Expression of CFTR in the Epididymis of Control Adult Rats
CFTR, a cAMP-dependent protein kinase—activated chloride channel, has been located primarily in the apical plasma membrane of polarized epithelial cells of a variety of different tissues (Crawford et al, 1991; Riordan, 1993). In the rat testis, the CFTR message has been associated with developing germ cells, Sertoli cells during early postnatal development, and cultured Sertoli cells (Boockfor et al, 1989; Trezise and Buchwald, 1991). In the epididymis, the presence of CFTR is not novel, as a functional role for CFTR has been well described for several years (Wong et al, 2002). However, in the present study, it is revealed that CFTR shows cell- and region-specific expression, with principal, clear, and peritubular cells of the corpus and cauda regions presenting the most prominent reaction. There was no evidence of CFTR expression in narrow, apical, or basal cells of the entire epididymis. Thus, CFTR appears to play an important role in the distal regions of the epididymis.
The presence of weak CFTR expression in the initial segment would support the finding that water in this region is driven mainly from the lumen to the intertubular space. This has been suggested to be of importance for concentrating sperm in the small luminal diameter of the initial segment so as to better facilitate interactions of the sperm surface with the secretion products of the epithelial cells lining this region of the duct (Robaire and Hermo, 1988; Hess et al, 1997, 2002; Hess, 2002). In fact, in the initial segment, intense expression of AQP-9 has been localized to the microvilli of the principal cells, which may aid the passage of water from the lumen to the underlying intertubular space (Badran and Hermo, 2002).
In the corpus epididymidis, where there is a progressive accumulation of sperm, and in the cauda region, where they are stored, a maximal expression of CFTR was observed in the principal cells, and a weak-to-moderate reaction was observed in the clear cells. It has been noted that under basal conditions, various transporters located in the basolateral plasma membranes of the epididymal epithelial cells take up Cl− into their cytoplasm so that intracellular chloride activity is held above its electrochemical equilibrium. When cells are stimulated, there is an increase in intracellular cAMP, which activates CFTR, allowing chloride efflux into the lumen (Wong, 1988; Chan et al, 1995). Under such stimulated conditions, the net secretory flux is increased to a level that exceeds the reabsorptive flux, resulting in a net secretion of water into the lumen. In the cauda epididymidis where most of the testicular fluid would have been reabsorbed, the secretion of electrolytes and fluid would be imperative to prevent luminal dehydration (Wong et al, 2002). Thus, sperm in the corpus and cauda regions, while encountering immobilin, a protein that gradually fills the lumen of the cauda epididymidis to eventually immobilize them (Hermo et al, 1992), would nevertheless be maintained in a fluid environment created by the input and output of water, the fine-tuning of which is exerted by various transporter proteins (Wong et al, 2002), including the presence of CFTR in the principal and clear cells as demonstrated in the present study.
In addition to being a chloride channel, CFTR acts as a regulator of other membrane transport proteins, such as the epithelial Na+ channels and the outwardly rectifying Cl− channels (Schwiebert et al, 1995; Schreiber et al, 1999). CFTR has also been shown to activate AQP-3 in Xenopus oocytes, and the water permeability of respiratory cells was enhanced with CFTR activation (Schreiber et al, 1997, 1999). The present localization of CFTR on the apical surface of the principal and clear cells of the corpus and cauda regions, along with previous findings of AQP-9 expression in these 2 cell types (Badran and Hermo, 2002), suggests that these 2 proteins functionally interact with one another in the epididymis. Recently, the interaction between rat epididymal CFTR and AQP-9 has been visualized by expressing both in Xenopus oocytes. The results demonstrated that AQP-9 alone resulted in an increase in oocyte water permeability, which was further augmented by CFTR, and these results were also confirmed in the rat epididymis by in vivo studies (Cheung et al, 2003). Thus, there appears to be a synergistic effect of the 2 proteins in conferring water permeability. In this way, the fluidity of the epididymal lumen appears to be fine-tuned by the presence of both AQPs and CFTR, resulting in a microenvironment that is conducive not only for sperm maturation but also for their passage down the duct and storage in the cauda epididymidis. The finding of CFTR expression in peritubular myoid cells suggests that they also aid in the movement of water from the circulation to the lumen of the tubules.
Regulation of CFTR in Orchidectomized and Efferent Duct—Ligated Adult Rats
In the present study, there was a dramatic alteration in the staining pattern of CFTR in the epididymis following orchidectomy at early and late time points, and this was also observed with testosterone treatment. The alteration was seen as the complete absence of the thick intense band of reaction product over the apical plasma membrane of the principal cells of the corpus and cauda epididymidis. However, under such conditions, numerous small CFTR-positive vesicles appeared in the apical and supranuclear cytoplasm of the principal cells, which were not prominent in control animals. It is suggested, therefore, that the synthesis of CFTR-positive secretory vesicles from the Golgi apparatus is ongoing and unaffected following orchidectomy but that these vesicles in the absence of testicular factors do not bind with the apical plasma membrane. The administration of testosterone to orchidectomized animals also failed to restore staining to the apical plasma membrane, excluding the possibility that androgens are responsible for the targeting and binding of CFTR- Golgi—derived vesicles with the apical plasma membrane. As efferent duct ligation demonstrated similar results at early and late time points, it is suggested that testicular factors are not essential for regulating the synthesis of CFTR and its presence in Golgi-derived secretory vesicles. However, it would appear that luminal factors emanating from the testis are essential for the targeting and binding of Golgi-derived CFTR-secretory vesicles with the apical cell surface of the principal cells.
In contrast to the principal cells, the clear cells of the corpus and cauda epididymidis did not exhibit changes to CFTR expression after any experimental treatment or at early or late time points. In addition, the layers of the peritubular myoid cells, which envelop each epididymal tubule (Robaire and Hermo, 1988), maintained their intense reactivity. Thus, regulation of CFTR in the epididymis appears to be cell-specific.
Although it is well established that many epididymal functions are under the control of androgens (Robaire and Hermo, 1988; Orgebin-Crist, 1996; Cornwall et al, 2001; Ezer and Robaire, 2002), several proteins have been shown to be unaffected in their protein or mRNA expression by androgen withdrawal (Hermo et al, 2000; Luedtke et al, 2000; Cornwall et al, 2002; Hermo and Andonian, 2003). However, in addition to the regulation mediated by androgens, factors emanating from the testis that enter the epididymis via the lumen of the duct, defined as lumicrine factors, play a role in regulating epididymal functions (Hinton et al, 1998; Cornwall et al, 2002; Ezer and Robaire, 2002). Lumicrine factors derived from the testis have been shown to regulate several proteins synthesized by the epididymis. Indeed, ligation of the efferent ducts induces changes in epididymal gene and protein expression (Cornwall et al, 2002). The luminal testicular factors that regulate gene expression in the initial segment include ions, solutes, proteins, steroids, and even the spermatozoa themselves. These factors can upregulate or down-regulate gene expression after efferent duct ligation (Brooks, 1983). In fact, spermatozoa have been suggested to regulate expression of proencephalin in the rat initial segment (Garrett et al, 1991). While orchidectomy and efferent duct ligation prevent the entry of spermatozoa into the epididymis, they continue to be present in the corpus and cauda epididymidis even at the later experimental time points. This suggests that spermatozoa are not the lumicrine factor involved in regulating CFTR expression; however, further experimentation would be needed to confirm this point. In addition, while the same cell type along the epididymal duct may express a given protein, different factors appear to regulate its expression in different epididymal regions (Cyr et al, 1992; Andonian and Hermo, 2003). In the present study, regulation of CFTR adds an additional twist. While its synthesis by principal cells is not regulated by testicular factors, it appears that its targeting and binding with the apical plasma membrane, where it is normally found, are regulated by a lumicrine testicular factor, the identity of which remains to be determined.
An alternative possibility to the targeting and binding of de novo Golgi-derived CFTR secretory vesicles with the apical membrane may be the inhibition of CFTR vesicles recycling from endosomes to the apical plasma membrane. However, the recycling of apical membrane receptors normally occurs via tubular structures emanating from endosomes, referred to as the CURL, located at the apex of the cell (Geuze et al, 1983). In the present study, numerous CFTR-positive vesicles, not tubules, were evident, and these were also prominent in the supranuclear region of the principal cells where recycling does not normally occur.
It is of interest to compare the expression and regulation of CFTR with AQP-9. Principal cells express AQP-9 in the initial segment and cauda regions, while CFTR is expressed in the corpus and cauda regions. Clear cells express AQP-9 in the cauda region, while CFTR is expressed in the corpus and cauda regions. Thus, region-specific differences exist for these 2 proteins. AQP-9 expression in principal cells of the initial segment and clear cells of the cauda region was substantially reduced after efferent duct ligation and orchidectomy. As its expression was not restored to control levels by testosterone replacement, it was concluded that AQP-9 expression was regulated by a lumicrine testicular factor (Badran and Hermo, 2002). However, in the case of AQP-9 regulation, no accumulation of AQP-9-reactive vesicles was observed in the cytoplasm. Furthermore, no changes to the expression of AQP-9 were noted in the case of the principal cells of the caput, corpus, or cauda regions after either treatment (Badran and Hermo, 2002). Thus, differences clearly exist in the regulation of CFTR and AQP-9 expression in the principal and clear cells of the epididymis.
It has been documented that multiple regulatory pathways control the secretion of electrolytes and water transport across the epididymal epithelium, such as neurotransmitters, prostaglandins, bradykinins, and other peptide hormones (Wong et al, 1999). Prostaglandins (PGE2) synthesized in basal cells diffuse out and act on prostaglandin receptors on the basolateral membrane of the principal cells. This causes an increase in intracellular cAMP through receptor-G protein-coupled adenylate cyclase, which then activates CFTR, resulting in the secretion of anions and water (Wong et al, 2002). The present study further suggests that in vivo, a lumicrine factor derived from the testis regulates the targeting and binding of CFTR- Golgi—derived vesicles with the apical plasma membrane and that, in its absence, such vesicles are maintained and accumulate in the cytoplasm. The nature of this regulatory factor (or factors) needs to be examined in future studies.
In summary, the present data show that, in the epididymis, CFTR is distributed in a cell- and region-specific manner and that, although its synthesis is not under the control of testicular factors, its targeting to the apical plasma membrane of the principal cells is regulated by a testicular luminal factor.