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X-ray fluorescence spectroscopy (XRF) is an analytical technique employed to determine the elemental composition of diverse materials. Due to its nondestructive nature and direct analysis that requires little or no sample preparation, it has been particularly useful for investigating the mineral composition of plants and soil. However, commercially available XRF benchtop equipment often restricts this type of experiment in plant science due to the volume of the sample chamber and the source–detector geometry. To overcome this problem, we developed an XRF setup that prioritizes in vivo-based experiments. The equipment is equipped with a 4 W Ag X-ray tube and a silicon drift detector. The detection limits are comparable to those of commercial instruments and suitable for evaluating plant tissues. Finally, a case study using tomato plants as a model species and rubidium (Rb+) and strontium (Sr2+) as tracers for potassium (K+) and calcium (Ca2+), respectively, demonstrated their feasibility for long-term in vivoanalysis. Therefore, the present XRF system stands out as a viable and cost-effective tool for assessing the absorption and transport of minerals in plant tissues probed by time-resolved in vivoX-ray spectroscopy.

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)+ macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE−/− mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.

Activation of soluble adenylyl cyclase (sAC) by bicarbonate causes local cAMP generation, indicating that sAC might act as a pH and/or bicarbonate sensor in kidney cells involved in acid-base homeostasis. Therefore, we examined the expression of sAC in renal acid-base transporting intercalated cells (IC) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all IC, sAC and V-ATPase showed considerable overlap under basal conditions, but sAC staining was also found in other cellular locations in the absence of V-ATPase. In type A-IC, both sAC and V-ATPase were apically and subapically located, whereas in type B-IC, significant basolateral colocalization of sAC and the V-ATPase was seen. When apical membrane insertion of the V-ATPase was stimulated by treatment of rats with acetazolamide, sAC was also concentrated in the apical membrane of A-IC. In mice that lack a functional B1 subunit of the V-ATPase, sAC was colocalized apically in A-IC along with V-ATPase containing the alternative B2 subunit isoform. The close association between these two enzymes was confirmed by coimmunoprecipitation of sAC from kidney homogenates using anti-V-ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in IC, that they are concentrated in the IC plasma membrane under conditions that “activate” these proton secretory cells, and that they are both present in an immunoprecipitated complex. This suggests that these enzymes have a close association and could be part of a protein complex that is involved in regulating renal distal proton secretion.

Mice deficient in the ATP6V1B1 (“B1”) subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 (“B2”) isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1−/− mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1−/− mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of “rod-shaped” intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28–40% of normal) V-ATPase function is preserved in medullary ICs from B1−/− mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.

The mammalian aquaporins (AQPs) are a family of 13 transmembrane channel proteins that are involved in the transport of water in numerous organs. In the male excurrent duct, the movement of fluid and solutes across the epithelium is essential for establishing the proper luminal environment in which sperm mature and are stored. AQP9 is abundantly expressed in the efferent ducts, the epididymis, and the vas deferens, where it could represent an important apical pathway for transmembrane water and solute movement. However, other organs in which water transport is critical, including the kidney, the lung, or the eye, express several different AQPs with a cell-specific pattern. To undertake a systematic analysis of the expression of known AQPs in the postnatal and adult rat epididymis, we examined the expression of their respective mRNAs in epithelial cells isolated by laser capture microdissection (LCM), and we determined their corresponding protein expression pattern by immunofluorescence and Western blotting. Our data show that, whereas AQP9 is the main AQP of the epididymis, the mRNA specific for Aqp2, 5, 7,and 11are also expressed in epididymal epithelial cells. AQP5 protein colocalizes with AQP9 in the apical membrane of a subpopulation of principal cells in the corpus and cauda regions. Aqp2mRNA was detected in epithelial cells after the second postnatal week and the amount significantly increased up to adulthood. However, AQP2 protein was detected only in the distal cauda of young rats (between the second and fourth postnatal week). No AQP2 protein was detected in the adult epididymis, indicating that posttranscriptional mechanisms are involved in the regulation of AQP2 expression. In addition, epididymal epithelial cells express significant amounts of the mRNAs coding for AQP7 and 11. No mRNA or protein for AQPs 0, 4, 6, and 8 were detectable in epithelial cells, and Aqp1 was detected in whole epididymal samples, but not in epithelial cells. Thanks to the recent development of microdissection technologies, our observations suggest that epididymal epithelial cells express several members of the AQP family with a region-specific pattern. AQPs may be involved not only in the transepithelial transport of water in the epididymis but also in the postnatal development of this organ, as suggested by the differential expression of AQP2.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the β2 integrin CD11c. In order to determine the cause of CD11cgene expression in HCL theCD11cgene promoter was characterized. Transfection of theCD11cpromoter linked to a luciferasereporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11cpromoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reducedCD11cpromoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11cpromoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal–regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11cpromoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11cpromoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11cgene characteristic of HCL is dependent upon activation of the proto-oncogenes rasand junD.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the β2 integrin CD11c. In order to determine the cause of CD11c gene expression in HCL theCD11c gene promoter was characterized. Transfection of theCD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reducedCD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal–regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11cpromoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11cgene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.

CD43 is an abundant, heavily glycosylated molecule expressed specifically on the surface of leukocytes and platelets. When leukocytes are at rest, CD43 acts to prevent both homotypic and heterotypic interactions. However, during leukocyte activation CD43 expression is repressed, facilitating the intercellular contact required for chemotaxis, phagocytosis, aggregation, adhesion to endothelium, and transendothelial migration. Consequently, CD43 repression plays a vital role both in innate and acquired immunity. Here we report that a dramatic down-regulation of CD43 mRNA levels occurs during activation of the leukocytic cell line K562. This repression coincides with repression of the transcriptional activity of the CD43 gene promoter. We have determined that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and Purα act together to mediate repression of the CD43 promoter during K562 activation. The hnRNP-K molecule and Purα bind single-stranded DNA. Therefore, exposure of single-stranded structures within theCD43 promoter probably plays a major role in effectingCD43 repression.

CD43 is an abundant, heavily glycosylated molecule expressed specifically on the surface of leukocytes and platelets. When leukocytes are at rest, CD43 acts to prevent both homotypic and heterotypic interactions. However, during leukocyte activation CD43 expression is repressed, facilitating the intercellular contact required for chemotaxis, phagocytosis, aggregation, adhesion to endothelium, and transendothelial migration. Consequently, CD43 repression plays a vital role both in innate and acquired immunity. Here we report that a dramatic down-regulation of CD43 mRNA levels occurs during activation of the leukocytic cell line K562. This repression coincides with repression of the transcriptional activity of the CD43gene promoter. We have determined that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and Purα act together to mediate repression of the CD43promoter during K562 activation. The hnRNP-K molecule and Purα bind single-stranded DNA. Therefore, exposure of single-stranded structures within theCD43promoter probably plays a major role in effectingCD43repression.

A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens.

The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.

We previously showed that basal cells send "antenna-like" body projections to the lumen and act as both sensors that survey the luminal environment, and transmitters that communicate their findings to neighboring proton-secreting clear cells. We have, thus, uncovered a novel luminal signal detection and intercellular interaction mechanism for the regulation of luminal acidification, a critical process for sperm maturation and viability. We investigated here whether or not this novel property of basal cells is triggered at puberty, and followed their appearance and morphology during postnatal development in the rat epididymis. Immunofluorescence labeling for the basal cell markers, claudin-1 and keratin-5, showed that basal cells are absent at birth, and appear initially in the vas deferens during the first postnatal week (PNW). They then gradually appear within the epididymal epithelium in a retrograde manner from the cauda to the initial segments. They form a continuous cell layer at the base of the epithelium at PNW3 in the cauda, and at PNW 5 in the initial segments. Interestingly, during the first and second PNWs, basal cells have different morphological characteristics: some are similar to adjacent epithelial cells, with their apical pole in contact with the lumen and nucleus located at the same height, some have their nucleus in the basal region of the epithelium but extend a narrow body projection towards the lumen, and some have the characteristic basal cell "dome-shape" appearance and are located underneath adjacent epithelial cells. Double-labeling for claudin-1 and the tight-junction protein ZO-1, followed by laser scanning confocal microscopy 3D reconstruction, confirmed that basal cells are in contact with the lumen as early as at PNW 1 in the vas deferens and PNW 2 in the cauda. In the trachea, basal cells are believed to be progenitors of other epithelial cell types. We, thus, examined whether they might be progenitors of clear cells, whose sequence of appearance in the epididymis is similar to that of basal cells. We found that clear cells (labeled for the V-ATPase) and basal cells appear simultaneously but independently within the epithelium. These results are not compatible with the hypothesis that basal cells are progenitors of clear cells. Previous studies proposed that basal cells might have immunological properties. We found that basal cells are distinct from the network of dendritic cells that populate the base of the epididymal epithelium (visualized in CX3CR1-GFP mice). Basal cells are also distinct from F4/80 and CD206 expressing macrophages, which are also present in rat and mouse epididymis. In summary, we show here that during early development basal cells can be in contact with the luminal border of the epithelium. Thus the apical-reaching property of basal cells does not depend on the appearance of spermatozoa or production of steroid hormones that occur during puberty. Basal cells are also distinct from dendritic cells, and do not express two macrophage markers, F4/80 and CD206. Further studies will be required to determine whether they have other immunological characteristics. This work was supported by an MGH Grant to WS and NIH DK085715 to SB.(poster)

The establishment and maintenance of male fertility requires close interactions between the reproductive, endocrine and immune systems. Yet, the cellular and molecular mechanisms that prevent the development of an autoimmune response against gametes, while maintaining the ability to fight pathogens and cancer cells, are still not well understood. We recently showed that the murine epididymis is populated by a dense network of dendritic cells (DCs). Isolated epididymal DCs (eDCs) take up, process and present ovalbumin to activate the proliferation of T cells in vitro, however, the functions of eDCs in vivo remain to be elucidated. The present study aims at characterizing the behavior of eDCs following a "danger signal" induced by 1) efferent duct ligation (EDL) and 2) vasectomy (Vx), to prevent sperm and lumicrine factors from entering or leaving the epididymis, respectively. The modulation of the eDC network was analyzed using CD11c-EYFP and CX3CR1-GFP mice, and fluorescence microscopy. We focused on the short-term effects of EDL and Vx (1-5 days after procedure). Under homeostatic conditions, the entire epididymal duct is surrounded by CD11c- and CX3CR1-positive eDCs that establish intimate interactions with the epithelium. Immunogold electron microscopy revealed that eDC bodies are located between the basement membrane and the basal pole of epithelial cells. In the initial segments (IS), eDCs project slender intraepithelial dendrites toward the lumen. Within 24 hours after efferent duct ligation, the eDC network was dramatically reorganized in the IS, but not in other epididymal regions. IS eDCs lost their stellate morphology, became larger and relocated deeper towards the lumen into the epithelium. Some intraluminal CD11c+ and CX3CR1+ cells were also visible. This modulation of eDC location and morphology was transient and temporally correlated with the EDL-triggered wave of epithelial apoptosis that has been described in the IS by other groups. Indeed, IS eDCs were loaded with TUNEL-positive material. eDCs returned to a "normal" dendriform appearance 4-5 days after EDL. Surprisingly, the behavior of eDCs following vasectomy was radically different. Vx induced a rapid disappearance of most peritubular DCs from cauda epididymidis. This was accompanied by a dramatic disruption of the blood-epididymis barrier: less than 24 hours after Vx, ZO-1 immunolabeling revealed that the tight-junction barrier is breached, and extravasated sperm were visible in the cauda and vas deferens, upstream of the ligation site. 5 days after Vx, peritubular eDCs remained virtually absent from the cauda, though interstitial CD11c+ and CX3CR1+ were visible. Corpus, caput and initial segments eDCs were not visibly affected by Vx. EDL and Vx affected eDCs in a totally different manner. Quickly following EDL, IS eDCs relocated into the epithelium and engulfed apoptotic cells, suggesting that these cells could be responsible for the rapid removal of apoptotic and dead epithelial cells and/or sperm. In contrast, eDCs from cauda epididymidis do not seem to have the ability to clean up the dramatic tissue damage caused by Vx. While our previous work had shown that the eDC population is characterized by a phenotypical and morphological heterogeneity, these new data indicate that eDCs have also striking region-specific functional characteristics. Study supported by NIH grants DK043351, DK038452 and DK085715.(platform)

In the epididymis, a luminal acidic pH maintains sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) located in the apical membrane of clear cells is a major contributor to proton secretion in this organ. We showed that luminal alkaline pH triggers the apical membrane accumulation of V-ATPase in a calcium-dependent manner. However, the link between alkaline pH and intracellular calcium is not completely elucidated. We examined the expression of some pH-sensitive purinergic receptors by RT-PCR in rat epithelial cells isolated by laser capture microdissection. mRNA transcripts for P1 adenosine receptors (subtypes A1, A2B and A3) and P2 ATP receptors (subtypes P2X1, P2X2, P2X4, P2X11, P2Y1 and P2Y2) were detected. mRNA transcripts for enzymes responsible for ATP hydrolysis such as ecto-ATPase, 5'-nucleotidase and alkaline phosphatase were also detected, indicating potential degradation of ATP into adenosine in the epididymal lumen. Alkaline phosphatase activity was detected in the apical pole of principal cells from the proximal cauda using a fluorescent phosphatase substrate (ELF 97) on fixed rat epididymal sections. In vivo luminal perfusion of rat and mouse epididymis showed that both ATP (600 μM) and adenosine (300 μM) induced apical membrane V-ATPase recruitment even in the absence of an alkaline pH stimulus. Addition of BAPTA-AM to the perfusate abolished the ATP-mediated effect, indicating the participation of calcium in this response. To evaluate the participation of cAMP in the adenosine-dependent V-ATPase activation pathway, rat epididymis was perfused in vivo with adenosine (300 μM) in the presence of inhibitors of downstream effectors of adenylyl cyclase. The myristoylated protein Kinase A inhibitor (mPKI) abolished the adenosine-mediated V-ATPase recruitment, indicating the participation of the cAMP/PKA pathway in this response. Altogether these data show that ATP and adenosine are sufficient to cause the accumulation of V-ATPase into the apical membrane of clear cells. The activation of purinergic receptors P1 and/or P2 might, therefore, play a significant role in the male reproductive tract by regulating proton secretion and, therefore, luminal acidification of the epididymal sperm environment. This work is supported by NIH grant HD40793.(platform)

All the components of the Kinin-Kallikrein system are present in the male reproductive system and regulate important functions related to reproductive physiology and fertility. A major protagonist of this system is the active nonapeptide bradykinin (BK), which is involved in pre-spermatogonial cell proliferation in the testis, in anion secretion in the epididymis, in smooth muscle contractions and prostaglandin production in the vas deferens, and in the regulation of motility and vitality of ejaculated spermatozoa. In the epididymis, water and solute transport is essential for establishing the proper luminal environment for sperm maturation and storage. Significant water re-absorption occurs in the proximal epididymis and participates in the concentration of spermatozoa, while water secretion occurs in the distal epididymis and helps to control the final fluidity of semen. In addition, glycerol - a sperm metabolic substrate is accumulated in the lumen of the epididymis. Both water and glycerol transport occur via the aquaglyceroporin AQP9, which is abundantly expressed in the apical membrane of principal cells. Most of the physiological activities induced by BK are mediated by two G protein-coupled receptors: BK1R, whose expression is induced by inflammation, and BK2R, which is constitutively expressed. Activation of these two receptors results in the release of intracellular calcium from internal compartments through phospholipase C (PLC) activation. The aim of this study was (1) to characterize the expression pattern of BK2R in the male reproductive tract, and (2) to investigate the role of BK on glycerol transport. mRNA expression of bk2r was examined during post-natal development by RT-PCR on RNA extracted from epithelial cells isolated by laser microdissection (LMD) from newborn, 1, 2, 3 and 4 week old pups and from adult rats. The bk2r transcript was expressed in increasing amounts during post-natal development up to the 3rd postnatal week. bk2r relative expression then decreased, but remained high, up to adulthood. The distribution of BK2R was examined by immunofluorescence on cryosections of adult rat efferent ducts and epididymis. Double staining of efferent ducts for AE2 a marker of ciliated cells and BK2R showed that BK2R is specifically expressed in the apical membrane of non-ciliated cells. Staining for BK2R showed a heterogeneous distribution in different epididymal regions with a moderate staining in the initial segment, absence of staining in the distal caput and corpus, and very intense staining in the cauda. Double-immunofluorescence for BK2R and the B1 subunit of the V-ATPase proton pump a marker of clear cells - showed that BK2R is exclusively expressed in the apical membrane of principal cells. Functional assays on isolated tubules perfused in vitro showed a 2.6 fold increase of apical membrane permeability to glycerol in the presence of luminal BK (1μM). This effect was abolished by pre-incubation with the calcium chelator BAPTA-AM (5μM). These data suggest a novel function of luminal BK in the epididymis as a regulator of AQP9-dependent glycerol transport in a calcium dependent manner via BK2R, a receptor abundantly expressed in the apical membrane of principal cells. In addition, the progressive increase in bk2r mRNA that occurs during the first three weeks of post-natal development indicates a potential role for BK2R in the maturation of the epididymal epithelium.