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Roles of 11β-Hydroxysteroid Dehydrogenase in Fish Spermatogenesis

Roles of 11β-Hydroxysteroid Dehydrogenase in Fish Spermatogenesis In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17α,20β-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11β-hydroxysteroid dehydrogenase short form (e11β-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11β-HSDsf in testis were induced by DHP. The recombinant e11β-HSDsf had 11β-dehydrogenase activity, metabolizing cortisol to cortisone, and 11β-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11β-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11β-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11β-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol. SPERMATOGENESIS IS DIVIDED into three major phases: spermatogonial proliferation, meiosis, and spermiogenesis. The sequential actions of various hormones and factors are involved in the control of the spermatogenesis. The Japanese eel (Anguilla japonica) has a unique spermatogenic pattern. Under aquaculture conditions, the male Japanese eel has an immature testis containing only nonproliferated type A and early type B spermatogonia (1), and the testis does not develop further without hormonal treatment. A single injection of human chorionic gonadotropin (hCG) stimulates complete spermatogenesis from spermatogonial proliferation through spermiogenesis (1). Spermatogenesis can also be induced by 11-ketotestosterone (11-KT), which is a major androgen in teleosts, in vitro using an organ culture system (2). In teleosts, it has been generally accepted that the main action of progestins in reproduction is the induction of final maturation of gametes. 17α,20β-Dihydroxy-4-pregnen-3-one (DHP) is a major teleost progestin and is related to the regulation of sperm maturation in the Japanese eel (3). In several fishes, DHP increases dramatically in blood during final maturation of males and induces the acquisition of sperm motility (4–7). Interestingly, serum DHP levels show a small peak early in the spermatogenic process in Japanese huchen (8). Recently, it has been found that DHP has the ability to induce the initiation of meiosis in male germ cells, using a testis organ culture system for the Japanese eel (9), suggesting that DHP plays crucial roles in early spermatogenesis. To elucidate the molecular mechanism underlying the action of DHP in early spermatogenesis, we carried out cDNA subtraction using testicular fragments cultured with or without DHP. We identified a factor whose expression was induced by DHP treatment and examined its function in eel spermatogenesis. Materials and Methods Testicular organ culture technique Testicular organ culture was performed as described previously (2) with minor modifications. Briefly, freshly removed testes were cut into pieces of 1 × 1 × 0.5 mm3 and placed on floats of 1% agarose covered with a nitrocellulose membrane in 24-well plastic tissue culture dishes. The basal medium consisted of Leibovitz L-15 medium supplemented with 1.7 mm proline, 0.1 mm aspartic acid, 0.1 mm glutamic acid, 0.5% BSA, 1 mg/liter bovine insulin, and 10 mm HEPES adjusted to pH 7.4. cDNA subtraction The testicular fragments were cultured for 6 d with or without DHP at a concentration of 100 ng/ml. Total RNA was extracted from the cultured testicular fragments by the acid guanidium isothiocyanate-phenol-chloroform extraction method using Sepasol-RNA I Super (Nacalai Tesque, Kyoto, Japan). Poly (A)+ RNA was subsequently isolated from total RNA with Oligotex-dT-30 (Takara, Shiga, Japan). The cDNA subtraction was carried out using Clontech PCR-Select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA). The cDNA fragments encoding only differentially expressed genes were amplified exponentially using suppression PCR. The amplified fragments were subcloned into a pGEM-T Easy Vector (Promega Corp., Madison, WI). Screening and cloning of full-length cDNA clone cDNA fragments obtained from cDNA subtraction were labeled with DIG-dUTP using PCR DIG Labeling Mixplus (Roche, Mannheim, Germany) and used as probes to screen a λZAPII cDNA library constructed from oligo(dT) primed mRNA extracted from testes of eels on d 12 after hCG injection. The positive clone obtained from the library screening was sequenced by the dideoxy chain termination method using the Dual CyDye Terminator sequencing kit (Amersham Biosciences, Piscataway, NJ). Sequence determination was performed on Long-Read Tower DNA sequencer (Amersham Biosciences). The homology search of the deduced amino acid sequence of the obtained cDNA was carried out using the FASTA in the DNA Data Bank of Japan web site (http://www.ddbj.nig.ac.jp/search/fasta-j.html). The deduced amino acid sequences were aligned using the clustal W (10) in the DNA Data Bank of Japan web site. Northern blot analysis Total RNA was extracted using Sepasol-RNA I super from the testicular fragments cultured for 6 d with the following steroids: 1, 10, or 100 ng/ml DHP; 10 ng/ml 11-KT, or 1 ng/ml estradiol-17β (E2). Testicular fragments before culture and testicular fragments cultured without steroids were used as initial control and control, respectively. After denaturing at 70 C for 10 min, 1 μg poly (A)+ RNA was electrophoresed on a 1% agarose gel containing 16% formaldehyde and then transferred onto a nylon membrane (Hybond-N+; Amersham Biosciences). The membrane was baked at 75 C for 2 h. The cDNA fragments obtained from cDNA subtraction were labeled with DIG-dUTP PCR DIG probe synthesis kit (Roche) and served as probes. The cDNA fragment encoding Japanese eel elongation factor 1 (EF1) was also labeled to use as internal standard. The membrane was prehybridized in Dig Easy Hyb (Roche) at 65 C for 3 h. After overnight hybridization at 65 C with the DIG-labeled probe in Dig Easy Hyb, the membrane was washed, immunostained with an antibody against DIG, and analyzed using a LAS-1000mini (Fujifilm, Tokyo, Japan). RT-PCR After DNase I treatment, poly (A)+-RNA was prepared from total RNA extracted from testicular fragments cultured as described above. To examine expression of eel 11β-hydroxysteroid dehydrogenase short form (e11β-HSDsf) cDNA isolated in the present study and eel 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) cDNA reported in a previous study (11) by RT-PCR using specific primers for each 11β-HSD, cDNA was synthesized from 1 μg poly (A)+ RNA primed with oligo(dT) using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The primers for e11β-HSDsf were 5′-GGGTGTGATGTCTGTGTTTG-3′ (nucleotide position 54–73) and 5′-TTGCTGGATTGGCCTGTCTT-3′ (nucleotide position 823–842). The primers for eel 11β-HSD2 were 5′-CACCGTTGTTTGTGAGTCAG-3′ (nucleotide position −57 to −38) and 5′-GCTGCTGGAAATGTTGTGAC-3′ (nucleotide position 778–797). EF1 transcripts were used as the internal standard. The PCR cycling parameters were as follows: 30 cycles of 94 C for 30 sec, 59 C for 30 sec, and 72 C for 60 sec. The negative control contained no template. The PCR products were resolved by electrophoresis on a 1.5% agarose gel, which was then stained with ethidium bromide. Cell-free protein synthesis To amplify the cDNA fragment encoding the open reading frame (ORF) of e11β-HSDsf by RT-PCR, the primers with SpeI site, 5′-TTACTAGTATGATTCCAAATGCCATTG-3′ and 5′-TTACTAGTTCCATCGAACTACTTATC-3′ were used. The amplified fragments were subcloned into a pGEM-T Easy vector, and the nucleotide sequence was confirmed. After digestion with SpeI, the digested fragments were subcloned into pEU3b expression vector (12). mRNA was prepared by in vitro transcription with SP6 RNA polymerase (Promega), and cell-free protein synthesis was performed with wheat germ extract as previously reported (13). Analysis of enzymatic activity of recombinant e11β-HSDsf Analysis of 11β-dehydrogenase activity of recombinant e11β-HSDsf was performed as in a previous study (14). Thirty microliters of resultant solution, in which recombinant e11β-HSDsf was produced with wheat germ cell-free protein synthesis system, were mixed with 470 μl incubation buffer [10 mm Tris, 100 mm KCl, 0.5 mm NAD+, pH 7.4] containing 1 μm 11β-hydroxytestosterone (11β-OHT) or cortisol at final concentration as substrates and then incubated for 3 h at 25 C. After incubation, steroids were extracted twice with 5 vol diethyl ether. The extract was dried and dissolved in the assay buffer (50 mm Tris-HCl, 50 mm NaCl, 20 mm diethylene triamine pentaacetic acid, 0.5 g/liter NaN3, 0.1 ml/liter Tween 20, and 0.5 g/liter BSA, pH 7.8) to measure the levels of 11-KT and cortisone using a time-resolved fluoroimmunoassay (TR-FIA) according to the methods reported previously (15). Cross-reactivity of the antibody against 11-KT to 11β-OHT was 1.7%, and that of the antibody against cortisone to cortisol was 3.4%. The minimal detection thresholds were 0.006 ng/ml for 11-KT and cortisone. 11-Oxo-reductase activity was determined in the same way as the 11β-dehydrogenase activity except that the incubation buffer consisted of 10 mm phosphate buffer (pH 7.4) containing 50 mm MgCl2, 0.5 mm NADPH and 1 μm cortisone as substrate. Cross-reactivity of the antibody against cortisol to cortisone was 4.0%. The minimal detection threshold was 0.024 ng/ml for cortisol. After measurements, the conversion rate was calculated and the value of cross-reactivity was subtracted. Results are expressed as means ± sem of three replicates. Data analysis was carried out using one-way ANOVA followed by unpaired t test. Significance was accepted at P < 0.05. Analysis of enzymatic activity of testicular fragments To assess the enzymatic activity of eel testis, testicular fragments were cultured for 5 d with or without 100 ng/ml DHP. Thereafter, 50 mg of the cultured fragments were transferred into 1 ml eel Ringer (150 mm NaCl, 3 mm KCl, MgCl2, 5 mm CaCl2, 10 mm HEPES, pH 7.4) containing 100 ng/ml 11β-OHT, cortisol, or cortisone as substrates. DHP (100 ng/ml) was added to the Ringer of some treatments. After incubation for 18 h at 20 C, concentrations of each steroid in the Ringer were measured using TR-FIA as described above. Results are expressed as means ± sem of five replicates. Data analysis was carried out using one-way ANOVA followed by Tukey’s test. Significance was accepted at P < 0.05. Effects of cortisol and cortisone on eel spermatogenesis in vitro Testicular fragments were cultured for 6 d with or without 11-KT (10 ng/ml). Cortisol or cortisone (0.01, 0.1, 1, 10, or 100 ng/ml) was further added to each well at d 0 and 3. Testicular fragments cultured without steroids were used as control. To detect cell division, germ cells were labeled with 5-bromo-2-deoxyuridine (BrdU) according to the manufacturer’s instructions (Amersham Bioscience, Little Chalfont, UK); testicular fragments were incubated with BrdU (1 μl/well) for the last 18 h of culture. The cultured fragments were fixed in Bouin’s solution, embedded in paraffin, and cut at 5 μm thickness. The sections were stained immunohistochemically using an antibody against BrdU and then counterstained with Delafield’s hematoxylin. The number of immunolabeled germ cells was counted and expressed as a percentage of the total germ cells. Results are expressed as means ± sem of five replicates. Data analysis was carried out using one-way ANOVA followed by Dunnett’s test. Significance was accepted at P < 0.05. Effects of cortisol on 11-KT production in testis Twenty milligrams of freshly removed eel testicular fragments were transferred into 400 μl eel Ringer. The fragments were incubated with or without cortisol (0.01, 0.1, 1, 10, or 100 ng/ml) and incubated with hCG (0.1 IU/ml) as positive control. After incubation for 18 h at 20 C, concentrations of 11-KT in the Ringer were measured using TR-FIA as described above. Results are expressed as means ± sem of five replicates. Data analysis was carried out using one-way ANOVA followed by Tukey’s test. Significance was accepted at P < 0.05. Results cDNA subtraction and cloning of full-length cDNA To clone the full-length cDNA of the cDNA fragments obtained from cDNA subtraction, a cDNA library was screened and a positive clone was obtained. The clone appeared to include the complete ORF based on the alignments with other fish 11β-HSDs, and the putative protein consisted of 417 amino acids. The deduced amino acid sequence of the positive clone corresponded closely to eel 11β-HSD2 reported previously (11). However, the deduced amino acid sequence of the positive clone had an additional 28 amino acids at the N terminus, which is not detected in eel 11β-HSD2, and 47 amino acids located centrally in eel 11β-HSD2 were absent. The amino acid sequence of the clone obtained in the present study was more similar to those of other fish 11β-HSDs than the eel 11β-HSD2 reported previously (Fig. 1). Therefore, the cDNA obtained in this study was designated as Japanese eel 11β-HSD short form (e11β-HSDsf). Fig. 1 Open in new tabDownload slide Alignments of the deduced amino acid sequence of e11β-HSDsf isolated in the present study with that of fish 11β-HSDs. GenBank accession nos. are AB252646 for e11β-HSDsf, AB061225 for Japanese eel, AB104415 for rainbow trout, and AY190043 for Nile tilapia. Fig. 1 Open in new tabDownload slide Alignments of the deduced amino acid sequence of e11β-HSDsf isolated in the present study with that of fish 11β-HSDs. GenBank accession nos. are AB252646 for e11β-HSDsf, AB061225 for Japanese eel, AB104415 for rainbow trout, and AY190043 for Nile tilapia. Transcripts of eel 11β-HSD A single transcript of about 3.8 kb in length was detected in all samples except the initial control by Northern blot analysis using a probe for eel 11β-HSDs. The transcript levels were correlated to the concentration of added DHP. The 11β-HSD transcript levels in testicular fragments cultured with E2 were not different from control, whereas increased e11β-HSDsf expression was observed after culture with 11-KT (Fig. 2). Fig. 2 Open in new tabDownload slide Northern blot analysis using probe for eel 11β-HSDs (A) and results of RT-PCR using specific primers for e11β-HSDsf and eel 11β-HSD2 (B). Testicular fragments were cultured with 1, 10, or 100 ng/ml DHP; 10 ng/ml 11-KT; or 1 ng/ml E2 for 6 d. C, Control sample cultured without steroids; IC, initial control sample taken before culture; NC, negative control including no template. EF1 transcripts were used as internal standard. Fig. 2 Open in new tabDownload slide Northern blot analysis using probe for eel 11β-HSDs (A) and results of RT-PCR using specific primers for e11β-HSDsf and eel 11β-HSD2 (B). Testicular fragments were cultured with 1, 10, or 100 ng/ml DHP; 10 ng/ml 11-KT; or 1 ng/ml E2 for 6 d. C, Control sample cultured without steroids; IC, initial control sample taken before culture; NC, negative control including no template. EF1 transcripts were used as internal standard. Changes in transcript levels of e11β-HSDsf assessed by RT-PCR showed a similar pattern to the results of the Northern blot analysis. In contrast, eel 11β-HSD2 transcript was slightly detectable only in testicular fragments cultured with 100 ng/ml DHP (Fig. 2). Enzymatic activity of recombinant e11β-HSDsf The recombinant e11β-HSDsf converted 11β-OHT to 11-KT (average 85.2%; P = 0.0013; Fig. 3A) and cortisol to cortisone (average 44.3%; P = 0.00032; Fig. 3B) with high efficiency. In contrast, the conversion of cortisone to cortisol was negligible (average 3.9%; P = 0.052; Fig. 3C). Fig. 3 Open in new tabDownload slide Enzymatic activity of recombinant e11β-HSDsf. A, Conversion rate of 11β-OHT to 11KT; B, conversion rate of cortisol to cortisone; C, conversion rate of cortisone to cortisol. HSD, Wheat germ cell extract with mRNA synthesis from pEU3b vector including e11β-HSDsf cDNA; pEU, wheat germ cell extract with mRNA synthesis from pEU3b vector including no e11β-HSDsf cDNA. Asterisks indicate values significantly different from pEU3b vector including no e11β-HSDsf cDNA; P < 0.05. Fig. 3 Open in new tabDownload slide Enzymatic activity of recombinant e11β-HSDsf. A, Conversion rate of 11β-OHT to 11KT; B, conversion rate of cortisol to cortisone; C, conversion rate of cortisone to cortisol. HSD, Wheat germ cell extract with mRNA synthesis from pEU3b vector including e11β-HSDsf cDNA; pEU, wheat germ cell extract with mRNA synthesis from pEU3b vector including no e11β-HSDsf cDNA. Asterisks indicate values significantly different from pEU3b vector including no e11β-HSDsf cDNA; P < 0.05. Enzymatic activity of testicular fragments The conversion of 11β-OHT to 11-KT, and cortisol to cortisone, in the testicular fragments was measurable and the conversion activities were enhanced by DHP treatment (Fig. 4, A and B). The concentrations of cortisol in the incubation medium from testicular fragments incubated with cortisone and/or DHP were slightly higher than those without cortisone (Fig. 4C). Fig. 4 Open in new tabDownload slide Enzymatic activity of testicular fragments cultured with or without DHP. A, Concentrations of 11-KT in incubation medium; B, concentrations of cortisone in incubation medium; C, concentrations of cortisol in incubation medium. Cont, Testicular fragments incubated without steroids; DHP, testicular fragments incubated with DHP; Sub, testicular fragments incubated with 11β-OHT (A), cortisol (B), or cortisone (C) as substrate; DHP+Sub, testicular fragments incubated with DHP and each substrate. Means with different letters are significantly different between groups; P < 0.05. Fig. 4 Open in new tabDownload slide Enzymatic activity of testicular fragments cultured with or without DHP. A, Concentrations of 11-KT in incubation medium; B, concentrations of cortisone in incubation medium; C, concentrations of cortisol in incubation medium. Cont, Testicular fragments incubated without steroids; DHP, testicular fragments incubated with DHP; Sub, testicular fragments incubated with 11β-OHT (A), cortisol (B), or cortisone (C) as substrate; DHP+Sub, testicular fragments incubated with DHP and each substrate. Means with different letters are significantly different between groups; P < 0.05. Effects of cortisol and cortisone on eel spermatogenesis in vitro Results of immunohistochemistry using testicular fragments cultured for 6 d showed that the percentage of BrdU-immunoreactive germ cells to total germ cells was increased by cortisol in a dose-dependent manner. The percentages of immunoreactive germ cells in the fragments cultured with combinations of 0.01–10 ng/ml cortisol and 11-KT were higher than those of the fragments cultured with only 11-KT; however, treatment with 100 ng/ml cortisol inhibited spermatogonial proliferation induced by 11-KT (Fig. 5A). The percentages of immunoreactive germ cells in the fragments cultured with cortisone were slightly but not significantly higher than that of the control (Fig. 5B). There was no difference between the percentage of immunoreactive germ cells in the fragments cultured with cortisone and 11-KT vs. 11-KT alone (Fig. 5B). Fig. 5 Open in new tabDownload slide Effects of cortisol and cortisone at each concentration (0.01, 0.1, 1, 10, or 100 ng/ml) on DNA replication in spermatogonia and spermatogonial proliferation induced by 10 ng/ml 11-KT. Cell division was assessed using incorporation of BrdU. BrdU index is the percentage of the number of germ cells immunoreactive with an antibody against BrdU to the total number of germ cells. C, Testicular fragments cultured without steroids; IC, testicular fragments before culture. Asterisks indicate values significantly different from testicular fragments cultured without steroids and a dagger indicates values significantly different from testicular fragments cultured with 10 ng/ml 11-KT alone; P < 0.05. Fig. 5 Open in new tabDownload slide Effects of cortisol and cortisone at each concentration (0.01, 0.1, 1, 10, or 100 ng/ml) on DNA replication in spermatogonia and spermatogonial proliferation induced by 10 ng/ml 11-KT. Cell division was assessed using incorporation of BrdU. BrdU index is the percentage of the number of germ cells immunoreactive with an antibody against BrdU to the total number of germ cells. C, Testicular fragments cultured without steroids; IC, testicular fragments before culture. Asterisks indicate values significantly different from testicular fragments cultured without steroids and a dagger indicates values significantly different from testicular fragments cultured with 10 ng/ml 11-KT alone; P < 0.05. Effects of cortisol on 11-KT production in testis Levels of 11-KT produced by testicular fragments increased with the concentration of added cortisol. 11-KT production was also induced by hCG (Fig. 6). Fig. 6 Open in new tabDownload slide Effects of cortisol (0.01, 0.1, 1, 10, or 100 ng/ml) and hCG (0.1 IU/ml) on 11-KT production in testicular fragments. C, Testicular fragments cultured without steroids. Means with different letters are significantly different between groups; P < 0.05. Fig. 6 Open in new tabDownload slide Effects of cortisol (0.01, 0.1, 1, 10, or 100 ng/ml) and hCG (0.1 IU/ml) on 11-KT production in testicular fragments. C, Testicular fragments cultured without steroids. Means with different letters are significantly different between groups; P < 0.05. Discussion The involvement of a progestin in early spermatogenesis in fish was first revealed in studies on the Japanese huchen: in vitro, DHP induced the proliferation of spermatogonia (8). In Japanese eel, DHP has been determined to be involved in the regulation of the initiation of meiosis during spermatogenesis (9). Thus, the mechanisms regulating the early stages of spermatogenesis would be better understood by clarifying the molecular events underlying the action of DHP. In the present study, one of the cDNA clones obtained from cDNA subtraction encoded a gene that shares high homology with eel 11β-HSD2 cDNA isolated previously (11). In mammals, two distinct types of 11β-HSD have been identified and characterized. 11β-HSD type 1 is a NADP(H)- dependent enzyme, and acts predominantly as 11-oxo-reductase and rarely as 11β-dehydrogenase, with a relatively low affinity for substrates. 11β-HSD type 2 is a high-affinity, NAD-dependent 11β-dehydrogenase that metabolizes bioactive glucocorticoids (cortisol and corticosterone) to inert 11-keto forms (cortisone and 11-dehydrocorticosterone), thereby protecting mineralocorticoid receptor (MR) from overstimulation by cortisol. Recently, teleost 11β-HSD homologs that are similar to mammalian 11β-HSD type 2 have been identified in rainbow trout (16), tilapia, and Japanese eel (11). In rainbow trout, 11β-HSD showed 11β-dehydrogenase activity, the conversion of cortisol to cortisone and 11β-OHT to 11-KT (16). Japanese eel 11β-HSD2 also metabolized cortisol to cortisone (11). In the present study, Northern blot and RT-PCR analysis showed that the eel 11β-HSD transcripts were induced by DHP. Moreover, e11β-HSDsf displayed 11β-dehydrogenase activity, corresponding to mammalian 11β-HSD2 activity. These results suggest that DHP modulates androgen and glucocorticoid production in eel testis and thereby regulates spermatogenesis. 11β-Dehydrogenase activity of the 11β-HSD is essential for biosynthesis of a major fish androgen, 11-KT. However, 11-KT production via the action of 11β-HSD has been demonstrated only in rainbow trout (16). Although the conversion of 11β-OHT to 11-KT was not confirmed in a previous study, eel 11β-HSD2 was shown to have the 11β-dehydrogenase activity (11), suggesting that eel 11β-HSD2 can convert 11β-OHT to 11-KT. The results of the present study revealed that e11β-HSDsf has the ability to convert 11β-OHT to 11-KT and that immature testes have the ability to produce 11-KT, if the substrate, 11-OHT, is present. These results suggest that 11-KT synthesis in immature testis is arrested earlier in the steroidogenic pathway than the step from 11β-OHT to 11-KT. Although the induction of 11-KT production by DHP in testes cultured without 11β-OHT was not evident, DHP enhanced 11-KT production from 11β-OHT, coincident with the up-regulation of 11β-HSD expression. Our previous study suggested that spermatogonial proliferation is initiated by the action of 11-KT, and thereafter, DHP, which is produced in response to 11-KT, induces meiotic division (9). Hence, the role of DHP in spermatogenesis could be to provide positive feedback control of 11-KT production by increasing 11β-HSD expression, thereby promoting spermatogenic progression. In the present study, although expression of both eel 11β-HSDs was induced by DHP, results of RT-PCR analysis indicate that e11β-HSDsf is the dominant 11β-HSD, suggesting that e11β-HSDsf is the major source of 11β-dehydrogenase activity. However, additional research is needed to characterize the functional differences between the two types of eel 11β-HSDs. Our data further suggest that DHP also modifies glucocorticoid metabolism by up-regulating 11β-HSD expression. In teleosts, cortisol is closely related with the stress response (17), and high levels of circulating cortisol are found after various stressors that inhibit gonadal function (18). Interestingly, a previous in vivo study reported that cortisol administration promoted testicular development during the early stages of spermatogenesis, while inhibiting spermatogenesis during the mature phase in a freshwater fish, Notopterus notopterus (19). However, the effects of normal levels of cortisol on spermatogenesis have rarely been investigated. Therefore, we examined the direct effects of cortisol and cortisone on eel spermatogenesis using an organ culture system. Effects of cortisone on spermatogenesis were not observed. In contrast, cortisol induced DNA replication in spermatogonia, and a potentiating effect of cortisol on the spermatogonial proliferation induced by 11-KT was also revealed. Moreover, our results showed that 11-KT production was induced in testicular fragments incubated with cortisol. The precise mechanisms underlying 11-KT production have not been determined. However, the conversion of cortisol to 11-oxygenated androgens has been suggested in several teleosts (20), raising the possibility that cortisol is metabolized to 11-KT in eel testis. These results suggest that cortisol induces spermatogonial mitosis by increasing 11-KT production and that regulation of 11-KT production involves complex, multiple mechanisms. However, treatment of testis with cortisol at relatively high doses inhibited spermatogonial proliferation induced by 11-KT. In teleosts, a gene corresponding to mammalian 11β-HSD type 1 has never been identified, and it remains unclear whether such a gene is expressed in testis. In the present study, the experiments determining enzymatic activity of testicular fragments showed that the concentrations of cortisol in media after incubations with cortisone were slightly higher than those without cortisone. However, given the cross-reactivity of the antibody against cortisol to cortisone (4%), the conversion of cortisone to cortisol could not be confirmed. In immature eel testis, expression of 21-hydroxylase, a key enzyme for the synthesis of glucocorticoid from cholesterol, was also not detected (21). Therefore, it remains unclear whether cortisol is produced by eel testis. However, cortisol is a principal corticoid and is produced mainly in the interrenal tissue in eel, as in other teleosts (21). Therefore, a possible role of DHP may be to modify cortisol levels in the testis, via up-regulating 11β-HSD, and thereby protecting testicular development from circulating cortisol. The physiological roles of 11β-HSDs in the testis have not been yet defined. In mammals, several deleterious effects of glucocorticoid on the Leydig cells have been demonstrated: inhibition of testosterone biosynthesis, suppression of LH receptor expression, and induction of Leydig cell apoptosis (22–24). Therefore, it has been assumed that 11β-HSD type 1 acts predominantly as an 11β-dehydrogenase in testis and protects testis from glucocorticoid stimulation. However, this assumption was questioned by Leckie et al. (25), who demonstrated that 11β-HSD type 1 acted predominantly as a reductase in rat Leydig cells in vitro. Moreover, it was suggested that the functions of 11β-HSD type 1 depended on the age of the rat (26); the expression of the 11β-HSD type 1 and reductase activity was highest in immature Leydig cells (27). It is possible that glucocorticoids may also play some roles in early spermatogenesis in mammals. Unlike mammalian species, rainbow trout Leydig cells express 11β-HSD type 2 homolog at relatively high levels, and it was also suggested that one role of this 11β-HSD was to protect testis from circulating cortisol in addition to catalyzing 11-KT production (16). Generally in mammals, 11β-HSD type 1 is widely distributed in glucocorticoid target tissues such as liver, adipose tissue, and gonads, whereas 11β-HSD type 2 is expressed predominantly in mineralocorticoid-responsive tissue such as kidney, colon, and placenta. In contrast, the expression of fish 11β-HSD type 2 homolog was found to be relatively widespread in various tissues, similar to mammalian 11β-HSD type 1 (11, 16). In rainbow trout, 11β-HSD was also expressed in Leydig cells and other steroidogenic tissues at relatively high levels (16). However, a recent study demonstrated that 11β-HSD type 2 was present in rat Leydig cells at 1000-fold lower levels than 11β-HSD type 1 (28). Mammalian 11β-HSD type 2 converts cortisol to cortisone and thereby prevents the binding of cortisol to MR and allows selective access of aldosterone to the MR in mineralocorticoid target tissues. Although the MR is also found in rainbow trout (29), aldosterone is believed to be absent in teleosts (30, 31). In rainbow trout, cortisol appears to be a major ligand for the MR (29), but the biological actions of cortisol via MR remain unclear. In teleosts, little is known about the factors regulating 11β-HSD expression, because teleost 11β-HSD genes have only recently been identified. In mammals, several studies suggest that LH suppresses 11β-HSD type 2 expression in granulosa cells while inducing 11β-HSD type 1 expression (32, 33). In rat Leydig cells, LH and epidermal growth factor also up-regulated the 11β-HSD type 1, whereas they decreased the 11β-dehydrogenase activity (34). In male Japanese eel, however, 11β-HSD transcript levels in testes were remarkably increased after hCG injection (11). In rainbow trout, ovarian 11β-HSD mRNA level increased in late vitellogenic stage (16), when the plasma LH level was elevated (35), suggesting that LH may increase 11β-HSD expression. These results suggest differences in the physiological roles of 11β-HSD type 2 between mammalian and teleost species. In conclusion, we identified a cDNA encoding a homolog of mammalian 11β-HSD type 2 from Japanese eel testis. DHP induced 11β-dehydrogenase activity in testis through the up-regulation of 11β-HSD expression, and optimal levels of cortisol induced spermatogonial mitosis through increasing 11-KT production and enhanced testicular development induced by 11-KT. However, excess cortisol inhibits testicular development. These results suggest two possible roles of DHP in eel spermatogenesis: positive feedback control of 11-KT production and the modulation of cortisol levels to protect the testis from circulating cortisol. Acknowledgments We thank Prof. Graham Young, University of Washington, for critical reading of the manuscript, and Dr. Makoto Kusakabe, University of Washington, for discussion. This work was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan and from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government. The sequence reported in this paper has been deposited in the GenBank database [accession no. AB252646 (Japanese e11β-HSDsf)]. Disclosure statement: The authors have nothing to disclose. Abbreviations: BrdU 5-Bromo-2-deoxyuridine DHP 17α,20β-dihydroxy-4-pregnen-3-one E2 estradiol-17β e11β-HSDsf eel 11β-hydroxysteroid dehydrogenase short form EF1 elongation factor 1 hCG human chorionic gonadotropin 11β-HSD 11β-hydroxysteroid dehydrogenase 11-OHT 11β-hydroxytestosterone 11-KT 11-ketotestosterone MR mineralocorticoid receptor NAD+ nicotinamide adenine dinucleotide ORF open reading frame TR-FIA time-resolved fluoroimmunoassay 1 Miura T , Yamauchi K , Nagahama Y , Takahashi H 1991 Induction of spermatogenesis in male Japanese eel, Anguilla japonica, by a single injection of human chorionic gonadotropin . Zool Sci 8 : 63 – 73 WorldCat 2 Miura T , Yamauchi K , Takahashi H , Nagahama Y 1991 Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica) . 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Roles of 11β-Hydroxysteroid Dehydrogenase in Fish Spermatogenesis

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Oxford University Press
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Copyright © 2006 by The Endocrine Society
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0013-7227
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1945-7170
DOI
10.1210/en.2006-0391
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Abstract

In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17α,20β-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11β-hydroxysteroid dehydrogenase short form (e11β-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11β-HSDsf in testis were induced by DHP. The recombinant e11β-HSDsf had 11β-dehydrogenase activity, metabolizing cortisol to cortisone, and 11β-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11β-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11β-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11β-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol. SPERMATOGENESIS IS DIVIDED into three major phases: spermatogonial proliferation, meiosis, and spermiogenesis. The sequential actions of various hormones and factors are involved in the control of the spermatogenesis. The Japanese eel (Anguilla japonica) has a unique spermatogenic pattern. Under aquaculture conditions, the male Japanese eel has an immature testis containing only nonproliferated type A and early type B spermatogonia (1), and the testis does not develop further without hormonal treatment. A single injection of human chorionic gonadotropin (hCG) stimulates complete spermatogenesis from spermatogonial proliferation through spermiogenesis (1). Spermatogenesis can also be induced by 11-ketotestosterone (11-KT), which is a major androgen in teleosts, in vitro using an organ culture system (2). In teleosts, it has been generally accepted that the main action of progestins in reproduction is the induction of final maturation of gametes. 17α,20β-Dihydroxy-4-pregnen-3-one (DHP) is a major teleost progestin and is related to the regulation of sperm maturation in the Japanese eel (3). In several fishes, DHP increases dramatically in blood during final maturation of males and induces the acquisition of sperm motility (4–7). Interestingly, serum DHP levels show a small peak early in the spermatogenic process in Japanese huchen (8). Recently, it has been found that DHP has the ability to induce the initiation of meiosis in male germ cells, using a testis organ culture system for the Japanese eel (9), suggesting that DHP plays crucial roles in early spermatogenesis. To elucidate the molecular mechanism underlying the action of DHP in early spermatogenesis, we carried out cDNA subtraction using testicular fragments cultured with or without DHP. We identified a factor whose expression was induced by DHP treatment and examined its function in eel spermatogenesis. Materials and Methods Testicular organ culture technique Testicular organ culture was performed as described previously (2) with minor modifications. Briefly, freshly removed testes were cut into pieces of 1 × 1 × 0.5 mm3 and placed on floats of 1% agarose covered with a nitrocellulose membrane in 24-well plastic tissue culture dishes. The basal medium consisted of Leibovitz L-15 medium supplemented with 1.7 mm proline, 0.1 mm aspartic acid, 0.1 mm glutamic acid, 0.5% BSA, 1 mg/liter bovine insulin, and 10 mm HEPES adjusted to pH 7.4. cDNA subtraction The testicular fragments were cultured for 6 d with or without DHP at a concentration of 100 ng/ml. Total RNA was extracted from the cultured testicular fragments by the acid guanidium isothiocyanate-phenol-chloroform extraction method using Sepasol-RNA I Super (Nacalai Tesque, Kyoto, Japan). Poly (A)+ RNA was subsequently isolated from total RNA with Oligotex-dT-30 (Takara, Shiga, Japan). The cDNA subtraction was carried out using Clontech PCR-Select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA). The cDNA fragments encoding only differentially expressed genes were amplified exponentially using suppression PCR. The amplified fragments were subcloned into a pGEM-T Easy Vector (Promega Corp., Madison, WI). Screening and cloning of full-length cDNA clone cDNA fragments obtained from cDNA subtraction were labeled with DIG-dUTP using PCR DIG Labeling Mixplus (Roche, Mannheim, Germany) and used as probes to screen a λZAPII cDNA library constructed from oligo(dT) primed mRNA extracted from testes of eels on d 12 after hCG injection. The positive clone obtained from the library screening was sequenced by the dideoxy chain termination method using the Dual CyDye Terminator sequencing kit (Amersham Biosciences, Piscataway, NJ). Sequence determination was performed on Long-Read Tower DNA sequencer (Amersham Biosciences). The homology search of the deduced amino acid sequence of the obtained cDNA was carried out using the FASTA in the DNA Data Bank of Japan web site (http://www.ddbj.nig.ac.jp/search/fasta-j.html). The deduced amino acid sequences were aligned using the clustal W (10) in the DNA Data Bank of Japan web site. Northern blot analysis Total RNA was extracted using Sepasol-RNA I super from the testicular fragments cultured for 6 d with the following steroids: 1, 10, or 100 ng/ml DHP; 10 ng/ml 11-KT, or 1 ng/ml estradiol-17β (E2). Testicular fragments before culture and testicular fragments cultured without steroids were used as initial control and control, respectively. After denaturing at 70 C for 10 min, 1 μg poly (A)+ RNA was electrophoresed on a 1% agarose gel containing 16% formaldehyde and then transferred onto a nylon membrane (Hybond-N+; Amersham Biosciences). The membrane was baked at 75 C for 2 h. The cDNA fragments obtained from cDNA subtraction were labeled with DIG-dUTP PCR DIG probe synthesis kit (Roche) and served as probes. The cDNA fragment encoding Japanese eel elongation factor 1 (EF1) was also labeled to use as internal standard. The membrane was prehybridized in Dig Easy Hyb (Roche) at 65 C for 3 h. After overnight hybridization at 65 C with the DIG-labeled probe in Dig Easy Hyb, the membrane was washed, immunostained with an antibody against DIG, and analyzed using a LAS-1000mini (Fujifilm, Tokyo, Japan). RT-PCR After DNase I treatment, poly (A)+-RNA was prepared from total RNA extracted from testicular fragments cultured as described above. To examine expression of eel 11β-hydroxysteroid dehydrogenase short form (e11β-HSDsf) cDNA isolated in the present study and eel 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) cDNA reported in a previous study (11) by RT-PCR using specific primers for each 11β-HSD, cDNA was synthesized from 1 μg poly (A)+ RNA primed with oligo(dT) using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The primers for e11β-HSDsf were 5′-GGGTGTGATGTCTGTGTTTG-3′ (nucleotide position 54–73) and 5′-TTGCTGGATTGGCCTGTCTT-3′ (nucleotide position 823–842). The primers for eel 11β-HSD2 were 5′-CACCGTTGTTTGTGAGTCAG-3′ (nucleotide position −57 to −38) and 5′-GCTGCTGGAAATGTTGTGAC-3′ (nucleotide position 778–797). EF1 transcripts were used as the internal standard. The PCR cycling parameters were as follows: 30 cycles of 94 C for 30 sec, 59 C for 30 sec, and 72 C for 60 sec. The negative control contained no template. The PCR products were resolved by electrophoresis on a 1.5% agarose gel, which was then stained with ethidium bromide. Cell-free protein synthesis To amplify the cDNA fragment encoding the open reading frame (ORF) of e11β-HSDsf by RT-PCR, the primers with SpeI site, 5′-TTACTAGTATGATTCCAAATGCCATTG-3′ and 5′-TTACTAGTTCCATCGAACTACTTATC-3′ were used. The amplified fragments were subcloned into a pGEM-T Easy vector, and the nucleotide sequence was confirmed. After digestion with SpeI, the digested fragments were subcloned into pEU3b expression vector (12). mRNA was prepared by in vitro transcription with SP6 RNA polymerase (Promega), and cell-free protein synthesis was performed with wheat germ extract as previously reported (13). Analysis of enzymatic activity of recombinant e11β-HSDsf Analysis of 11β-dehydrogenase activity of recombinant e11β-HSDsf was performed as in a previous study (14). Thirty microliters of resultant solution, in which recombinant e11β-HSDsf was produced with wheat germ cell-free protein synthesis system, were mixed with 470 μl incubation buffer [10 mm Tris, 100 mm KCl, 0.5 mm NAD+, pH 7.4] containing 1 μm 11β-hydroxytestosterone (11β-OHT) or cortisol at final concentration as substrates and then incubated for 3 h at 25 C. After incubation, steroids were extracted twice with 5 vol diethyl ether. The extract was dried and dissolved in the assay buffer (50 mm Tris-HCl, 50 mm NaCl, 20 mm diethylene triamine pentaacetic acid, 0.5 g/liter NaN3, 0.1 ml/liter Tween 20, and 0.5 g/liter BSA, pH 7.8) to measure the levels of 11-KT and cortisone using a time-resolved fluoroimmunoassay (TR-FIA) according to the methods reported previously (15). Cross-reactivity of the antibody against 11-KT to 11β-OHT was 1.7%, and that of the antibody against cortisone to cortisol was 3.4%. The minimal detection thresholds were 0.006 ng/ml for 11-KT and cortisone. 11-Oxo-reductase activity was determined in the same way as the 11β-dehydrogenase activity except that the incubation buffer consisted of 10 mm phosphate buffer (pH 7.4) containing 50 mm MgCl2, 0.5 mm NADPH and 1 μm cortisone as substrate. Cross-reactivity of the antibody against cortisol to cortisone was 4.0%. The minimal detection threshold was 0.024 ng/ml for cortisol. After measurements, the conversion rate was calculated and the value of cross-reactivity was subtracted. Results are expressed as means ± sem of three replicates. Data analysis was carried out using one-way ANOVA followed by unpaired t test. Significance was accepted at P < 0.05. Analysis of enzymatic activity of testicular fragments To assess the enzymatic activity of eel testis, testicular fragments were cultured for 5 d with or without 100 ng/ml DHP. Thereafter, 50 mg of the cultured fragments were transferred into 1 ml eel Ringer (150 mm NaCl, 3 mm KCl, MgCl2, 5 mm CaCl2, 10 mm HEPES, pH 7.4) containing 100 ng/ml 11β-OHT, cortisol, or cortisone as substrates. DHP (100 ng/ml) was added to the Ringer of some treatments. After incubation for 18 h at 20 C, concentrations of each steroid in the Ringer were measured using TR-FIA as described above. Results are expressed as means ± sem of five replicates. Data analysis was carried out using one-way ANOVA followed by Tukey’s test. Significance was accepted at P < 0.05. Effects of cortisol and cortisone on eel spermatogenesis in vitro Testicular fragments were cultured for 6 d with or without 11-KT (10 ng/ml). Cortisol or cortisone (0.01, 0.1, 1, 10, or 100 ng/ml) was further added to each well at d 0 and 3. Testicular fragments cultured without steroids were used as control. To detect cell division, germ cells were labeled with 5-bromo-2-deoxyuridine (BrdU) according to the manufacturer’s instructions (Amersham Bioscience, Little Chalfont, UK); testicular fragments were incubated with BrdU (1 μl/well) for the last 18 h of culture. The cultured fragments were fixed in Bouin’s solution, embedded in paraffin, and cut at 5 μm thickness. The sections were stained immunohistochemically using an antibody against BrdU and then counterstained with Delafield’s hematoxylin. The number of immunolabeled germ cells was counted and expressed as a percentage of the total germ cells. Results are expressed as means ± sem of five replicates. Data analysis was carried out using one-way ANOVA followed by Dunnett’s test. Significance was accepted at P < 0.05. Effects of cortisol on 11-KT production in testis Twenty milligrams of freshly removed eel testicular fragments were transferred into 400 μl eel Ringer. The fragments were incubated with or without cortisol (0.01, 0.1, 1, 10, or 100 ng/ml) and incubated with hCG (0.1 IU/ml) as positive control. After incubation for 18 h at 20 C, concentrations of 11-KT in the Ringer were measured using TR-FIA as described above. Results are expressed as means ± sem of five replicates. Data analysis was carried out using one-way ANOVA followed by Tukey’s test. Significance was accepted at P < 0.05. Results cDNA subtraction and cloning of full-length cDNA To clone the full-length cDNA of the cDNA fragments obtained from cDNA subtraction, a cDNA library was screened and a positive clone was obtained. The clone appeared to include the complete ORF based on the alignments with other fish 11β-HSDs, and the putative protein consisted of 417 amino acids. The deduced amino acid sequence of the positive clone corresponded closely to eel 11β-HSD2 reported previously (11). However, the deduced amino acid sequence of the positive clone had an additional 28 amino acids at the N terminus, which is not detected in eel 11β-HSD2, and 47 amino acids located centrally in eel 11β-HSD2 were absent. The amino acid sequence of the clone obtained in the present study was more similar to those of other fish 11β-HSDs than the eel 11β-HSD2 reported previously (Fig. 1). Therefore, the cDNA obtained in this study was designated as Japanese eel 11β-HSD short form (e11β-HSDsf). Fig. 1 Open in new tabDownload slide Alignments of the deduced amino acid sequence of e11β-HSDsf isolated in the present study with that of fish 11β-HSDs. GenBank accession nos. are AB252646 for e11β-HSDsf, AB061225 for Japanese eel, AB104415 for rainbow trout, and AY190043 for Nile tilapia. Fig. 1 Open in new tabDownload slide Alignments of the deduced amino acid sequence of e11β-HSDsf isolated in the present study with that of fish 11β-HSDs. GenBank accession nos. are AB252646 for e11β-HSDsf, AB061225 for Japanese eel, AB104415 for rainbow trout, and AY190043 for Nile tilapia. Transcripts of eel 11β-HSD A single transcript of about 3.8 kb in length was detected in all samples except the initial control by Northern blot analysis using a probe for eel 11β-HSDs. The transcript levels were correlated to the concentration of added DHP. The 11β-HSD transcript levels in testicular fragments cultured with E2 were not different from control, whereas increased e11β-HSDsf expression was observed after culture with 11-KT (Fig. 2). Fig. 2 Open in new tabDownload slide Northern blot analysis using probe for eel 11β-HSDs (A) and results of RT-PCR using specific primers for e11β-HSDsf and eel 11β-HSD2 (B). Testicular fragments were cultured with 1, 10, or 100 ng/ml DHP; 10 ng/ml 11-KT; or 1 ng/ml E2 for 6 d. C, Control sample cultured without steroids; IC, initial control sample taken before culture; NC, negative control including no template. EF1 transcripts were used as internal standard. Fig. 2 Open in new tabDownload slide Northern blot analysis using probe for eel 11β-HSDs (A) and results of RT-PCR using specific primers for e11β-HSDsf and eel 11β-HSD2 (B). Testicular fragments were cultured with 1, 10, or 100 ng/ml DHP; 10 ng/ml 11-KT; or 1 ng/ml E2 for 6 d. C, Control sample cultured without steroids; IC, initial control sample taken before culture; NC, negative control including no template. EF1 transcripts were used as internal standard. Changes in transcript levels of e11β-HSDsf assessed by RT-PCR showed a similar pattern to the results of the Northern blot analysis. In contrast, eel 11β-HSD2 transcript was slightly detectable only in testicular fragments cultured with 100 ng/ml DHP (Fig. 2). Enzymatic activity of recombinant e11β-HSDsf The recombinant e11β-HSDsf converted 11β-OHT to 11-KT (average 85.2%; P = 0.0013; Fig. 3A) and cortisol to cortisone (average 44.3%; P = 0.00032; Fig. 3B) with high efficiency. In contrast, the conversion of cortisone to cortisol was negligible (average 3.9%; P = 0.052; Fig. 3C). Fig. 3 Open in new tabDownload slide Enzymatic activity of recombinant e11β-HSDsf. A, Conversion rate of 11β-OHT to 11KT; B, conversion rate of cortisol to cortisone; C, conversion rate of cortisone to cortisol. HSD, Wheat germ cell extract with mRNA synthesis from pEU3b vector including e11β-HSDsf cDNA; pEU, wheat germ cell extract with mRNA synthesis from pEU3b vector including no e11β-HSDsf cDNA. Asterisks indicate values significantly different from pEU3b vector including no e11β-HSDsf cDNA; P < 0.05. Fig. 3 Open in new tabDownload slide Enzymatic activity of recombinant e11β-HSDsf. A, Conversion rate of 11β-OHT to 11KT; B, conversion rate of cortisol to cortisone; C, conversion rate of cortisone to cortisol. HSD, Wheat germ cell extract with mRNA synthesis from pEU3b vector including e11β-HSDsf cDNA; pEU, wheat germ cell extract with mRNA synthesis from pEU3b vector including no e11β-HSDsf cDNA. Asterisks indicate values significantly different from pEU3b vector including no e11β-HSDsf cDNA; P < 0.05. Enzymatic activity of testicular fragments The conversion of 11β-OHT to 11-KT, and cortisol to cortisone, in the testicular fragments was measurable and the conversion activities were enhanced by DHP treatment (Fig. 4, A and B). The concentrations of cortisol in the incubation medium from testicular fragments incubated with cortisone and/or DHP were slightly higher than those without cortisone (Fig. 4C). Fig. 4 Open in new tabDownload slide Enzymatic activity of testicular fragments cultured with or without DHP. A, Concentrations of 11-KT in incubation medium; B, concentrations of cortisone in incubation medium; C, concentrations of cortisol in incubation medium. Cont, Testicular fragments incubated without steroids; DHP, testicular fragments incubated with DHP; Sub, testicular fragments incubated with 11β-OHT (A), cortisol (B), or cortisone (C) as substrate; DHP+Sub, testicular fragments incubated with DHP and each substrate. Means with different letters are significantly different between groups; P < 0.05. Fig. 4 Open in new tabDownload slide Enzymatic activity of testicular fragments cultured with or without DHP. A, Concentrations of 11-KT in incubation medium; B, concentrations of cortisone in incubation medium; C, concentrations of cortisol in incubation medium. Cont, Testicular fragments incubated without steroids; DHP, testicular fragments incubated with DHP; Sub, testicular fragments incubated with 11β-OHT (A), cortisol (B), or cortisone (C) as substrate; DHP+Sub, testicular fragments incubated with DHP and each substrate. Means with different letters are significantly different between groups; P < 0.05. Effects of cortisol and cortisone on eel spermatogenesis in vitro Results of immunohistochemistry using testicular fragments cultured for 6 d showed that the percentage of BrdU-immunoreactive germ cells to total germ cells was increased by cortisol in a dose-dependent manner. The percentages of immunoreactive germ cells in the fragments cultured with combinations of 0.01–10 ng/ml cortisol and 11-KT were higher than those of the fragments cultured with only 11-KT; however, treatment with 100 ng/ml cortisol inhibited spermatogonial proliferation induced by 11-KT (Fig. 5A). The percentages of immunoreactive germ cells in the fragments cultured with cortisone were slightly but not significantly higher than that of the control (Fig. 5B). There was no difference between the percentage of immunoreactive germ cells in the fragments cultured with cortisone and 11-KT vs. 11-KT alone (Fig. 5B). Fig. 5 Open in new tabDownload slide Effects of cortisol and cortisone at each concentration (0.01, 0.1, 1, 10, or 100 ng/ml) on DNA replication in spermatogonia and spermatogonial proliferation induced by 10 ng/ml 11-KT. Cell division was assessed using incorporation of BrdU. BrdU index is the percentage of the number of germ cells immunoreactive with an antibody against BrdU to the total number of germ cells. C, Testicular fragments cultured without steroids; IC, testicular fragments before culture. Asterisks indicate values significantly different from testicular fragments cultured without steroids and a dagger indicates values significantly different from testicular fragments cultured with 10 ng/ml 11-KT alone; P < 0.05. Fig. 5 Open in new tabDownload slide Effects of cortisol and cortisone at each concentration (0.01, 0.1, 1, 10, or 100 ng/ml) on DNA replication in spermatogonia and spermatogonial proliferation induced by 10 ng/ml 11-KT. Cell division was assessed using incorporation of BrdU. BrdU index is the percentage of the number of germ cells immunoreactive with an antibody against BrdU to the total number of germ cells. C, Testicular fragments cultured without steroids; IC, testicular fragments before culture. Asterisks indicate values significantly different from testicular fragments cultured without steroids and a dagger indicates values significantly different from testicular fragments cultured with 10 ng/ml 11-KT alone; P < 0.05. Effects of cortisol on 11-KT production in testis Levels of 11-KT produced by testicular fragments increased with the concentration of added cortisol. 11-KT production was also induced by hCG (Fig. 6). Fig. 6 Open in new tabDownload slide Effects of cortisol (0.01, 0.1, 1, 10, or 100 ng/ml) and hCG (0.1 IU/ml) on 11-KT production in testicular fragments. C, Testicular fragments cultured without steroids. Means with different letters are significantly different between groups; P < 0.05. Fig. 6 Open in new tabDownload slide Effects of cortisol (0.01, 0.1, 1, 10, or 100 ng/ml) and hCG (0.1 IU/ml) on 11-KT production in testicular fragments. C, Testicular fragments cultured without steroids. Means with different letters are significantly different between groups; P < 0.05. Discussion The involvement of a progestin in early spermatogenesis in fish was first revealed in studies on the Japanese huchen: in vitro, DHP induced the proliferation of spermatogonia (8). In Japanese eel, DHP has been determined to be involved in the regulation of the initiation of meiosis during spermatogenesis (9). Thus, the mechanisms regulating the early stages of spermatogenesis would be better understood by clarifying the molecular events underlying the action of DHP. In the present study, one of the cDNA clones obtained from cDNA subtraction encoded a gene that shares high homology with eel 11β-HSD2 cDNA isolated previously (11). In mammals, two distinct types of 11β-HSD have been identified and characterized. 11β-HSD type 1 is a NADP(H)- dependent enzyme, and acts predominantly as 11-oxo-reductase and rarely as 11β-dehydrogenase, with a relatively low affinity for substrates. 11β-HSD type 2 is a high-affinity, NAD-dependent 11β-dehydrogenase that metabolizes bioactive glucocorticoids (cortisol and corticosterone) to inert 11-keto forms (cortisone and 11-dehydrocorticosterone), thereby protecting mineralocorticoid receptor (MR) from overstimulation by cortisol. Recently, teleost 11β-HSD homologs that are similar to mammalian 11β-HSD type 2 have been identified in rainbow trout (16), tilapia, and Japanese eel (11). In rainbow trout, 11β-HSD showed 11β-dehydrogenase activity, the conversion of cortisol to cortisone and 11β-OHT to 11-KT (16). Japanese eel 11β-HSD2 also metabolized cortisol to cortisone (11). In the present study, Northern blot and RT-PCR analysis showed that the eel 11β-HSD transcripts were induced by DHP. Moreover, e11β-HSDsf displayed 11β-dehydrogenase activity, corresponding to mammalian 11β-HSD2 activity. These results suggest that DHP modulates androgen and glucocorticoid production in eel testis and thereby regulates spermatogenesis. 11β-Dehydrogenase activity of the 11β-HSD is essential for biosynthesis of a major fish androgen, 11-KT. However, 11-KT production via the action of 11β-HSD has been demonstrated only in rainbow trout (16). Although the conversion of 11β-OHT to 11-KT was not confirmed in a previous study, eel 11β-HSD2 was shown to have the 11β-dehydrogenase activity (11), suggesting that eel 11β-HSD2 can convert 11β-OHT to 11-KT. The results of the present study revealed that e11β-HSDsf has the ability to convert 11β-OHT to 11-KT and that immature testes have the ability to produce 11-KT, if the substrate, 11-OHT, is present. These results suggest that 11-KT synthesis in immature testis is arrested earlier in the steroidogenic pathway than the step from 11β-OHT to 11-KT. Although the induction of 11-KT production by DHP in testes cultured without 11β-OHT was not evident, DHP enhanced 11-KT production from 11β-OHT, coincident with the up-regulation of 11β-HSD expression. Our previous study suggested that spermatogonial proliferation is initiated by the action of 11-KT, and thereafter, DHP, which is produced in response to 11-KT, induces meiotic division (9). Hence, the role of DHP in spermatogenesis could be to provide positive feedback control of 11-KT production by increasing 11β-HSD expression, thereby promoting spermatogenic progression. In the present study, although expression of both eel 11β-HSDs was induced by DHP, results of RT-PCR analysis indicate that e11β-HSDsf is the dominant 11β-HSD, suggesting that e11β-HSDsf is the major source of 11β-dehydrogenase activity. However, additional research is needed to characterize the functional differences between the two types of eel 11β-HSDs. Our data further suggest that DHP also modifies glucocorticoid metabolism by up-regulating 11β-HSD expression. In teleosts, cortisol is closely related with the stress response (17), and high levels of circulating cortisol are found after various stressors that inhibit gonadal function (18). Interestingly, a previous in vivo study reported that cortisol administration promoted testicular development during the early stages of spermatogenesis, while inhibiting spermatogenesis during the mature phase in a freshwater fish, Notopterus notopterus (19). However, the effects of normal levels of cortisol on spermatogenesis have rarely been investigated. Therefore, we examined the direct effects of cortisol and cortisone on eel spermatogenesis using an organ culture system. Effects of cortisone on spermatogenesis were not observed. In contrast, cortisol induced DNA replication in spermatogonia, and a potentiating effect of cortisol on the spermatogonial proliferation induced by 11-KT was also revealed. Moreover, our results showed that 11-KT production was induced in testicular fragments incubated with cortisol. The precise mechanisms underlying 11-KT production have not been determined. However, the conversion of cortisol to 11-oxygenated androgens has been suggested in several teleosts (20), raising the possibility that cortisol is metabolized to 11-KT in eel testis. These results suggest that cortisol induces spermatogonial mitosis by increasing 11-KT production and that regulation of 11-KT production involves complex, multiple mechanisms. However, treatment of testis with cortisol at relatively high doses inhibited spermatogonial proliferation induced by 11-KT. In teleosts, a gene corresponding to mammalian 11β-HSD type 1 has never been identified, and it remains unclear whether such a gene is expressed in testis. In the present study, the experiments determining enzymatic activity of testicular fragments showed that the concentrations of cortisol in media after incubations with cortisone were slightly higher than those without cortisone. However, given the cross-reactivity of the antibody against cortisol to cortisone (4%), the conversion of cortisone to cortisol could not be confirmed. In immature eel testis, expression of 21-hydroxylase, a key enzyme for the synthesis of glucocorticoid from cholesterol, was also not detected (21). Therefore, it remains unclear whether cortisol is produced by eel testis. However, cortisol is a principal corticoid and is produced mainly in the interrenal tissue in eel, as in other teleosts (21). Therefore, a possible role of DHP may be to modify cortisol levels in the testis, via up-regulating 11β-HSD, and thereby protecting testicular development from circulating cortisol. The physiological roles of 11β-HSDs in the testis have not been yet defined. In mammals, several deleterious effects of glucocorticoid on the Leydig cells have been demonstrated: inhibition of testosterone biosynthesis, suppression of LH receptor expression, and induction of Leydig cell apoptosis (22–24). Therefore, it has been assumed that 11β-HSD type 1 acts predominantly as an 11β-dehydrogenase in testis and protects testis from glucocorticoid stimulation. However, this assumption was questioned by Leckie et al. (25), who demonstrated that 11β-HSD type 1 acted predominantly as a reductase in rat Leydig cells in vitro. Moreover, it was suggested that the functions of 11β-HSD type 1 depended on the age of the rat (26); the expression of the 11β-HSD type 1 and reductase activity was highest in immature Leydig cells (27). It is possible that glucocorticoids may also play some roles in early spermatogenesis in mammals. Unlike mammalian species, rainbow trout Leydig cells express 11β-HSD type 2 homolog at relatively high levels, and it was also suggested that one role of this 11β-HSD was to protect testis from circulating cortisol in addition to catalyzing 11-KT production (16). Generally in mammals, 11β-HSD type 1 is widely distributed in glucocorticoid target tissues such as liver, adipose tissue, and gonads, whereas 11β-HSD type 2 is expressed predominantly in mineralocorticoid-responsive tissue such as kidney, colon, and placenta. In contrast, the expression of fish 11β-HSD type 2 homolog was found to be relatively widespread in various tissues, similar to mammalian 11β-HSD type 1 (11, 16). In rainbow trout, 11β-HSD was also expressed in Leydig cells and other steroidogenic tissues at relatively high levels (16). However, a recent study demonstrated that 11β-HSD type 2 was present in rat Leydig cells at 1000-fold lower levels than 11β-HSD type 1 (28). Mammalian 11β-HSD type 2 converts cortisol to cortisone and thereby prevents the binding of cortisol to MR and allows selective access of aldosterone to the MR in mineralocorticoid target tissues. Although the MR is also found in rainbow trout (29), aldosterone is believed to be absent in teleosts (30, 31). In rainbow trout, cortisol appears to be a major ligand for the MR (29), but the biological actions of cortisol via MR remain unclear. In teleosts, little is known about the factors regulating 11β-HSD expression, because teleost 11β-HSD genes have only recently been identified. In mammals, several studies suggest that LH suppresses 11β-HSD type 2 expression in granulosa cells while inducing 11β-HSD type 1 expression (32, 33). In rat Leydig cells, LH and epidermal growth factor also up-regulated the 11β-HSD type 1, whereas they decreased the 11β-dehydrogenase activity (34). In male Japanese eel, however, 11β-HSD transcript levels in testes were remarkably increased after hCG injection (11). In rainbow trout, ovarian 11β-HSD mRNA level increased in late vitellogenic stage (16), when the plasma LH level was elevated (35), suggesting that LH may increase 11β-HSD expression. These results suggest differences in the physiological roles of 11β-HSD type 2 between mammalian and teleost species. In conclusion, we identified a cDNA encoding a homolog of mammalian 11β-HSD type 2 from Japanese eel testis. DHP induced 11β-dehydrogenase activity in testis through the up-regulation of 11β-HSD expression, and optimal levels of cortisol induced spermatogonial mitosis through increasing 11-KT production and enhanced testicular development induced by 11-KT. However, excess cortisol inhibits testicular development. These results suggest two possible roles of DHP in eel spermatogenesis: positive feedback control of 11-KT production and the modulation of cortisol levels to protect the testis from circulating cortisol. Acknowledgments We thank Prof. Graham Young, University of Washington, for critical reading of the manuscript, and Dr. Makoto Kusakabe, University of Washington, for discussion. This work was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan and from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government. The sequence reported in this paper has been deposited in the GenBank database [accession no. AB252646 (Japanese e11β-HSDsf)]. Disclosure statement: The authors have nothing to disclose. Abbreviations: BrdU 5-Bromo-2-deoxyuridine DHP 17α,20β-dihydroxy-4-pregnen-3-one E2 estradiol-17β e11β-HSDsf eel 11β-hydroxysteroid dehydrogenase short form EF1 elongation factor 1 hCG human chorionic gonadotropin 11β-HSD 11β-hydroxysteroid dehydrogenase 11-OHT 11β-hydroxytestosterone 11-KT 11-ketotestosterone MR mineralocorticoid receptor NAD+ nicotinamide adenine dinucleotide ORF open reading frame TR-FIA time-resolved fluoroimmunoassay 1 Miura T , Yamauchi K , Nagahama Y , Takahashi H 1991 Induction of spermatogenesis in male Japanese eel, Anguilla japonica, by a single injection of human chorionic gonadotropin . Zool Sci 8 : 63 – 73 WorldCat 2 Miura T , Yamauchi K , Takahashi H , Nagahama Y 1991 Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica) . 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Endocrinology – Oxford University Press

Published: Nov 1, 2006

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