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Male Reproductive Tract |
College of Life Sciences,3 Northeast Agricultural University, Harbin 150030, China
Department of Physiology,4 Harbin Medical University, Harbin 150086, China
Department of Biology,5 Texas Woman's University, Denton, Texas 76204
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFFERENCES |
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epididymis, male reproductive tract, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFFERENCES |
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L-PGDS, a bifunctional protein, can act as a PGD2-producing enzyme that catalyzes the conversion of PGH2 to PGD2, or in addition, as a member of the lipocalin family, L-PGDS binds and transports small lipophilic ligands such as retinoids, steroids, thyroid hormones, biliverdin, and bilirubin [5]. L-PGDS is expressed in a wide range of tissues, such as brain [6], retina [7], cochlea [8], and male genital organs [9]. L-PGDS is also abundant in biological fluids such as cerebrospinal fluid, ascites, seminal plasma, serum, urine, and amniotic fluid [10–13].
L-PGDS is found in significant concentration in male reproductive organs of several mammalian species. In the human male, concentration of L-PGDS is significantly lower in both reproductive organs and seminal plasma of the oligozoospermic group when compared with the normozoospermic group [14]. L-PGDS protein in the seminal plasma of bulls with above-average fertility is 3.5-fold greater than that in bulls of average and below-average fertility [12]. In mouse testis, PGDS mRNA appeared to be expressed only in the tubules of neonatal mouse testes and only in the interstitial tissue of the adult testis by in situ hybridization studies [15]. L-PGDS concentration in rat testis and epididymis during sexual maturation and castration was briefly examined by radioimmunoassay [9]. However, no localization for L-PGDS mRNA and protein was performed. L-PGDS expression was also investigated in the adult epididymis of rat, hamster, and cynomolgus monkey by in situ hybridization and immunohistochemistry [16]. The aim of the present study was to investigate the regionalization and regulation of L-PGDS expression in rat testis and epididymis under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments using in situ hybridization and immunohistochemical approaches.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFFERENCES |
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Mature male rats (Sprague-Dawley) were caged in a controlled environment (14L:10D) with food and water provided ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. At least three rats were used in each stage or treatment in this study.
Sexual Maturation
The male rats were killed by stunning and cervical dislocation on Postnatal Days 1, 10, 20, 30, 40, and 70. The testis and epididymis were collected. The epididymis was divided into three segments: the caput, corpus, and cauda. The testis and epididymis from one side were fixed in Bouin solution for 24 h and embedded in paraffin for immunohistochemistry, whereas the contralateral testis and epididymis were frozen in liquid nitrogen and stored at -70°C for in situ hybridization.
Castration and Hormonal Replacement
Mature male rats weighing 300–350 g were bilaterally orchidectomized under anesthesia. Surgical procedures were performed under sterile condition. Then the epididymides were returned into the scrotum. Three days after surgery, the experimental rats were injected i.m. with testosterone propionate (TP) dissolved in sesame seed oil (3 mg/kg body weight/day; Sigma, St. Louis, MO) and the sham-operated control group was injected with a comparable volume of sesame seed oil (the vehicle) for 5 days. For study, the caput, corpus, and cauda epididymis were collected and treated as above.
EDS Treatment
Mature male rats weighing 300–350 g were treated with a single i.p. injection of EDS (75 mg/kg body weight) dissolved in dimethylsulfoxide:water (1:3) at 37.5 mg/ml to eliminate Leydig cells in the testicular interstitium [17]. One week later, the rats were injected i.m. with TP (3 mg/kg body weight per day; Sigma) for 5 days. For control, sesame seed oil was injected for 5 days. The tissues were collected and treated 24 h after the last injection.
Hybridization Probes
Total RNA from mouse uterus was reverse transcribed and amplified with forward primer 5'-CG
ATGCTGTGGATGGGTTTG and reverse primer 5'-GC
AGTGACAGAGCAAGGGAG designed from mouse L-PGDS (75–704 bp, GenBank accession number AB006361). In these primers, protection bases (CG) and EcoRI sites (GAATTC, underlined) were added at the 5' end of the forward primer, and protection bases (CG) and BamHI sites (GGATCC, underlined) at the 5' end of the reverse primer. The polymerase chain reaction (PCR) fragment (630 bp + 16 bp from protection bases and EcoRI/BamHI sites) was recovered from the agarose gel and cloned into pGEM-3Zf (+) plasmid through EcoRI and BamHI sites, respectively. The cloned L-PGDS fragment was further verified by sequencing. These plasmids were linearized with appropriate enzymes for digoxigenin labeling. (DIG)-labeled antisense or sense cRNA probes were transcribed in vitro using a DIG RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany).
In Situ Hybridization
Tissue was flash frozen in liquid nitrogen. Frozen sections (10 µm) were mounted on 3-aminopropyltriethoxy-silane-coated (Sigma) slides and fixed in 4% (w/v) paraformaldehyde solution in PBS. The sections were washed twice in PBS, treated with 1% (v/v) Triton X-100 for 20 min and washed again in PBS three times. Following prehybridization in 50% (v/v) formamide and 5x SSC (1x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) at room temperature for 15 min, the sections were hybridized in the hybridization buffer (5x SSC, 50% [v/v] formamide, 0.02% [w/v] BSA, 250 µg/ml yeast tRNA, 10% [w/v] dextran sulfate, 1 g/ml denatured DIG-labeled antisense or sense RNA probe for mouse L-PGDS) at 55°C for 16 h. After hybridization, the sections were washed in 50% (v/v) formamide/5x SSC at 55°C for 15 min, 50% (v/v) formamide/2x SSC at 55°C for 30 min, 50% (v/v) formamide/0.2x SSC at 55°C twice for 30 min each, and 0.2x SSC at room temperature for 5 min. After nonspecific binding was blocked in 1% (w/v) block mix (Roche Diagnostics GmbH) for 1 h, the sections were incubated in sheep anti-DIG antibody conjugated with alkaline phosphatase (1:5000, Roche Diagnostics GmbH) in 1% block mix overnight at 4°C. The signal was visualized with 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.4 mM nitroblue tetrazolium in the buffer containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2. Endogenous alkaline phosphatase activity was inhibited with 2 mM levamisole (Sigma). All of the sections were counterstained with 1% (w/v) methyl green in 0.12 M glacial acetic acid and 0.08 M sodium acetate for 30 min.
Immunohistochemistry
L-PGDS immunostaining was performed as previously described [18]. Briefly, sections (7 µm) were cut and mounted onto 2% 3-aminopropyltriethoxysilane-coated slides, deparaffinized, and rehydrated. Nonspecific binding was blocked in 10% normal horse serum in PBS for 1 h. The sections were incubated with rabbit anti-human L-PGDS (Cayman Chemical, Ann Arbor, MI) in 10% horse serum overnight at 4°C. After washing in PBS three times for 5 min each, the sections were incubated with biotinylated goat anti-rabbit IgG followed by an avidin-alkaline phosphatase complex and Vector Red according to the manufacturer's protocol (Vectastain ABC-AP kit; Vector Laboratories, Burlingame, CA). Vector Red was visualized as a red color. Endogenous alkaline phosphatase activity was inhibited with levamisole (Sigma). In some sections, rabbit anti-human L-PGDS was replaced with normal rabbit IgG as a negative control. The sections were counterstained with hematoxylin and mounted. The degree of staining was assessed subjectively by blinded examination of the slides by two investigators.
Histochemistry for 3ß-Hydroxysteroid Dehydrogenase
The activity of 3ß-hydroxysteroid dehydrogenase (HSD) was shown as previously described [19]. Frozen sections (10 µm) were stored at -70°C. Immediately before use, they were thawed and incubated for 4 h at 37°C with 0.25 mg/ml 4-nitro blue tetrazolium chloride, 0.28 mg/ml nicotinamide, 0.60 mg/ml nicotinamide adenine dinucleotide, and 0.05 mg/ml dehydroepiandrosterone. The 3ß-HSD-positive cells were dark blue.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFFERENCES |
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In developing rat testes, no detectable L-PGDS mRNA was observed on Days 1, 10, 20, 30, and 40 after birth (only Day 30 is shown in Fig. 1A). A weak signal for L-PGDS mRNA was seen only in the spermatogonia or Sertoli in the seminiferous tubules on Day 70 after birth (Fig. 1B). L-PGDS immunostaining was not detected in rat testis on any of the Days 1, 10, and 20 after birth (data not shown). There was a basal level of L-PGDS immunostaining in the peritubular cells on Day 30 after birth (Fig. 1C). However, by 70 days postpartum, there was a strong L-PGDS immunostaining in the testicular Leydig cells (Fig. 1D).
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Expression of L-PGDS mRNA and Protein in Rat Epididymis During Sexual Maturation
In caput epididymis, L-PGDS mRNA was not detected on Days 1, 10, and 20 after birth (data not shown). An intermediate level of L-PGDS mRNA signal was seen in the epididymal epithelium on Days 30 (Fig. 2A) and 40 (data not shown). There was an increase of L-PGDS mRNA signal at Day 70 (Fig. 2B). In corpus epididymis, L-PGDS mRNA expression was not detected on Days 1, 10, 20, 30, and 40 after birth (only Day 30 is shown in Fig. 2E). On Day 70, L-PGDS expression was highly detected in the epididymal epithelium (Fig. 2F). In cauda epididymis, no expression of L-PGDS was seen on Days 1, 10, and 20 postpartum (data not shown). L-PGDS signal was at a basal level on Day 30 (Fig. 2I), gradually increased on Day 40 (data not shown), and significantly increased on Day 70 after birth (Fig. 2J). However, no positive signals were observed in cauda epididymis on Day 70 after birth when a DIG-labeled sense probe was used to replace DIG-labeled antisense probe (data not shown).
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During early sexual maturation, L-PGDS immunostaining was not observed in caput, corpus, or cauda epididymis on Days 1, 10, and 20 after birth (data not shown). In the caput segment, L-PGDS immunostaining was at a basal level in the epididymal epithelium on Day 30 (Fig. 2C) and at a low level on Days 40 and 70 after birth (only Day 70 is shown in Fig. 2D). In corpus segment, L-PGDS immunostaining was seen in only a few of the principal cells on Day 30 (Fig. 2G) but was strongly evident in the principal cells on Day 70 after birth (Fig. 2H). Moreover, there was no detectable immunostaining in corpus epididymis on Day 70 after birth when rabbit antihuman L-PGDS was replaced with normal rabbit IgG (data not shown). In caudal segments, L-PGDS immunostaining was at a basal level in the epididymal epithelium on Day 30 (Fig. 2K) and was at a low level on Day 70 (Fig. 2L).
Effect of Castration and Hormone Supplementationon L-PGDS mRNA and Protein Expression in Epididymis
In this experiment, castrated rats were treated with either vehicle or TP for 5 days after 3 days of surgery. In caput epididymis, there was a basal level of L-PGDS mRNA expression in the controls treated with vehicle for 5 days (Fig. 3A). In contrast, a strong level of L-PGDS mRNA was seen in the epididymal epithelium treated with TP for 5 days (Fig. 3B). For the immunostaining, there was no detectable signal in the control group (Fig. 3C), whereas a low level of L-PGDS immunostaining was seen in some of the principal cells after 5-day treatment with TP (Fig. 3D).
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In corpus epididymis, there was no detectable L-PGDS mRNA in the epididymal epithelium of the control group (Fig. 3E), but a high level of L-PGDS mRNA signal was seen after 5 days of androgen replacement (Fig. 3F). L-PGDS immunostaining was not observed in the control (Fig. 3G). However, L-PGDS immunostaining was strongly detected in some of the principal cells after 5-day treatment with TP (Fig. 3H).
In caudal epididymis, L-PGDS mRNA signal was at a basal level in the control (Fig. 3I). However, treatment with TP for 5 days significantly stimulated L-PGDS expression in the epididymal epithelium (Fig. 3J). There was no detectable L-PGDS immunostaining in the epididymal tubules in the control (Fig. 3L). TP treatment induced a significant increase of L-PGDS immunostaining in some of the principal cells (Fig. 3L).
Effects of EDS Treatment on L-PGDS mRNA and Protein Expression in Testis
In this experiment, a single dose of EDS (75 mg/kg body weight) given i.p. was used to selectively eliminate Leydig cells from mature rat testicular interstitium. In normal adult rat testis, seminiferous tubules and interstitium were tightly packed (Fig. 4A), whereas the structures of seminiferous tubules and interstitium were distorted 8 days after EDS treatment (Fig. 4B). Because Leydig cells are the cells that synthesize testosterone in the male gonad and 3ß-HSD is a key enzyme in that process, 3ß-HSD was used to confirm that Leydig cells had been depleted from the testicular interstitium by histochemistry. The staining for 3ß-HSD was seen in the Leydig cells of testicular interstitium in normal adult testis (Fig. 4C), but no 3ß-HSD staining was detectable in the interstitium 3 days after EDS injection (Fig. 4D). EDS-treated rats were injected with TP to replace endogenous androgen, and controls were injected with vehicle for 1 and 5 days, beginning 7 days post-EDS injection. There was no detectable L-PGDS mRNA in the control groups treated with vehicle for 1 and 5 days (Fig. 4E for 1 day). However, L-PGDS mRNA signals were seen in a few of the cells of seminiferous tubules after 1-day or 5-day treatment with TP (Fig. 4F for 1 day). There was no detectable L-PGDS immunostaining either in the controls or in the TP-treated groups (data not shown).
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Effects of EDS Treatment on L-PGDS mRNA and Protein Expression in the Epididymis
In caput epididymis, there was no detectable L-PGDS mRNA in the control group treated with vehicle for 5 days (Fig. 5A). However, TP treatment for 5 days significantly increased L-PGDS expression in the epididymal epithelium (Fig. 5B). L-PGDS immunostaining was not observed in the control (Fig. 5C), whereas a very low level of L-PGDS immunostaining was detected in the epididymal epithelium after TP treatment for 5 days (Fig. 5D).
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In corpus epididymis, a basal level of L-PGDS expression was observed in the control (Fig. 5E), whereas there was an increase of L-PGDS expression after TP treatment for 5 days (Fig. 5F). No immunostaining was detected in the control (Fig. 5G), but there was an increase of L-PGDS immunostaining in the epididymal epithelium after TP treatment for 5 days (Fig. 5H).
In cauda epididymis, there was no detectable L-PGDS expression in the control (Fig. 5I). However, a very low level of L-PGDS expression was seen after TP treatment for 5 days (Fig. 5J). No L-PGDS immunostaining was detected in the control (Fig. 5K). But a low level of L-PGDS immunostaining was seen in the epididymal epithelium after TP treatment for 5 days (Fig. 5L).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFFERENCES |
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Both L-PGDS mRNA and immunostaining in rat testis during sexual maturation were examined in this study. L-PGDS mRNA expression was weakly detected only in the testicular peritubular cells. These positive cells were near the basement membrane inside seminiferous tubules and should be Sertoli cells or spermatogonia. In the rat, L-PGDS mRNA was expressed in both Sertoli and germs shown by reverse transcription-PCR (RT-PCR) [9]. Moreover, L-PGDS mRNA was localized in Sertoli cells, spermatogonia, and spermatocyte in human testis by in situ hybridization [20]. However, in mouse testis, PGDS mRNA appeared to be expressed only in the tubules of neonatal mouse testes and only in the interstitial tissue of the adult testis by in situ hybridization [5]. This localization difference of L-PGDS mRNA expression in the testis between mouse and rat may be species related. The mRNA for L-PGDS was found to be expressed in the heart of humans, monkeys, and mice. However, in the rat heart, the expression was not detected by Northern blot analysis or RT-PCR analysis [21]. Because we found that L-PGDS immunostaining was highly detected in the Leydig cells, it appears that L-PGDS protein produced by Sertoli cells or spermatogonia may be secreted to testicular interstitium regions. In ovine epididymis, it has been shown that there was L-PGDS of testicular origin in the lumen of the anterior caput [22]. Additionally, no L-PGDS mRNA expression was detected in the Leydig cells of adult rat testis in our study. Conversely, in the mouse, L-PGDS mRNA expression was mainly seen within the tubules of neonatal testis and in the Leydig cells of adult testis [15]. This difference in L-PGDS mRNA expression may also indicate a low sensitivity of our situ hybridization because L-PGDS immunostaining was highly detected in the Leydig cells in our study. Additionally, a basal level of L-PGDS immunostaining was seen in the peritubular cells, although no L-PGDS mRNA signal was seen in the testis on Day 30 after birth. Thereafter, a low level of L-PGDS mRNA signal was only seen in the testicular peritubular cells on Day 70 after birth, suggesting that the L-PGDS expression in these cells might be not detected on Day 30 after birth. Nevertheless, our methods of in situ hybridization gave a strong signal in the epididymis. Whether the Leydig cells or other spermatogenic cells can express L-PGDS remains to be further determined.
Both L-PGDS mRNA and immunostaining in the caput, corpus, and cauda epididymis during the sexual maturation were examined in this study. L-PGDS expression was not detected on Days 1, 10, and 20 after birth. On Day 20, L-PGDS mRNA expression was detected only in the caput and cauda epididymis. But L-PGDS mRNA expression was strongly observed in all of the caput, corpus, and cauda epididymis on Day 70 after birth. Our results were similar to another study in rat [9]. By RT-PCR analysis, it was shown that L-PGDS mRNA level was the highest in the caput, followed by the cauda and corpus of rat epididymis. PGDS concentration also increased steadily during maturation and reached its highest level on Day 60 after birth [9]. In the epididymis of rat, hamster, and cynomolgus monkey, L-PGDS mRNA expression was also detected in caput, corpus, and cauda by in situ hybridization, whereas the PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry [16]. However, L-PGDS immunostaining was strongly detected only in the principal cells of corpus epididymis, and was at a low level in the epithelial cells of both caput and cauda epididymis. In both mouse and bull epididymis, L-PGDS immunostaining was also located in the epithelial principal cells [23, 24]. Our results also suggest that there may be a region-specific regulation on L-PGDS translation, although L-PGDS mRNA expression was highly detected in all of the caput, corpus, and cauda epididymis. This regional specificity of L-PGDS protein expression should be related to corresponding functions in each part of the epididymis. The acquisition of the sperm motility and zona pellucida-binding capacity mainly occurs in the caput and corpus epididymis, whereas the cauda epididymis is specialized in sperm storage [25, 26]. The three regions of the epididymis are different in their epithelial cell morphology and specific pattern gene expression [27]. In human epididymis, human epididymal protein 2 (HE2) is specifically expressed in distal caput epididymis with no expression in other segments [28]. But in cynomolgus monkey, the maximum expression of HE2-1 mRNA occurs in proximal corpus epididymis, with almost no expression appearing in other parts of the epididymis [27]. However, the significance of the region-specific expression of L-PGDS in rat epididymis is still not known.
We found that both L-PGDS mRNA and protein expression in rat epididymis was significantly reduced in all of the epididymal regions after castration, whereas L-PGDS expression was significantly induced by testosterone propionate treatment for 5 days, compared with controls. Additionally, there was a significant decrease of L-PGDS expression in the epididymis of EDS-treated rats, compared with adult rat epididymis. Similarly, testosterone propionate treatment could also increase L-PGDS expression in the epididymis of EDS-treated rats. EDS, a unique testicular toxin with cytotoxic action, specifically destroys mature Leydig cells [29]. One week after EDS treatment, the serum testosterone concentration and testicular testosterone production were reduced to undetectable levels [30]. Our data suggest that L-PGDS expression can be up-regulated by testosterone. Sorrentino et al. [9] also reported that orchiectomy induced a drastic reduction of L-PGDS concentration in all three epididymal compartments of rat epididymis. They also found that dihydrotestosterone treatment for 5 days induced a significant increase of L-PGDS concentration in caput epididymis after castration but only a slight increase of L-PGDS in corpus and cauda epididymis. However, in our study, although testosterone propionate induced a similar increase of L-PGDS expression in all of the caput, corpus, and cauda epididymis of the castrated rats, TP had differential effects on the caput, corpus, and cauda epididymis of EDS-treated rats, having a slight increase especially in cauda epididymis. This suggests that the epididymal L-PGDS level is not entirely regulated by androgen and that another yet-to-be-identified testicular factor(s) may be also involved in its regulation. Sorrentino et al. [9] found that germ cell-conditioned medium also stimulated L-PGDS expression in the Sertoli cells.
In the present study, L-PGDS immunostaining was strongly detected only in the Leydig cells of rat testis. There was a strong level of L-PGDS mRNA and immunostaining in the adult rat epididymis. In the seminal fluid, the concentration of PGD2 is positively correlated with L-PGDS [14]. In the human, L-PGDS concentration is significantly lower in both reproductive organs and seminal plasma of the oligozoospermic group when compared with the normozoospermic group [14]. Moreover, L-PGDS protein in the seminal plasma of bulls with above-average fertility is 3.5-fold greater than that in bulls of average and below-average fertility [12]. Additionally, as an important carrier of bile pigments, retinoids, thyroid hormones, and essential fatty acids, the major function of seminal L-PGDS does not seem to be the synthesis of PGD2 from its precursor PGH2, which is very unstable in aqueous solution. Instead, seminal L-PGDS would contribute to providing thyroid hormones and retinoids to the developing germ cells in the seminiferous tubules and the maturing spermatozoa in the epididymis beyond the blood-testis barrier [31]. It has been established that retinoids (vitamin A derivatives) are crucial for male infertility [32]. These data suggest that L-PGDS secreted by both epididymis and testis may play an important role during the spermatogenesis and sperm maturation.
In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.
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FOOTNOTES |
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2 Correspondence: Zeng-Ming Yang, College of Life Sciences, Northeast Agricultural University, Harbin 150030, China. FAX: 86 451 55103336; zmyang{at}mail.neau.edu.cn
Received: 7 August 2003.
First decision: 2 September 2003.
Accepted: 1 December 2003.
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REFFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFFERENCES |
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