isoenzymes (5R1 and 5R2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5R isoenzymes and their relationship to the production of 5-reduced androgens in the postnatal rat testis. Both 5R1 and 5R2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5R1 and 5R2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60–160 days of age. The developmental pattern of both 5R isoenzyme activity levels was mirrored by the testicular production of 5-reduced metabolites. Although 5R1 was greater than 5R2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5-reduced androgen biosynthesis. The peak in 5R isoenzymes and 5-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5R inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5R activity during puberty by 75%–86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5R inhibitors. We conclude that both 5R1 and 5R2 isoenzymes are involved in the peak of 5-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.
pituitary hormones, spermatogenesis, steroid hormones, testis, testosterone
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-reduced metabolites dihydrotestosterone (DHT) and 5-androstane-3,17ß-diol (Adiol) predominate in the pubertal testis [2–4]. Because DHT is a more potent androgen than testosterone and serves to amplify androgen action when testosterone levels are low [5–7], it seems likely that increased production of 5-reduced metabolites may be important for this androgen-dependent initiation of spermatogenesis and/or other aspects of testicular function during puberty.
The conversion of testosterone to DHT is an irreversible reaction catalyzed by the 5-reductase (5R) enzyme, of which there are two isoenzymes, 5R1 and 5R2, encoded by different genes (for review, see [8]). The two isoenzymes possess different biochemical properties, with 5R1 having a micromolar affinity for steroid substrates and peak enzyme activity in cell lysates extending over a broad neutral pH range and 5R2 having a nanomolar affinity for steroid substrates and peak enzyme activity in cell lysates at pH 5.0 [9, 10]. The regulatory factors that control 5R expression and activity are unclear, but they may include androgens [8, 10, 11], gonadotropins [11], and other hormones [12, 13].
The pubertal peak in testicular 5-reduced metabolites seems likely to be caused by an increase in testicular 5R activity. Various studies during the 1970s showed that immature testes had higher levels of 5R enzyme activity than mature testes [2, 4, 14, 15]. However, these studies were performed before the identification of the second 5R isoenzyme (5R2); thus, the isoenzyme responsible for the pubertal peak of 5-reduced androgen synthesis is unclear. A more recent study investigating 5R isoenzyme expression by Northern blot analysis during testicular development in the rat suggested that the immature testis had higher levels of 5R1 compared to the adult, with 5R2 expression being barely detectable at any age [16]. Although this study concluded that 5R1 was the major isoenzyme expressed in the testis at all ages, other studies have suggested that 5R2 is, in fact, the predominant isoenzyme present in this tissue [10, 17, 18]. Thus, discrepancy exists in the literature concerning the relative expression and activity levels of the 5R isoenzymes in the testis.
The ability of the testis to 5-reduce testosterone to DHT has been shown to be important for maintaining or restoring sperm production in adult rats in a setting of low testicular testosterone [19, 20]. Because testosterone levels are lower in the pubertal testis than in the adult [3], it is reasonable to speculate that formation of DHT may also be important for initiating spermatogenesis during puberty. We recently developed specific assays to measure the activity levels of 5R1 and 5R2 isoenzymes in the rat testis [21] and showed that 5R1 activity in the testis predominates over 5R2 in both immature and adult testes. However, we also found clear evidence for the 5R2 isoenzyme in the testis.
To address the discrepancies in the literature regarding testicular 5R isoenzyme expression as well as to understand the basis for the pubertal peak in testicular 5-reduced androgen biosynthesis, the present study aimed to investigate the developmental regulation of both 5R1 and 5R2 isoenzymes in the rat testis. Real-time polymerase chain reaction (PCR) methods were used to quantitate 5R1 and 5R2 mRNAs, and specific isoenzyme activity assays [21] were used to study the ontogeny of 5R isoenzyme mRNA and protein. Patterns of testicular levels of 5R isoenzymes were compared to circulating gonadotropins and the concentration of testicular androgens. These endpoints were examined in prepubertal (10-day-old [d.o.]), pubertal (20- to 40-d.o.), and adult (60- to 160-d.o.) rats. Finally, in an effort to investigate the role of 5-reduced androgens during the initiation of spermatogenesis, a 5-reductase inhibitor was administered to pubertal rats.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
4-Androsten-17ß-ol-3-one (testosterone), 5-androstan-17ß-ol-3-one (DHT), and 5-androstane-3,17ß-diol (Adiol) were purchased from Sigma (St Louis, MO). [1,2-3H(N)]Testosterone (53.5 Ci/mmol) and [4-14C]DHT (58.3 mCi/mmol) were purchased from NEN Life Sciences (Boston, MA). [4-14C]Testosterone (57.3 mCi/mmol) was purchased from Amersham Pharmacia Biotech, Inc. (Castle Hill, New South Wales, Australia). [4-14C]Adiol was prepared by enzymatic reduction of [4-14C]DHT using 35-d.o. rat testis (10 000 x g supernatant) as a source of 3-hydroxysteroid dehydrogenase activity. The [4-14C]Adiol produced was isolated by thin-layer chromatography (TLC; see below). [4-14C]Adiol and [1,2-3H(N)]testosterone were purified by TLC before use [21]. Analytical-grade chloroform, toluene, ethanol, and diethyl ether (Merck, Kilsyth, Victoria, Australia) were used for TLC. The coenzyme, ß-NADPH, and analytical-grade sodium acetate and isopropanol were obtained from Sigma.
Animals
Male Sprague-Dawley rats (10, 20, 25, 30, 35, 40, 60, 80, 120, and 160 d.o.; n = 11 per age group) were obtained from the Monash Central Animal House (Clayton, Victoria, Australia) for the analysis of testicular 5R isoenzyme and androgen levels. Six animals from each age group were used for the measurement of serum gonadotropins and testicular 5R mRNA and enzyme activity. The remaining five animals were used for the determination of testicular androgen concentrations. For the 5R-inhibitor experiments, 20-, 30-, and 40-d.o. rats (n = 16 per group) were used. The study was approved by the Monash Medical Centre Animal Ethics Committee.
5R-Inhibitor (L685,273) Experiment
Eight rats per age group (20, 30, and 40 d.o.) received 20 mg/kg twice daily (i.e., 40 mg kg-1 day-1) of the competitive 5R-inhibitor L685,273 (21,21-pentamethylene-4-aza-5-pregn-1-ene-3,20-dione; a kind gift from Dr. Gary Rasmusson, Merck Sharpe and Dohme, Rahway, NJ) [10, 20] for 6 days, whereas a further eight rats in each age group received twice-daily injections of vehicle. The L685,273 and vehicle were prepared as described previously [20]. At the end of the 6-day treatment period, animals were administered heparin and anesthetized. One testis was removed, weighed, and then frozen in liquid nitrogen and stored at -80°C for testicular androgen analysis. Blood was then collected by cardiac puncture and the remaining testis fixed in situ by whole-body perfusion with Bouin fixative [20]. Fixed testes and ventral prostates were then removed and weighed. Testes from the 36-day-old (d.o.) group were processed into methacrylate resin [20], and germ cell numbers, from pachytene spermatocytes through to elongated spermatids, expressed per testis were determined using the optical disector stereological method as detailed elsewhere [22].
Analysis of 5R Isoenzymes and Testicular Androgens Experiment
Animals were killed by CO2 inhalation, and blood was collected by cardiac puncture and stored at 4°C overnight, after which serum was collected. Both testes were quickly removed, trimmed of fat, weighed, snap-frozen in liquid nitrogen, and then stored at -80°C until measurement of testicular androgens, mRNA, or 5R activity levels.
5R1 and 5R2 Isoform Activity Assay
The method for the measurement of 5R1 and 5R2 activity levels in rat testis, utilizing the different pH optima of each isoform, has been described in detail elsewhere [21]. Briefly, testes were decapsulated, homogenized in 0.25 M sucrose, and centrifuged at 10 000 x g, and then the supernatant was stored at -80°C. Testicular supernatants, assayed in duplicate, were incubated with radioactive ([3H]testosterone, 0.5 µCi) and nonradioactive (9.5 µM) testosterone and 0.5 mM ß-NADPH in assay buffer (0.1 M Tris-citrate) for 60 min at 37°C. The pH of the assay buffer was varied depending on the isoenzyme being measured (pH 5.0 for 5R2 or 7.0 for 5R1). Steroids were then extracted and separated by TLC [21]. The recovery of steroids was monitored by the inclusion of 14C-labeled steroids (testosterone, DHT, and Adiol) in the assay. Steroids were quantified by dual-isotope liquid scintillation counting to measure both 3H (conversion of testosterone to 5-reduced metabolites) and 14C radioactivity.
The recoveries of steroids as indicated by the recovery of [14C]steroids were 45.2% ± 5.7% for testosterone, 40.7% ± 4.6% for DHT, and 37.3% ± 5.9% for Adiol (mean ± SD, n = 45). The average background for the 5R isoenzyme assay was 0.021 ± 0.008 pg/min (mean ± SD, n = 7). Detection limits were set so that any value below two SD above the average background would be classified as nondetectable; however, all samples measured were above this detection limit. The between-assay variation as estimated by repeated measurements of quality-control (QC) samples was 11.9% (n = 7) for 5R1 and 24.2% (n = 7) for 5R2.
In each assay a recombinant 5R1 enzyme preparation [21] was measured at both pH 5 and pH 7 to give a pH 5:7 ratio. This ratio allows the correction for the fact that the broad peak of 5R1 enzyme activity across the neutral range leads to some 5R1 activity being detected at pH 5 in the 5R2 assay; thus, using the recombinant ratio, the amount of 5R1 activity at pH 5 is subtracted, allowing measurement of 5R2 activity only [21]. The average pH 5:7 ratio for recombinant 5R1 was 0.063 ± 0.009 (mean ± SD, n = 7), with a 14.3% between-assay variation.
The 5R activity levels were calculated as the sum of the 5-reduced metabolites (DHT + Adiol) formed from [3H]testosterone after corrections for procedural losses. The testicular concentrations of 5R1 and 5R2 were calculated according to formulae described previously [21] and were expressed as nanograms of 5R per minute per gram testis.
Quantitation of 5R Isoform mRNA Using Real-Time PCR
Total RNA was isolated using an RNeasy Mini extraction kit (Qiagen, Clifton Hill, Victoria, Australia), and an isopropanol purification procedure was included. Two micrograms of RNA were reverse transcribed using random primers for 5R1 and oligo dT primers for 5R2 and Expand Reverse Transcriptase (Roche Molecular Biochemicals, Mannheim, Germany). Real-time PCR was then performed using the LightCycler (Roche; for review, see [23]) using SYBR Green I fluorescence detection of amplified products. The forward primer (5'-TCC TGG TCA CCT TTG TCT TGG C-3') and reverse primer (5'-GTT TCC CCT GGT TTT CTC AGA TTC-3') for 5R1 were designed to produce a 128-base pair (bp) amplicon. The forward primer (5'-ACA TCC ACA GTG ACT ACA CCC TGC-3') and reverse primer (5'-TCC ATT CAA TAA TCT CGC CCA G-3') for 5R2 were designed to produce a 207-bp amplicon. A 20-µl PCR reaction was used and included 10.8 µl of PCR-grade water, 5 mmol/L of magnesium chloride, specific reverse and forward primers (0.5 µmol/L final concentration for 5R1 and 0.375 µmol/L final concentration for 5R2), 2 µl of SYBR Green dye, and 2 µl of reverse-transcribed cDNAs (diluted 1:10). An initial denaturation step of 10 min was used for both 5R1 and 5R2. The PCR amplification for 5R1 consisted of 38 cycles at 95°C for 15 sec (denaturation), 55°C for 5 sec (annealing), and 72°C for 10 sec (elongation). The PCR amplification for 5R2 consisted of 40 cycles at 95°C for 15 sec (denaturation), 62°C for 5 sec (annealing), and 72°C for 10 sec (elongation). The standards used as a reference preparation were highly purified and sequence-verified cDNAs that were identical to the real-time PCR products to ensure equal amplification efficiency between standards and PCR products. Standard curves for 5R1 (0.125–2 fg) and 5R2 (0.0125–0.2 fg) were performed in 2-fold dilutions, and QC samples were included in each assay. Samples were assayed in duplicate. Following PCR, melting-curve analysis was performed on the amplified product to ensure that only specific PCR amplicons were obtained and quantitated. This was also confirmed by nucleotide sequencing. The mRNA data obtained from the LightCycler was normalized against 28S ribosomal RNA, which was quantitated by electrophoresing equivalent RNA on an agarose gel and the 28S ribosomal RNA fluorescence measured using a CCD camera with Quantity One 5.1 software (Bio-Rad, Hercules, CA). All measurements were done within the linear range under nonsaturating pixel conditions. The electrophoresed RNA was also used to confirm the integrity of all RNAs used in the real-time PCR analysis. Using the QC samples, the between-assay variation was 8.6% (n = 7) for 5R1 and 20.9% (n = 10) for 5R2. Concentrations of 5R1 and 5R2 mRNAs were expressed as picograms per gram testis.
Serum Gonadotropins
Serum LH was measured by immunofluorometric assay as previously described [24], with an assay sensitivity of 12 pg/ml. Samples were assayed at 25 µl in duplicate. Serum FSH was measured by RIA as previously described [20], with an assay sensitivity of 2.5 ng/ml. Samples were assayed at 100 µl in duplicate.
Testicular Androgens
Testicular testosterone, DHT, and Adiol were separated by solvent extraction and high-performance liquid chromatography and then quantified by specific RIAs as described previously [20]. Average detection limits were 40, 42, and 120 pg/ml and between-assay variations of 12%, 22%, and 6% for testosterone, DHT, and Adiol, respectively.
Statistics
Data are shown as the mean ± SEM. Statistical comparisons were performed using SigmaStat 2.03 (SPSS Science, Chicago, IL). All data were log10 transformed before statistical analysis. Data from the 5R isoenzymes study were analyzed by one-way ANOVA. When a P value of less than 0.05 was achieved, differences compared to prepubertal (10-d.o.) or adult (120-d.o.) rats were assessed by Dunnett post-hoc comparison, whereas differences between all groups were assessed by Student-Newman-Keuls post-hoc comparison. Data satisfied the requirements for parametric testing in all cases except for the analysis of 5R1 enzyme activity levels, in which case nonparametric one-way ANOVA on ranks was used. Differences between vehicle and L685,273 groups were determined by unpaired Sudent t-test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
R Isoforms During the Initiation of SpermatogenesisTestis weights Testis weights in 10-d.o. (prepubertal) rats were 35.2 mg and increased gradually to reach 1694 mg in adult rats at 120 d.o. (data not shown).
Gonadotropins Serum LH levels were 0.33 ng/ml in 10-d.o. (prepubertal) rats, rising significantly by 20 d.o. (Fig. 1A). A broad peak of LH was observed between Days 20 and 60; however, these values were not different from 120-d.o. (adult) levels. Serum FSH levels rose significantly between 10 and 20 d.o. (Fig. 1B). A broad peak of circulating FSH was observed between Days 20 and 60 before the levels declined at later ages.
|
Testicular androgens Testicular testosterone levels were 21 ng/g in 10-d.o. (prepubertal) rats (Fig. 2A). Values remained around this level between Days 20 and 40 until a marked increase at Day 60, when adult levels were achieved.
|
Testicular DHT (Fig. 2B) and Adiol (Fig. 2C) showed an inverse relationship to testicular testosterone, in that both 5-reduced steroids were very low in the 10-d.o. testis, rising significantly by 20 d.o. A broad peak was observed in 5-reduced androgen levels between Days 20 and 60 before the concentrations declined to reach the low levels seen at Days 80–160.
5R isoenzyme expression and activity levels
Very low but detectable levels of 5R1 mRNA and activity were found in the 10-d.o. rat testis (Fig. 3, A and B). A significant rise in 5R1 mRNA expression by 20 d.o. (Fig. 3A) was coincident with a marked increase in 5R1 activity levels at this time (Fig. 3B). The 5R1 mRNA was significantly greater in the pubertal testis at 20 and 30 d.o. compared to testis at 10 d.o. (prepubertal) or 120 d.o. (adult) (Fig. 3A). A rapid decrease in 5R1 mRNA expression occurred between Days 30 and 35, such that the low adult levels of expression were seen in the testis from 35 d.o. onward. The peak of 5R1 mRNA (Fig. 3A) was coincident with a peak in 5R1 enzyme activity levels (Fig. 3B) between 20 and 35 d.o., followed by a marked decline to reach adult levels. Note that the drop in 5R1 mRNA expression between 30 and 35 d.o. (Fig. 3A) appeared to result in a drop in 5R1 activity levels between 35 and 40 d.o. (Fig. 3B).
|
Like 5R1, 5R2 mRNA expression (Fig. 3C) and activity levels (Fig. 3D) were very low in the 10-d.o. (prepubertal) testis. A significant rise in 5R2 activity levels (Fig. 3D) was seen after 20 and 25 d.o.; however, this was not accompanied by a significant rise in 5R2 mRNA (Fig. 3C), which rose 2-fold from 10 to 20 d.o. The 5R2 mRNA was significantly higher than the prepubertal (10 d.o.) testis from 35 d.o. onward. The 5R2 enzyme activity levels peaked between Days 20 and 40 and then declined to reach the very low levels seen in the adult (Fig. 3D).
The production of 5-reduced metabolites (i.e., the ratio of the 5-reduced metabolites to the substrate testosterone) as an estimation of testicular 5-reductase activity levels was compared to the activity of each 5R isoenzyme (Fig. 3E). Like 5R isoenzyme activity levels, the production of 5-reduced metabolites from testosterone was highest between Days 20 and 40, being significantly greater than the levels in the 10-d.o. (prepubertal) and 120-d.o. (adult) testis.
Comparison of 5R Isoenzyme Activity Levels with Testicular Androgens
To relate the changes in the concentration of testicular 5R1 and 5R2 enzyme activity levels with the levels of testosterone and DHT in the testis, the data were expressed as a percentage of that found in the prepubertal (10-d.o.) testis (Fig. 4), in which the levels of 5R isoenzymes and testicular androgens were lowest. Between Days 10 and 20, parallel increases occurred in the testicular concentration of DHT as well as the 5R1 and 5R2 activity levels. The pubertal peak in 5R1 and 5R2 activity levels between Days 20 and 40 was coincident with a peak in DHT. At this time, testicular testosterone levels were very low. The decline in testicular levels of DHT after 40 d.o. more closely followed 5R1 than 5R2 activity. After 40 d.o., testicular testosterone levels markedly increased.
|
The timing of the initiation of the first spermatogenic cycle (or first wave of spermatogenesis) was compared to isoenzyme activity and androgen levels (Fig. 4). Entry into meiosis occurs after Day 15 in the rat [25], and one cycle is completed at approximately Day 44 [26]. Thus, the timing of the first wave of spermatogenesis coincided with the peak in testicular DHT and 5R isoenzyme activity levels yet with low levels of testicular testosterone.
Effects of L685,273 During the Initiation of Spermatogenesis
Organ weights Acute (6-day) administration of L685,273 to rats at 20, 30, and 40 d.o. did not change testis weights but did cause significant decreases in ventral prostate weights (Table 1).
|
Serum Gonadotropins and Testicular Androgens
Administration of L685,273 did not significantly alter serum LH or FSH concentrations at any age (Table 2). However, testicular testosterone levels were significantly elevated in L685,273-treated rats at all ages, being 5- to 9-fold higher than in the corresponding vehicle-treated rats (Table 2). Testicular 5-reduced androgens (DHT and Adiol) were suppressed by L685,273 only in the 36-d.o. age group (Table 2) but were unchanged at the other ages. Although the absolute levels of 5-reduced androgens were reduced by L685,273 only in the 36-d.o. group, the production of 5-reduced metabolites from testosterone (i.e., the ratio of DHT + Adiol to testosterone) was significantly reduced to 14%–25% of that of vehicle-treated rats in all age groups (Table 2).
|
Spermatogenesis The impact of L685,273 on spermatogenesis was assessed in 36-d.o. rats that had received either vehicle or L685,273 in the preceding 6 days. No significant changes were seen in any germ cell type assessed, from early pachytene spermatocytes in stages I–III through to elongating spermatids in stage XIV, which were the most mature germ cell type at this age (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-reduced androgens DHT and Adiol predominate during puberty and that testosterone concentrations are comparatively low at this time [3]. These findings are supported in the present study. It is also established that immature testes have higher levels of 5R enzyme activity than mature testes [2, 4, 14]. However, the contribution of the 5R1 and 5R2 isoenzymes to pubertal androgen metabolism has been unclear, because many of the studies concerning testicular 5R were performed before the identification of 5R2. Since the discovery of the two 5R isoenzymes, the literature has contained a discrepancy as to which isoenzyme predominates in the testis, with some studies suggesting a predominance of 5R2 [2, 4, 14] and others suggesting that 5R1 is the major isoenzyme present [16, 21]. The present study used more sensitive methods to assess and quantify specific isoenzyme mRNA expression and enzyme activity levels to clarify some of the discrepancies in the literature and to investigate the developmental regulation of 5R1 and 5R2. The present study 1) demonstrates that both 5R1 and 5R2 mRNAs and activity are present in the postnatal testis, with 5R1 activity levels being higher than 5R2; 2) describes the relationship between mRNA and activity levels of each isoenzyme during postnatal development; 3) compares the expression of each isoenzyme with testicular androgen and circulating gonadotropin levels during postnatal development; and 4) acutely administers a competitive 5R inhibitor at key stages of postnatal development.
The pubertal peak in the testicular production of 5-reduced androgens between Days 20 and 40 coincided with increased transcription of both 5R1 and 5R2 genes and subsequent increases in activity levels of both isoenzymes. Although 5R2 activity levels were approximately 10-fold lower than 5R1 between Days 20 and 40 as measured by specific isoenzyme assay, 5R2 likely is at least equally important for the pubertal rise in 5-reduced metabolites. This is because the pubertal testis contains testosterone concentrations in the nanomolar range (30–120 nM in the present study), which is in the range of the Km (75 nM) for 5R2 [10]. Interestingly, our kinetic analysis of 5R isoenzymes in the 30-d.o. rat testis suggested that although 5R1 activity levels were 13-fold those of 5R2, the Vmax:Km ratio (an indication of the potential in vivo isoenzyme activity) of the two isoenzymes suggested that both would contribute equally to the 5-reduction of testosterone in the pubertal testis [21]. Although we believe 5R1 and 5R2 likely are of equal importance between 20 and 40 d.o., after this time 5R1 likely is the predominant isoenzyme involved in testicular testosterone metabolism, because testosterone levels increase to micromolar concentrations at approximately Day 60 (1.2 µM in the present study), which is comparable to the Km value (1.02 µM) for 5R1 [10]. Thus, we conclude that the peak of 5-reduced metabolites between Days 20 and 40 in the rat testis is caused by increases in the expression of both 5R1 and 5R2 isoenzymes whereas the 5R1 isoenzyme likely is more important for 5-reduced androgen biosynthesis in the postpubertal testis.
In general, the pubertal increase and postpubertal decrease in 5R isoenzyme activity levels were consistent with changes in 5R isoenzyme mRNA levels. Two notable discrepancies were observed, however. First, the pubertal increase in 5R2 activity levels at 20 and 25 days was accompanied by a nonsignificant, 2-fold increase in mRNA. Whereas a doubling of mRNA transcripts may be enough to promote significant increases in the activity levels of the 5R2 isoenzyme, it was interesting that we observed a marked increase in mRNA levels after Day 25 yet no further increases in activity levels. This could perhaps be explained by the appearance of cell types in the testis that express 5R2 mRNA (e.g., spermatocytes, spermatids, or pubertal Leydig cells). It should be noted that whereas evidence exists for 5R activity in Leydig cells [16, 27–29] and seminiferous tubules [2, 14, 30], including Sertoli cells and germ cells [15, 31, 32], the localization of 5R2 in the rat testis is unknown. A second unexpected finding was that both 5R1 and 5R2 activity declined to very low levels in the adult, which were comparable to the low levels seen in the prepubertal testis, yet the levels of 5R1 and 5R2 mRNAs remained high in adult rats when compared to the prepubertal testis. This observation suggests maintenance of 5R1 and 5R2 gene transcription yet low levels of protein production/activity. This contention is supported by various observations in the literature. For example, Viger and Robaire [16] showed that 5R1 mRNA expression as analyzed by Northern blotting was detectable in the adult testis yet 5R1 protein as assessed by immunohistochemistry was undetectable in adult testes compared to immature rat testes. Those authors noted that a change in mRNA transcript size from 2.5 to 2.7 kilobases (kb) accompanied the decrease in protein expression, leading them to conclude that the 2.7-kb transcript may not be translated. Because the sequence of the 2.7-kb transcript is unknown, it is not clear which transcript (or transcripts) will be detected by the PCR methods used to measure 5R1 in the current study. Other studies comparing 5R mRNA and protein noted discrepancies in expression patterns as well as tissue-specific differences in 5R mRNA transcript sizes (for review, see [8]), suggesting that 5R isoenzyme activity may be controlled by translational repression and/or posttranslational modification of the enzyme. In the present study, we noted that the postpubertal decline in both 5R1 and 5R2 mRNAs was followed approximately 5 days later by a decline in activity levels. However, this observation likely is explained by the exceptionally long half-lives (>30 h) of the 5R1 and 5R2 proteins [9].
The pubertal onset of 5R isoenzyme gene transcription and protein expression coincided with the onset of the pubertal peak in FSH levels, perhaps suggesting a role for this gonadotropin in stimulating 5R. This contention is consistent with our previous observations in which we showed that FSH can stimulate 5R1 and 5R2 activity levels in adult rats in which gonadotropins have been manipulated [33] and with earlier studies that showed administration of FSH to hypophysectomized rats stimulated testicular 5R activity [34]. We and others have also reported that 5R1 may be negatively regulated by testosterone [11, 33], which may partially explain the peak in 5R1 isoenzyme mRNA and activity levels at a time when testicular testosterone levels are lowest.
The first wave of meiotic and postmeiotic germ cell development in the rat occurs after Sertoli cell division has ceased after Day 15, and one cycle is complete by approximately Day 44, with the release of the first mature spermatids from the testis [25, 26]. The present study shows that increases in the transcription of both 5R isoenzyme genes, with subsequent increases in isoenzyme activity levels and a predominance of 5-reduced androgens in the testis, coincide with initiation of the first spermatogenic cycle. Because spermatogenesis can be initiated by administration of either testosterone or DHT [1], it is reasonable to speculate that the increase in the potent androgen DHT, in the presence of low testosterone levels, may be required for the onset of spermatogenesis. A previous study [35] chronically administered the 5R-inhibitor finasteride to rats from birth to 4–7 wk of age and showed that whereas DHT levels were modestly decreased, testicular testosterone levels were markedly elevated, presumably because of the effects on pituitary gonadotrophins [35], and that no effects on spermatogenesis were seen.
We thus chose to utilize an acute model of 5R-inhibitor administration to limit effects on the pituitary and to prevent rises in LH. Although changes in LH or FSH were not seen using this acute administration model, testicular testosterone was significantly elevated. The reasons for this elevation of testosterone levels are unclear but could include 1) possible effects on LH pulsatility [36], which may influence testosterone production; 2) effects on the Leydig cells to perhaps change their maturational status and/or ability to produce testosterone [37]; and/or 3) an accumulation of substrate when high 5R activity is suppressed. Although L685,273 was clearly effective in suppressing the conversion of testosterone to 5-reduced metabolites, the marked increase in substrate concentration meant that reductions in the absolute levels of 5-reduced metabolites were not seen. Thus, the net effect was to essentially elevate the total amount of androgens within the testis; hence, it is not surprising that no effects on testis weight or spermatogenesis were seen. Recently, a double 5R1 and 5R2 knockout mouse model was used to test the involvement of these isoenzymes in male gonadal pre- and postnatal development [5]. Similar to finasteride and L685,273 treatment, this transgenic mouse study also showed that in the absence of 5R, testosterone levels were elevated, which were apparently responsible for the maintenance of male virilization and the onset of fertility [5]. Taking together the results from the present study, previous 5R-inhibitor studies [35], and the transgenic mice experiments [5], it is reasonable to conclude that the pubertal peak of DHT normally serves to amplify androgen action on spermatogenesis in the face of low testicular testosterone, but if testosterone levels are elevated, as is the case in the absence of 5R, then testosterone can overcome its weaker androgenic potency and act directly to initiate spermatogenesis.
In summary, the present study has shown that the increased production of 5-reduced metabolites in the pubertal testis is caused by an increase in mRNA and activity levels of both 5R1 and 5R2 isoenzymes in the testis. The data support the contention that both 5R isoenzymes would contribute to the high levels of DHT and Adiol in the pubertal testis but that the 5R1 isoenzyme may be relatively more important in the postpubertal testis when testosterone levels are higher. The peak of 5R isoenzyme expression and activity levels, as well as the peak in the levels of 5-reduced metabolites, coincided with the timing of the first wave of spermatogenesis. When the role of the pubertal peak of DHT in the onset of spermatogenesis was evaluated using the 5R-inhibitor L685,273, then, like chronic 5R-inhibitor administration and genetic ablation of 5R isoenzymes, compensatory increases in testosterone were seen. Therefore, 5R-inhibitor administration to immature rats cannot be used to investigate whether DHT formation, in the presence of the normally low pubertal testosterone concentrations, is required for the androgen-dependent initiation of spermatogenesis. Nevertheless, acute administration of L685,273 to pubertal rats caused a 75%–86% decrease in the production of 5-reduced androgens from testosterone, highlighting the importance of 5R isoenzymes in pubertal 5-reduced androgen biosynthesis. We conclude that increased transcription of both the 5R1 and 5R2 isoenzymes is responsible for the peak in 5-reduced metabolites in immature rats, and we suggest that the role of DHT during puberty is to amplify the normally low levels of testosterone in the testis at this time.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Liza O'Donnell, Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton 3168, Australia. FAX: 61 03 9594 6125; liza.odonnell{at}med.monash.edu.au
Received: 7 July 2002.
First decision: 12 August 2002.
Accepted: 2 December 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-Reduction of testosterone in vitro by rat seminiferous tubules and whole testes at different stages of development. Endocrinology 1973 93:253-255[Medline]-reductase enzymes. Endocrinology 2001 142:4652-4662-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Mol Cell Endocrinol 1992 88:15-22[CrossRef][Medline]-reductase: two genes/two enzymes. Annu Rev Biochem 1994 63:25-61[Medline]-reductase isozymes. J Biol Chem 1993 268:17404-17412-reductase isozymes. Evidence for distinct physiological functions. J Biol Chem 1992 267:19548-19554-reductase cDNA: cloning, expression and regulation by pituitary implants and dihydrotestosterone. Biochim Biophys Acta 1996 1305:139-144[Medline]-reductase may be mediated via insulin-like growth factor-I. Endocrinology 1993 133:447-451[Abstract]-reductase activity in genital skin fibroblasts. Mol Cell Endocrinol 1995 107:99-104[CrossRef][Medline]-dihydrotestosterone and other 5-reduced metabolites of 3H-testosterone by the seminiferous tubules and interstitial tissue from immature and mature rat testes. Endocrinology 1973 92:41-47[Medline]-reductase and 3-hydroxysteroid dehydrogenase in the seminiferous tubule of the rat testis. Endocrinology 1975 96:879-889-reductase messenger ribonucleic acid levels and immunocytochemical localization of the type 1 protein in the rat testis during postnatal development. Endocrinology 1995 136:5409-5415[Abstract]-reductase isozyme expression. J Clin Invest 1993 92:903-910-reductase isozyme genes in epididymis, testis, and liver of the adult rat. J Androl 1997 18:372-377-reductase inhibitor. J Androl 1999 20:109-117-reductase activity impairs the testosterone-dependent restoration of spermiogenesis in adult rats. Endocrinology 1996 137:2703-2710[Abstract]-reductase type 2 activity in the presence of 5-reductase type 1 activity in rat testis. J Steroid Biochem Mol Biol 2000 75:75-82[CrossRef][Medline]
4-5-reductase in immature rat testes. Endocrinology 1978 102:918-925[Abstract]-reductase activity in cultured immature Leydig cells by human chorionic gonadotropin. J Steroid Biochem 1990 35:715-721[CrossRef][Medline]-androstan-3-17ß-14C. Endocrinology 1972 90:618-623[Medline]-reductase isoenzymes in human tissues. Acta Anat (Basel) 1996 156:241-252[Medline]-reductase type 1 and type 2 activity in rat testis. In: 11th International Congress of Endocrinology; 2000; Sydney, Australia. Abstract P197 -reductase. J Steroid Biochem 1975 6:329-335[CrossRef][Medline]-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology 1989 125:2434-2438[Abstract]-reductase deficiency and decreased dihydrotestosterone production. J Clin Endocrinol Metab 1994 78:916-921[Abstract]This article has been cited by other articles:
|
|
 |
|
  H.-G. Li, X.-F. Ding, A.-H. Liao, X.-B. Kong, and C.-L. Xiong Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility Mol. Hum. Reprod., May 1, 2007; 13(5): 299 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Sluka, L O'Donnell, J R Bartles, and P G Stanton FSH regulates the formation of adherens junctions and ectoplasmic specialisations between rat Sertoli cells in vitro and in vivo. J. Endocrinol., May 1, 2006; 189(2): 381 - 395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wistuba, C. M. Luetjens, R. Wesselmann, E. Nieschlag, M. Simoni, and S. Schlatt Meiosis in Autologous Ectopic Transplants of Immature Testicular Tissue Grafted to Callithrix jacchus Biol Reprod, April 1, 2006; 74(4): 706 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Dufresne, N. St-Pierre, R. S. Viger, L. Hermo, and D. G. Cyr Characterization of a Novel Rat Epididymal Cell Line to Study Epididymal Function Endocrinology, November 1, 2005; 146(11): 4710 - 4720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Obermann, I. Raabe, M. Balvers, B. Brunswig, W. Schulze, and C. Kirchhoff Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation Mol. Hum. Reprod., January 1, 2005; 11(1): 53 - 64. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||
Significant difference (P < 0.05) compared to 120-d.o. (adult) rats
