Postnatal Expression and Androgen Regulation ... - Wiley Online Library

Journal of Andrology, Vol. 28, No. 5, September/October 2007 Copyright E American Society of Andrology

Postnatal Expression and Androgen Regulation of HOXBES2 Homeoprotein in Rat Epididymis ESAKKI PRABAGARAN,* UMA C. HEGDE,{ SUDHIR B. MOODBIDRI,* ATMARAM H. BANDIVDEKAR,* AND VIJAYA P. RAGHAVAN* From the *Department of Biochemistry and ÀHybridoma Laboratory, National Institute for Research in Reproductive Health, Mumbai, India.

ABSTRACT: The multifunctional and androgen-regulated epididymis is known to provide a conducive microenvironment for the maturation and storage of mature spermatozoa. HOXB2 homeodomain-containing epididymis-specific sperm protein (HOXBES2), a molecule first reported by our group, exhibits cell- and regionspecific expression. It was found in the cytoplasm of the principal epithelial cells with maximum in the distal segments of the rat epididymis. The present study was undertaken to determine whether HOXBES2 expression is regulated by androgens and postnatal epididymal development. Toward this, the epididymis was disallowed access to circulating androgens either by chemical or biologic castration. In bilaterally orchidectomized animals, the levels of immunoreactive HOXBES2 declined to ,5 % of those seen in sham-operated animals. Exogenous dihydrotestosterone (DHT) supplementation (250mg/kg body weight) for 7 days restored the expression levels to $ 90 % of that observed in intact animals. Ethylene dimethane sulfonate (EDS) administration completely

abolished HOXBES2 expression in the epididymis, and supplementation with DHT or DHT + estradiol for 10 days re-established HOXBES2 expression to near normalcy. However, in the estradiol alone-supplemented EDS-treated group, HOXBES2 remained undetected. The unaltered HOXBES2 expression following efferent duct ligation suggested that HOXBES2 is not critically dependent on testicular factors. During postnatal development, protein expression in the epididymis begins to appear from day 40 and 50 and increased from day 60 onward, coinciding with the mature levels of circulating androgens and the well-differentiated epididymis. Thus, the data obtained from this study suggests that HOXBES2 expression could be regulated by androgens, and its expression level is closely associated with the postnatal development of the epididymis. Key words: Castration, EDS treatment, DHT supplementation, efferent duct ligation, postnatal development. J Androl 2007;28:755–771

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storage entail exposure and interaction of spermatozoa with luminal fluid proteins of the epididymis, which involves either tight binding or surface attachment of epididymal proteins to the sperm surface (Cornwall and Hann, 1995). Since sperm maturation has been shown to be an orchestrated sequence of multiple protein interactions with respect to region-specific gene expression (Brooks, 1987), it is of utmost importance to study the secretions and fate of proteins along the length of the epididymis. This is also of relevance to the identification of novel targets for male contraception and fertility. Several studies have earlier demonstrated that many of the individual processes that contribute to the creation of an optimal microenvironment within the epididymis are regulated by circulating luminal androgens and to some extent by other testicular factors that originate from the testis itself including basic fibroblast growth factor, androgen-binding protein, retinoids, etc (Ezer and Robaire, 2002, 2003). Androgen-regulated regionalized gene expression is complex in the epididymis and has been discussed in a recent review (Orgebin-Crist, 1996). Segment-specific down-regulation of gene expression following withdrawal of androgens or testicular factors is a hallmark characteristic of the epididymis (Cornwall et

he epididymis is a multifunctional male accessory organ of the testicular excurrent duct system. As a highly convoluted tubule, the epididymis is anatomically and physiologically divided into four major regions, the initial segment, caput, corpus, and cauda, based on the distribution of four major cell types (Orgebin-Crist, 1969; Jervis and Robaire, 2001). The structural and functional architecture of the epididymis is demarcated by the expression of a constellation of specific proteins (Berube et al, 1996) and regionalized gene expression pattern (Xu et al, 1997). The epididymis provides a continuously changing unique luminal microenvironment conducive for the transport, maturation, and storage of mature spermatozoa (Robaire and Hermo, 1988). Sperm maturation and This work (NIRRH/MS/20/2006) was supported by grants from the Indian Council of Medical Research (ICMR). E.P. is a recipient of the ICMR Senior Research Fellowship. Correspondence to: Dr Vijaya P Raghavan, Department of Biochemistry, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel – 400 012, Mumbai, India (e-mail: [email protected]). Received for publication December 11, 2006; accepted for publication May 7, 2007. DOI: 10.2164/jandrol.106.002394

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al, 2001). Androgens such as testosterone (T), 5a-reduced T, and dihydrotestosterone (DHT) exert their effects by binding and subsequently activating the androgen receptor (AR) protein with the help of a carrier molecule, androgen-binding protein (ABP). It is less clear whether the metabolites of T—namely estradiol—formed by the action of aromatase or the major metabolites of DHT— androstan-3a, 3a-diol, and 17b-diol—also have any significant role to play in epididymal differentiation or maturation. DHT, the most abundant and active form of androgen in the epididymis (Hinton et al, 1998), is produced by the reducing action of 5a reductase and is indispensable for forward progressive and vibrant motility, capacitation (Holland et al, 1992), sperm-egg interaction (Lakoski et al, 1988), zona pellucida (ZP) binding and penetration (Boue et al, 1994), vitellous fusion and penetration (Saling, 1982), and fertilizing ability (Amann et al, 1993) of the spermatozoa. The functional role of androgens in the synthesis and secretion of several epididymal proteins such as CD52 (Kirchhoff et al, 2000) and transcription factors such as ets-like factor PEA3 (Brooks, 1987) and reproductive homeobox on chromosome X 5 protein (previously known as placenta and embryonic expression protein; Lindsay and Wilkinson, 1996) was widely demonstrated earlier using the castrated rat model and DHT supplementation studies (Jones et al, 1980; Holland and Orgebin-Crist, 1988; Ghyselinck et al, 1989; Gould and Young, 1990). Further, the withdrawal of androgen by castration or by chemical suppression leads to involution of epididymal epithelium, gene down-regulation, and resultant arrest of sperm maturation. It has been suggested that incomplete sperm maturation is responsible for total failure of sperm binding to the ZP in unsuccessful in vitro fertilization treatment in humans (Bedford and Kim, 1993). In fact, a high percentage of male infertility in humans is believed to originate from malfunction of the epididymis (Lunde et al, 1990). We previously demonstrated the presence of HOXB2 homeodomain containing epididymis-specific sperm protein (HOXBES2) in the cytoplasm of the principal epithelial cells in a region-specific manner showing maximal expression in the distal segments of the rat epididymis (Prabagaran et al, 2007). Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segment of rat caudal and ejaculated human and monkey spermatozoa, respectively. The aim of the present study was first to determine whether androgens and testicular factors are required to maintain the regional expression patterns of HOXBES2 protein in the adult rat epididymis and second to study how changes in HOXBES2 expression correlate with specific morphologic and biochemical changes that have been reported during the development of the epididymis.

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Materials and Methods Animals and Organ Sampling For the studies described, 5- to 200-day-old male rats (Rattus norvegicus) of the Holtzmann strain weighing 10 to 450 g were used as the animal model. The rats were housed under conditions of a 12:12-hour light:dark cycle with access to rat chow and water ad libitum. All experimental procedures were conducted in accordance with the guidelines of the Animal Ethics Committee of the National Institute for Research in Reproductive Health for the use and care of animals for biomedical research.

Tissue Samples Experimental rats were anesthetized with ether, and the tissues were dissected out (testis, prostate, seminal vesicle, and epididymis). The epididymis was divided into initial segment, proximal and distal caput, proximal and distal corpus, and proximal and distal cauda (Robaire and Viger, 1995) and processed under sterile conditions for immunohistochemistry, Western blot analysis, and reverse transcriptase polymerase chain reaction (RT-PCR).

Castration and DHT Supplementation Three-month-old male rats weighing approximately 300 g were divided into 4 groups of 6 animals each. Group I rats were sham operated (control); the testes and epididymides were manipulated similar to castration except for the excision of testes. Group II rats were bilaterally castrated; following exposure of the scrotal contents, the testicular vascular supplies were ligated without compromising epididymal blood supplies and the testes were separated from the epididymides and associated fat pads and then excised. The epididymides and fat pads were returned, caput first, into the tunica vaginalis and the incisions sutured. Group III rats were sham operated; this vehicle-treated control group received 100 mL of sesame seed oil subcutaneously daily postsham operation for 2 weeks. Group IV rats were bilaterally castrated and received supplementations of 250 mg DHT/kg body weight in sesame oil for 7 days in a delayed T repletion regimen that began on day 7 postcastration. Groups I and II were sacrificed on day 7 postoperation. Groups III and IV were sacrificed on day 15. Blood was collected from all groups. From Groups I and II, blood was collected on days 0 and 7 before sacrifice; blood was collected on days 7 and 14 following castration for Groups III and IV. Serum T levels were measured in blood samples collected prior to castration or sham operation. The serum samples were stored at 220uC until they were assayed for circulatory levels of T determined by radioimmunoassay (RIA) as described earlier (Gupta et al, 1975; Corker and Davidson, 1978). At the time of sacrifice, epididymides, ventral prostrates, and seminal vesicles were removed, trimmed from associated fat and connective tissues, and weighed. The epididymides were dissected into initial segment, proximal and distal caput, proximal and distal corpus, and proximal and distal cauda (Teerds et al, 1988), fixed in Bouin fixative, and subsequently processed for histology and immunohistochemical analyses.

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Androgen Regulation of HOXBES2 Homeoprotein

Chemical Castration and Hormone Supplementation To study the androgen-dependent expression of HOXBES2 protein in the epididymis, 3-month-old male rats were administered a single dose of the Leydig cell–specific toxicant, ethylene dimethane sulfonate (EDS), which is reported to cause transient infertility. A single dose of EDS was sufficient to destroy a mature Leydig cell population and to ensure that no new Leydig cells were formed during the first 10 days of treatment (Girotti et al, 1992). The rats were divided into 7 groups of 5 animals each (groups I–VII). Groups I, II, and III served as controls and were administered intraperitoneally the vehicle alone (DMSO-H2O, 1:3 vol/vol), sesame oil, and saline, respectively. Group IV received a single intraperitoneal injection of EDS (75 mg/kg body weight) dissolved in DMSO-H2O (1:3 wt/vol) on day 0. Group V was administered EDS and in addition received 250 mg DHT/kg body weight dissolved in sesame oil daily for 10 days. Group VI was EDS treated and received DHT and 100 mg of b-estradiol (SigmaAldrich, St Louis, Mo) in saline subcutaneously for 10 days. The EDS-treated rats in group VII were administered 100 mg of b-estradiol alone subcutaneously for 10 days. Blood samples were collected on days 0, 5, and 10 from the controls (groups I–III), EDS-treated (group IV), and hormone-supplemented groups (groups V–VII) to measure circulating levels of T. Serum T levels obtained from untreated control animals or animals prior to treatment were considered basal T levels. The control and experimental groups were sacrificed on day 10, and testes, epididymides, and prostates were removed and processed for immunohistochemistry and Western blot analyses.

Unilateral Efferent Duct Ligation To determine whether factor(s) of testicular origin are required for the regionalized expression of HOXBES2 protein in the epididymis, unilateral efferent duct ligation (EDL) studies were performed on 3-month-old male rats. Efferent ductules of the right testes were ligated at their junctions with the extratesticular rete testes without compromising the testicular or epididymal blood supplies (Hermo et al, 1992b). As controls, the left testes were manipulated similarly to the right testes, but the efferent ductules were not ligated. Each testis was returned to its tunica vaginalis, and incisions were sutured. The rats were sacrificed on days 10 and 15 postligation. The durations of the experiment were scheduled to simulate the time scales previously followed for EDS treatment and castration studies, respectively. Five rats per time point of EDL treatment were used in this study. Ligated and control epididymides were dissected out and processed for immunohistochemistry and Western blot analyses.

Developmental Study To determine the influence of T, if any, on the interrelationship between the expression pattern of HOXBES2 protein and the postnatal development of the rat epididymis, normally developing pups (Hermo et al, 1992a) were included in the study. The number of animals used at each postnatal age was optimized to obtain sufficient tissue for RNA isolation,

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immunohistochemistry, and Western blot. The animals (n) were sacrificed on postnatal days 5(15), 10(12), 15(10), 20(7), 30(7), 40(7), 50(6), 55(6), 60(6), 70(6), 80(6), 90(6), 100(6), 120(6), and 200(6). Epididymal tissues obtained from each postnatal age were divided into 3 groups to analyze samples in triplicate. The body weights of all animals used, as well as the testicular and epididymal weights, were within the ranges reported earlier (Scheer and Robaire, 1980; Hermo et al, 1992a; Hermo et al, 1998). The time points were chosen to almost coincide with known developmental events: postnatal days 0 to 15 represent the proliferative phase in which undifferentiated cells undergo mitotic activity, and days 16 to 44 represent the period of differentiation when the blood-epididymal barrier is formed and the columnar cells differentiate into principal cells, followed by a period of expansion (days 44 to 91) during which sperm enter the epididymis and are stored in the lumen of the cauda epididymis (Cyr et al, 1993). In addition, postnatal days 20 and 40 reflect the period before and after the rise in serum androgens; on day 42 specific 5a reductase enzyme activity is at its peak. Postnatal days 49 and 56 are marked by the first appearance of spermatozoa in the caput and cauda epididymis, respectively. By day 77, specific 5a reductase enzyme activity declines rapidly. Finally, postnatal days 90 to 200 were used to represent the adult animal (Viger and Robaire, 1992).

RNA Extraction Total RNA was extracted from immature (15-day) and mature (60-day) epididymal tissues using the single-step acid guanidium thiocyanate–phenol-chloroform extraction method as described by Chomozynski and Sacchi (1987); quantitation and purity were determined by absorbance at 260 and 280 nm (Smartspec; BioRad, Hercules, Calif). The integrity of the isolated RNA was confirmed by electrophoresis on a 1% agarose (Amersham Biosciences, Piscataway, NJ), 2.2 M formaldehyde gel using formaldehyde-containing buffer, stained with 0.1 mg/mL ethidium bromide (Sigma-Aldrich) and visualized under UV (Lehrach et al, 1977).

RT-PCR Five micrograms each of total RNA extracted from immature and mature rat epididymides were subjected to RT-PCR analysis by a single-step RT-PCR system (Roche Diagnostics, Mannheim, Germany) using Hoxbes2 gene-specific primers, forward primer 59-CGGCACGAGGACTGCCGG-39 and reverse primer-59-GGGTTCTCTCGACAGCCCC-39, which encompass the 581-bp fragment including the conserved region showing homology to Hoxb2 (Prabagaran et al, 2007). The PCR conditions used were as follows: initial denaturation at 94uC for 2 minutes; 35 cycles of denaturation at 94uC for 45 seconds, annealing at 55uC for 30 seconds, and extension at 60uC for 45 seconds; and a final extension at 72uC for 7 minutes. Amplification of a 580 bp b-actin housekeeping gene served as the internal control.

Immunohistochemistry The immunohistochemical analysis of the HOXBES2 protein in the epididymis followed by image analysis of immunoreac-

758 tivity was performed based on the protocols published earlier by our laboratory (Prabagaran et al, 2007). In brief, the epididymal tissue sections were immunostained with a goat polyclonal HOXB2 antibody diluted 1:25 in blocking buffer for 1 hour at room temperature. Controls were incubated with nonimmune goat serum at a dilution similar to that used for the primary antibody. All other protocols were followed according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, Calif). Finally, the sections were dehydrated through an alcohol gradation (10%–100%), cleaned in xylene, mounted in p-xylene-bis (pyridinium bromide) Permount, and observed under a microscope (Leitz, Oberkochen, Germany). Immunoreactivity to the HOXB2 antibody was analyzed using Biovis Image Plus 2.0 software (Expert Vision Labs, Mumbai, India) based on the intensity of the color reactions observed in the epididymal tissue sections.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blots For determination of the expression pattern of HOXBES2 protein in the epididymides of control and experimental rats, the epididymal segments were dissected, minced, and incubated in 10 mL of 0.1 M phosphate-buffered saline (pH 7.4) containing 0.1 M phenylmethylsulphonyl fluoride for 30 minutes at room temperature to release the sperm, followed by extraction of protein as described earlier (Hu et al, 2003). The protein concentrations of the extracts were estimated by the method of Folin-Lowry (Lowry et al, 1951). Aliquots of 40 mg protein/lane of each tissue extract were analyzed on a 10% sodium dodecyl sulfate polyacrylamide gel (Laemmli, 1970) followed by Western blot analysis (Towbin et al, 1979). For identification of the immunoreactive protein, the blot was incubated with an affinity-purified monospecific goat polyclonal primary antibody (HOXB2 [P-20], SC-17165; Santa Cruz-Biotechnology) generated by epitope mapping within an internal region of the human HOXB2 protein. This was followed by incubation with a 1:1000 diluted horseradish peroxidase (HRP)–conjugated rabbit anti-goat secondary antibody (Bangalore Genei, Bangalore, India). For developmental expression of AR in the epididymis, another blot was probed using a rat monoclonal primary antibody to AR (Upstate Inc, Chicago, Ill) at a dilution of 1:2000. This was followed by incubation with a 1:2000 diluted HRP-conjugated rabbit anti-rat secondary antibody. The peroxidase activity was detected using enhanced chemiluminescence (Amersham Biosciences). The blots were stripped and reprobed with a bactin monoclonal antibody to confirm equal loading of protein samples on the gel.

Results Effect of Castration on HOXBES2 Expression In the castrated animals, as seen in Figure 1, a significant decrease in the luminal diameter of the epididymis was observed. This effect was more pronounced in the initial segment, caput, corpus, and followed by cauda epidid-


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ymis. HOXBES2 levels in the epididymis also decreased to $90% of that observed in the sham-operated animals and further declined to 5% 1 week postcastration. Circulating T levels following castration decreased within 5 days when compared with levels in the shamoperated controls (data not shown). Since bilateral orchiectomy lowers blood T concentrations to undetectable levels, the dramatic decrease in HOXBES2 levels following castration suggests that T may play a role in the regulation of Hoxbes2 gene expression. This was further shown when DHT supplementation to rats 1 week postcastration resulted in restoration of HOXBES2 expression in the epididymis to levels 50% to 70% of that observed in the sham-operated animals (Figure 2). The specificity of the immunoreaction was confirmed by immunolocalizing the sham-operated and castrated epididymal tissue sections independently using normal goat serum at a dilution similar to the primary antibody or with secondary antibody alone.

HOXBES2 Expression in the EDS-Treated and Supplemented Rats In the EDS-treated epididymis, HOXBES2 expression decreased drastically to almost undetectable levels as compared with that seen in the vehicle-treated controls as visualized immunohistochemically (Figure 3). Expression of the protein was restored following supplementation with DHT alone and/or DHT + estradiol combined. In the DHT + estradiol–supplemented group, HOXBES2 expression was comparatively higher than that observed in the DHT alone group. In the EDStreated and estradiol alone–administered rats, the presence of nodules and complete absence of HOXBES2 expression were observed in the epididymis (Figure 4). Western blot analysis of HOXBES2 expression in the control, EDS-treated, and supplemented rats further corroborated the findings obtained by immunohistochemistry (Figure 5). Quantitative analysis of the microscopic observations on protein localization was enabled using Biovis Image Plus software. The histogram derived from the protein localization data confirmed the immunohistochemistry findings (Figure 6). Circulating levels of T were assayed by RIA on days 0, 5, and 10 following EDS treatment, and the concentrations, measured in ng/mL, were expressed as means 6 SEM from 5 animals (Figure 7). A significant decrease in T levels was observed in the EDS-treated and EDS + estradiol–injected rats when compared with the vehicle alone–treated animals.

Influence of Testicular Factors on HOXBES2 Expression At 1 to 15 days following unilateral efferent duct ligation, HOXBES2 levels in the ligated and unligated

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Figure 1. Immunohistochemical localization of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) in the castrated rat epididymis. Sections of the epididymal segments from various groups of rats were immunolocalized with a HOXB2 antibody and nonimmune goat serum or secondary antibody alone (negative control) as described under ‘‘Materials and Methods’’ and counterstained with hematoxylin. The data depicted represents staining for the HOXBES2 protein in epididymis of day 7 postcastration rats with DHT supplementation (day 14). The maximum expression of HOXBES2 in the distal segments of epididymis is also shown. P, principal cells; L, lumen; IT, intertubular connective tissue space; S, spermatozoa. Arrows indicate HOXBES2 localization in the principal cells. Bar 5 25 mm. Color figure available online at www.andrologyjournal.org.

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Figure 3. HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) expression in the ethylene dimethane sulfonate (EDS)–treated and supplemented rats. Epididymal segments (labeled at the bottom) from various groups of rats (seen on the right) were immunolocalized with a HOXB2 antibody and nonimmune goat serum or a secondary antibody alone (negative control) as described under ‘‘Materials and Methods’’ and counterstained with hematoxylin. P-A, negative control; P-B, vehicle treated; P-C, EDS treated; P-D, EDS + dihydrotestosterone (DHT) supplemented; P-E, EDS + DHT + estradiol injected; P, principal cells; L, lumen; IT, intertubular connective tissue space; S, spermatozoa. HOXBES2 expression in the EDS-treated epididymis (P-C) decreased drastically to undetectable levels. In the DHT alone and DHT + estradiol–supplemented group, expression of the protein increased gradually to near normal. In the DHT + estradiol– supplemented group, HOXBES2 expression levels were comparatively higher than those in the DHT alone group. An increase in intensity of the immunostaining beginning from the distal caput was visualized. Arrows indicate HOXBES2 localization in the principal cells. Bar 5 25 mm. Color figure available online at www.andrologyjournal.org.

epididymis were almost identical to each other and to those in the sham-operated rats. This was made evident both by Western blot analysis and immunohistochemistry, indicating that expression of the HOXBES2 protein is not dependent on the presence of testicular factors other than T in 15-day postligated epididymis (Figures 8 and 9).

Postnatal Expression of HOXBES2 in the Epididymis Developmental changes in the levels of immunoreactive HOXBES2 protein were evaluated by immunohistochemistry and Western blot analyses. In 5-day postnatal

rats, there was no immunostaining for HOXBES2 in the epithelial cells of the epididymis along its entire length. In the epididymis, a lumen was already present at day 5 but without any apparent content. Similarly, no staining was seen on postnatal days 10, 15, 20, 30, and 40. By postnatal day 50, principal cells with complete structural differentiation and the first appearance of peroxidase staining for the HOXBES2 protein were observed in the proximal regions of the epididymis. Postnatal days 55 and 60 were characterized by the presence of spermatozoa, and HOXBES2 staining increased gradually with a dramatic increase in the number of stained principal epithelial cells.

r Figure 2. The graphs represent the intensity of immunostaining (peroxidase reaction) observed in various segments of epididymis from different groups of rat. Significant increases in the integrated optical density (IOD) beginning from the distal caput were visualized in the sham-operated epididymis compared with the castrated group (P , .05). The values for HOXB2 homeodomain–containing epididymis-specific sperm protein expression levels in each segment of the epididymis are expressed as percentages of the IOD values of the sham-operated group. Data are means 6 SEM. **Significantly different than sham-operated animals; *Significantly different than castrated animals.

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Figure 4. Immunolocalization of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) in ethylene dimethane sulfonate (EDS) + estradiol–supplemented epididymis. Panels I, II, and III represent localization of HOXBES2 in the normal and EDS- and EDS + estradiol–treated epididymis with normal goat serum (negative control), and Panels I’, II’, and III’ depict localization with a HOXB2 antibody. Arrows indicate HOXBES2 localization in the principal cells. P, principal cells; L, lumen; IT, intertubular connective tissue space; S, spermatozoa. Color figure available online at www.andrologyjournal.org.

The intensity of the immunoperoxidase reaction products appeared similar to that observed in adults by postnatal day 70 onward, concomitant with markedly elevated circulating androgen levels. Although the epithelial cells were stained at 200 days, most of the immunoreactivity was localized in the lumen (Figure 10). In the epididymal tissue sections immunoreacted with normal goat serum or secondary antibody alone (negative control), there was no immunoreactivity in the epitheli-


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um, intertubular space, and spermatozoa. In tissues treated with an anti-HOXB2 antibody, HOXBES2 localization was observed in the apical region of the principal epithelial cells. The spread of staining from the apical region on day 50 to the entire cytoplasm from 55 days onward was demonstrated by image analysis (Figure 11). The differences in the localization of the HOXBES2 protein in the epididymis of different age groups were comprehensively complemented by Western blot analysis (Figure 12). Equal loading of the protein samples in all lanes was confirmed by stripping and reprobing the blot using a monoclonal antibody to bactin as an internal control. Comparison of the developmental profile of AR and HOXBES2 expression by Western blot analysis indicated that AR was present at all stages of epididymal development with a slight elevation in the later stages of development (Figure 13).

RT-PCR Analysis of Hoxbes2 in Rat Epididymis To determine the differential expression of the Hoxbes2 transcript in the immature and mature epididymis, RTPCR analysis was carried out using gene-specific primers in 2 different stages (15 days and 60 days) of development of the epididymis. Day 15 signifies the immature status of the epididymis and is accompanied by very low levels of T (10 ng/g tissue) and undifferentiated cells undergoing mitosis, and day 60 indicates the presence of adult levels of androgens (21 ng/g tissue) together with a fully differentiated epididymal epithelium and presence of sperm in all segments of the epididymis. RT-PCR analysis indicated the presence of the expected 581-bp Hoxbes2 product amplified from the 60-day-old rat epididymal RNA but not from the 15-

Figure 5. Western blot analysis of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) expression in ethylene dimethane sulfonate (EDS)–treated rats. A single band of approximately 30 kDa representing the HOXBES2 protein was identified when the blot was probed with a specific antibody. Protein samples (40 mg/lane) were prepared separately from pooled epididymis of control and treated (EDS-treated, EDS + dihydrotestosterone [DHT]–, EDS + DHT + estradiol–, and EDS + estradiol–supplemented) groups. The blot was reprobed with a b-tubulin (55 kDa) monoclonal antibody to confirm equal loading of proteins in all lanes. The blot indicates the presence of HOXBES2 in all lanes except the negative control and in the EDS-treated group.

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day-old immature epididymis. These results suggest that transcription of the Hoxbes2 gene may be regulated by androgens in the adult rat epididymis (Figure 14), and its expression pattern at these time points was in agreement with its protein expression.

Discussion We previously reported the identification and characterization of a novel 30-kDa homeoprotein from epididymis, HOXBES2. This epididymis-specific protein displayed cell type–specific expression with intense immunostaining in the supranuclear cytoplasm of the principal and basal epithelial cells. We also demonstrated that the HOXBES2 protein is coated onto the luminal spermatozoa and on the stereocilia layering the luminal epithelium (Prabagaran et al, 2007). In the present report, we conclusively demonstrated that the epididymal expression of the HOXBES2 homeoprotein is regulated by androgens. This inference was drawn using the approaches of biologic and chemical castration and anatomic manipulation such as unilateral EDL and postnatal developmental studies. Following castration, which eliminates both circulating androgens and testicular factors, T levels decreased to ,10 % of baseline values within 24 hours (data not shown), whereas HOXBES2 protein levels decreased gradually to $90% of the control levels by day 5 postcastration and disappeared completely by day 7. In the current study, the critical dependence of HOXBES2 expression on circulating T was demonstrated by a delayed T repletion regimen in which the castrated rats were injected daily with DHT beginning day 7 postcastration. The DHT supplementation restored the expression level of HOXBES2 gradually to approximately 70% of that measured in the sham-operated rats and maintained the circulating T to levels similar to those reported for caput fluid in the rat (Turner et al, 1994). Therefore, it could be inferred that the concentration of DHT used in this study was sufficient to restore HOXBES2 expression in the castrated epididymis. Nevertheless, the gradual retention in luminal morphology from caput to cauda and the progressive reappearance of HOXBES2 expression in principal epithelial cells following castration and DHT supplementation could be explained by the fact that the delivery of exogenous DHT through the general circulation may affect regionalized Hoxbes2 gene expression differently than T reaching the caput from the testis. Viger and Robaire (1992) reported that the expression of 5a reductase can be maintained by normal plasma T concentrations in the corpus and cauda but not in the initial segment, where it requires supraphys-

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iologic concentrations of plasma T. Presumably the genes that respond to a decline in the expression in all segments of the epididymis by 1 week following orchiectomy are regulated by circulating androgens and not testicular factors because the latter have been implicated in the regulation of gene expression in the proximal epididymis but not in the distal segments (Hinton et al, 1998). Altogether, the ablation of HOXBES2 expression within 1 week following castration and its re-establishment by DHT supplementation in caput, corpus, and followed by cauda epididymis indicated that the expression of the protein is under the control of circulating androgens. The EDS-treated rat model (Jackson, 1973) was used to establish the interrelationship between the intratesticular distribution of T and its influence on the expression of HOXBES2 in the adjacent epididymis. EDS treatment of adult male rats destroyed the T-secreting mature Leydig cell populations completely, and that resulted in the subsequent depletion of T in the epididymis (Kerr et al, 1985; Molenaar et al, 1985; Jackson et al, 1986; Molenaar et al, 1986; Morris et al, 1986). Immunohistochemical analysis indicated a significant decrease in the expression of the HOXBES2 protein in different segments of the epididymis following EDS treatment. Similar studies have previously shown that exposure of epididymal epithelial cells and sperm to EDS results in a significant decline in the secretion of proteins in the range of 34 to 38 kDa and decrease in progressive motility and velocity of spermatozoa (Klinefelter et al, 1992). Klinefelter et al (1994) reported that both castration and EDS treatment significantly compromise the fertilizing ability of sperm from the proximal cauda after 4 days of exposure. Most Leydig cells exhibit degenerative changes 12 hours after treatment, and all Leydig cells showed gross degenerative changes after 24 and 48 hours; these changes disappear completely by 4 and 14 days (Morris et al, 1986). In the current study, the absence of mature Leydig cells during the first 10 days following EDS treatment indicated that the development of new Leydig cells takes a longer time under this regimen. It was also suggested that the advanced precursor cells, capable of rapid proliferation into Leydig cells, are killed by EDS or inhibited from differentiation; the complete repopulation of Leydig cells was established approximately 49 days following a single dose of EDS. These changes were accompanied by a decrease in the levels of serum T and epididymal AR. When EDS-treated rats were subjected to an immediate T repletion regimen with DHT supplementation, HOXBES2 expression was restored gradually, concordant with the increase in the levels of T. Results similar to this have been reported for proteins such as 80-kDa human sperm antigen (Khobarekar et al, 2007)

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Figure 7. Serum testosterone levels in ethylene dimethane sulfonate (EDS)–treated and supplemented rats. Circulating levels of serum testosterone were assayed by radioimmunoassay in the various groups and expressed in ng/mL serum. Each group consisted of 5 animals, and the results were expressed as means 6 SEM. A significant decrease in testosterone level was observed in the EDStreated and EDS + estradiol–injected rats.

and lipocalin-type prostaglandin D synthase (Zhu et al, 2004) in the castrated and EDS-treated rat epididymis. In the case of DHT and estradiol combined supplementation, HOXBES2 expression was comparatively higher than that observed in the DHT alone–supplemented group. This could be attributed to the response from estrogen receptors a (ERa) and b (ERb) in the efferent ducts and epididymis (Meistrich et al, 1975; Fisher et al, 1997; Hess et al, 2002). Given that the postnatal epididymis contains receptors for ERb (Atanassova et al, 2001) and that the major circulating steroid during postnatal development is 3a-diol (Moger, 1977), a putative ligand for ERb, an argument can be proposed in favor of a role for estrogen in the expression of HOXBES2. Earlier reports indicated that the combined treatment of rats with estradiol (0.02 mg) and androgens maintained normal motility and transport of spermatozoa (Bandopadhyaha et al, 1974). Li et al (2003) reported that the expression of proteinase inhibitors such as cystatin E1 and E2 were up-regulated by estrogens in the mouse epididymis. However, HOXBES2 expression was not restored in the estradiol alone–administered EDS-treated rats. This observation was similar to the report by Gupta et al (1991) on the expression of certain glycolytic enzymes in the rhesus monkey epididymis. These data may possibly explain the differential response in epididymal gene expression to androgens and estrogens in different mammalian species. On the other hand, it emphasizes the importance of the androgen:estrogen balance in epididymal func-

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Figure 8. Effect of testicular factors on HOXB2 homeodomain– containing epididymis-specific sperm protein (HOXBES2) expression. Western blot analysis showed the presence of HOXBES2 in ligated and unligated epididymis when the blot was probed using a specific antibody. Protein samples (40 mg/lane) extracted from epididymis were pooled individually from both groups (n 5 5). The blot was stripped and reprobed with a b-tubulin (55 kDa) monoclonal antibody to confirm equal loading of protein samples.

tion. Disturbance in this balance, particularly lowering the androgen and simultaneously elevating the estrogen, could result in epididymal abnormalities described in the present study. The fact that these abnormalities are associated with changes in the expression of both AR and ERa reinforces the close functional relationship between androgen and estrogen in the maintenance of HOXBES2 expression in the epididymis. Further, the duality of epididymal response to the 2 sex steroids with respect to HOXBES2 expression suggests that these 2 hormones could exert their action in concert at the physiologic level as regulators of epididymal secretory function. Testicular factors are known to influence the expression of several proteins in the epididymis (Hinton et al, 1998; Hermo et al, 2000). In this study, the efferent ductules of 1 of the testes were ligated to determine whether the testicular factors are necessary to maintain HOXBES2 expression in the epididymis. EDL, which prevents the flow of testicular fluid into the epididymis, did not elicit any effect on epididymal HOXBES2 expression. Therefore, it could be inferred that the expression of HOXBES2 is dependent mainly on circulating and luminal androgens. Similar results have

r Figure 6. The histogram was derived from image analysis of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) localization represented by integrated optical density (IOD) of the immunostaining (peroxidase reaction) observed in the epididymal segments of different experimental groups. The values for HOXBES2 levels in each segment of the epididymis are expressed as percentages of the IOD values of the vehicle-treated control. Data are expressed as means 6 SEM (n 5 5; P , .05). **Significantly different than vehicle-treated control; *Significantly different than ethylene dimethane sulfonate–treated rats.

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Figure 9. Immunolocalization of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) in ligated and unligated epididymis. Localization of HOXBES2 (arrow) was seen in the principal epithelial cells of both ligated and unligated rat epididymis. The figure depicts HOXBES2 localization in the epididymis of a rat sacrificed on postnatal day 15. The fluid volume and sperm count were either reduced or absent in the lumen of the ligated epididymis. P, principal cells; L, lumen; IT, intertubular connective tissue space; S, spermatozoa. Bar 5 25 mm. Color figure available online at www.andrologyjournal.org.

been reported for the androgen-dependent, tight-junction epididymal glycoprotein cadherins (Cyr et al, 1995), epididymal protein B/C (Brooks and Higgins, 1980), and proenkephalin (Garrett et al, 1990). The gradual and significant increase in HOXBES2 expression was observed during postnatal development of the rat epididymis. Epididymal proteins such as acidic epididymal glycoprotein (Charest et al, 1989) and protein SP (Faye et al, 1980) are also known to exhibit similar postnatal expression patterns. This is in tandem with the acquisition of hormonal maturation of the epididymis with increases in age. As early as 2 weeks after birth, the epididymis consists of narrow cells in the initial segment and clear cells in the remainder of the epididymis. After 3 weeks, the epididymis acquires detectable activity of the enzyme 5a reductase, although the testicular production of T still remains low. Nevertheless, the immunoreactivity for HOXBES2 was not observed in the epididymis during that time point. By day 39, the principal cells of the epididymis attain adult-like structural features (Hermo et al, 1992a) along with high levels of luminal androgens (Scheer and Robaire, 1980). The initiation of HOXBES2 expression between day 40 and 50 coincides with the complete differentiation of principal cells from columnar cells and their further differentiation to principal cells and apical cells. The fact that the HOXBES2 protein is barely detectable in the epididymis at 10 days and between postnatal days 21 and 40, despite high concentrations of androgens and AR mRNA (Charest et al, 1989), indicates that the differentiating principal cells may simply be unable to express Hoxbes2 mRNA, which in turn supports our earlier observation that the localization of the HOXBES2 protein is mainly restricted to the principal cells that appear only on day 30 onward.

The increase in the number of HOXBES2-positive cells and the intensity of HOXBES2 immunostaining observed between postnatal days 60 and 70 onward coincides with the increasing T levels in the epididymis to mature levels (21 ng/g tissue) and a concomitant rise in AR protein levels. The Hoxbes2 transcript (581-bp RT-PCR–amplified product) could not be detected in epididymal RNA on day 15 but was present in 60-dayold epididymis, which is in agreement with the results obtained by immunohistochemistry and Western blot analyses. Calandra et al (1974) reported that the AR protein in rat epididymis is not detectable until 20 days. A parallel study that we carried out to compare the ontogeny of AR to HOXBES2 expression among different age groups indicated that AR was present at all stages with a slight elevation in its expression during the later stages of epididymal development. Although our observation was contrary to earlier reports, several studies have suggested that the expression of AR is essential for the early development and differentiation of the male reproductive tract, particularly the epididymis from the wolffian duct. Moreover, the developmental increase in the levels of HOXBES2 is of functional importance because it occurs simultaneously with the first appearance of sperm in the epididymis and immediately after the significant rise in levels of ABP and 5a reductase. The dramatic increase in HOXBES2 expression following this period could be due to the appearance of critical factors regulating the expression of the transcript for HOXBES2. Several factors can be proposed to address the maturation-dependent regulation of the HOXBES2 protein. Garrett et al (1990) suggested that sperm may be an essential factor for the expression of proenkephalin mRNA that appears on day 49 in the proximal initial segment and day 56 in the

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Figure 10. Developmental regulation of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) in the epididymis. Sections of epididymis from rats aged 5, 10, 15, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 120, and 200 days were immunolocalized using normal goat serum (negative control; Panel IA–O) and HOXB2 antibody (Panel IIA9–O9). Immunohistochemistry demonstrated that the staining (block arrow) for HOXBES2 begins to appear in the apical compartment of the principal epithelial cells between day 40 and 50 and increases gradually from day 60 onward. At day 90, HOXBES2 immunoreactivity was more intense and was prominent in the lumen of the epididymis. P, principal cells; L, lumen; IT, intertubular connective tissue space; S, spermatozoa are shown. Bar 5 25 mm. Color figure available online at www.andrologyjournal.org.

corpus and cauda regions coinciding with that of adult ABP expression. However, this cannot be the case for HOXBES2 because the expression of this protein occurs even in the absence of testicular factors as demonstrated

by unilateral EDL studies. Further, the expression of the HOXBES2 protein was found to be maximal in the corpus and caudal regions of the epididymis. These data reiterated our earlier findings that the HOXBES2

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Figure 11. Postnatal HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) expression in the developing epididymis. The histogram obtained from image analysis as described under ‘‘Materials and Methods’’ was derived based on the level (percent of area stained or total integrated optical density [IOD] of immunostaining) of HOXBES2 localization in the epididymis during development of rats. The values for HOXBES2 levels are given as percentages of the IOD values of the 200-day-old epididymis. **Significantly different than days 90–200; *Significantly different than days 5–40. Results are expressed as means 6 SEM (P , .05).

protein is maximally expressed in the distal regions of the epididymis from where the protein is transferred to the transiting testicular spermatozoa (Prabagaran et al, 2007). In addition, in silico analysis of the 1657-bp Hoxbes2 gene (GenBank accession number DQ399532) indicated the presence of a putative functional androgen response element in the 39 untranslated region at positions 1578 to 1592 bp. This observation suggests

that the Hoxbes2 gene could be regulated directly by the circulating and luminal androgens. Taken together, this finding concurs with the observations from orchiectomized, EDS-treated, and EDL adult rats, revealing the dependence on T or its metabolites for HOXBES2 expression in the epididymal principal cells. Finally, the outcome of our study suggests that HOXBES2 expression is regulated by androgens and

Figure 12. Western blot analysis of HOXB2 homeodomain–containing epididymis-specific sperm protein (HOXBES2) expression in the epididymis during postnatal development. Proteins extracted from adult rat epididymis of different age groups were pooled separately, and 40 mg/lane was analyzed. HOXBES2 expression first appeared between days 40 and 50 and began to increase gradually from day 60 onward. No band was observed when the blot was probed with normal goat serum (negative control; data not shown). The blot was stripped and reprobed with a b-actin monoclonal antibody to confirm equal loading of proteins in all lanes.

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Figure 13. Western blot analysis of androgen receptor (AR) protein during postnatal development. Expression of AR protein was observed in the epididymis of all age groups from day 5 to 200. The AR level was comparatively higher in mature animals than in prepubertal rats. Equal loading of proteins in all lanes was confirmed by stripping and reprobing the blot with a monoclonal antibody to b-actin.

the developmental status of the epididymis and attains considerable significance in the realm of epididymal sperm maturation. Being a member of the homeobox gene cluster as a conserved HOXB2 homeodomaincontaining protein, the androgen regulation of the HOXBES2 protein indicates that the Hoxbes2 gene might be involved in a cascade of androgen-regulated events in the epididymis. Therefore, future studies would

be directed to further delineate the physiologic role of this androgen-dependent, developmentally regulated, and conserved HOXBES2 homeoprotein in sperm function.

Acknowledgments The authors gratefully acknowledge Dr C.P. Puri, Director of ICMR, for his constant support and encouragement. We are grateful to Professor A.J. Rao (Indian Institute of Science, Bangalore, India) for the kind gift of EDS used in this study. The authors express sincere thanks to Dr Geetanjali Sachdeva, Senior Research Officer, for assisting in editing the manuscript and Mr Ravi B. Kadam, Mr Hemant C. Karekar, and the institute’s animal house staff for their technical assistance.

References

Figure 14. Reverse transcriptase polymerase chain reaction (RTPCR) analysis of the Hoxbes2 gene in rat epididymis. An expected 581-bp RT-PCR product of the Hoxbes2 gene was obtained from 60day-old adult rat epididymal RNA using Hoxbes2-specific nested primers, forward 59-CGGCACGAGGACTGCCGG-39 and reverse 59GGGTTCTCTCGACAGCCCC-39, which encompass the 581-bp fragment that includes the conserved region showing homology to Hoxb2. No band was seen in the lane of 15-day-old epididymis and the negative control (in which reverse transcriptase or the cDNA template was omitted). A 580-bp b-actin amplified transcript served as the internal control.

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