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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 region-specific 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 (250μg/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.
The 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 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 al, 2001). Androgens such as testosterone (T), 5α-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-3α, 3α-diol, and 17β-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 5α 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 × 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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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 5α reductase can be maintained by normal plasma T concentrations in the corpus and cauda but not in the initial segment, where it requires supraphysiologic 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) 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 α (ERα) and β (ERβ) 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 ERβ (Atanassova et al, 2001) and that the major circulating steroid during postnatal development is 3α-diol (Moger, 1977), a putative ligand for ERβ, 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 μg) 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 function. 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 ERα 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 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 5α 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-day-old 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 5α 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 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 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 3′ 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 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 homeodomain-containing 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.