Immunolocalisation of 11β-HSD-1 and -2, glucocorticoid receptor, mineralocorticoid receptor and Na+K+-ATPase during the postnatal development of the rat epididymis

Authors


Peter Roberts, School of Medical Sciences, Edith Cowan University, 270 Joondalup Drive, Joondalup, Perth, WA 6027, Australia. T: 61 8 6304 5455; F: 61 8 6304 5717; E:p.roberts@ecu.edu.au

Abstract

Glucocorticoids have been implicated in male reproductive function and 11β-HSD-1 and -2, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), all of which are known to modulate glucocorticoid action, have been localised in the adult rat epididymis, but their developmental expression has not been investigated. Na+K+-ATPase activity, responsible for sodium transport, is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, and has been localised in epididymal epithelium. This study examined the immunolocalisation of 11β-HSD-1 and -2, GR, MR and Na+K+-ATPase in rat epididymal epithelium (= 5) at postnatal days (pnd) 1, 7, 15, 28, 40, 60, 75 and 104, and relative mRNA expression of 11β-HSD-1 and -2, and GR at pre-puberty (pnd 28) and post-puberty (pnd 75). 11β-HSD-1, GR and MR were localised in the epididymal epithelium from pnd 1, and 11β-HSD-2 and Na+K+-ATPase reactivity from pnd 15. At pnd 28 there was maximal immunoreactivity for both the GR and MR and 11β-HSD-1 and -2. 11β-HSD-1 mRNA expression in the caput increased from pre- to post-puberty, whereas 11β-HSD-2 mRNA expression fell over the same period (< 0.01). GR mRNA expression was similar at pre- and post-puberty in both caput and cauda. Developmental changes in expression of 11β-HSD-1 and -2 suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but cell-specific expression of the 11β-HSD enzymes still provides a capacity for intricate local control of glucocorticoid exposure.

Introduction

The mammalian epididymis is essential for the transport, maturation, storage and protection of spermatozoa, which acquire their fertilisation potential during their passage through the epididymis (Robaire & Hermo, 1988; Turner, 1995; Hinton et al. 1996; Jones, 1998; Dacheux et al. 2003; Toshimori, 2003). The epididymal epithelium and luminal fluid are therefore highly specialised to maintain a suitable environment for the maturation of sperm (Hinton et al. 1988).

Glucocorticoid and mineralocorticoid hormones can modulate a range of functions in the epididymis, including carbohydrate and lipid metabolism (Balasubramanian et al. 1983, 1987), expression of secretory proteins (Courty, 1991), and ion and fluid transport (Munck et al. 1984). Glucocorticoids have also been linked to oxidative stress through their effects on the antioxidant defence system (Dhanabalan et al. 2010), and excess glucocorticoid exposure has been implicated in the overproduction of reactive oxygen species (Iuchi et al. 2003; Ozmen, 2005; Bjelakovic et al. 2007). As sperm have limited antioxidant defences of their own (Aitken & Delulis, 2010), the epididymis functions to protect sperm from the detrimental effects of oxidative stress through specialised antioxidant defence mechanisms (Veri et al. 1994; Latchoumycandane et al. 2002; Vernet et al. 2004; Martin-DeLeon, 2006).

Glucocorticoid and mineralocorticoid actions in target tissues are dependent on the local expression of specific glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Furthermore, glucocorticoids bind with equal affinity to both the GR and MR, and access of glucocorticoids and mineralocorticoids to their receptors can be modulated locally by the two 11β-hydroxysteroid dehydrogenase enzymes (11β-HSD-1 and -2; Krozowski, 1999). The 11β-HSD-1 enzyme acts both as a reductase (which activates inert cortisone to cortisol in humans and 11-dehydrocortisone to corticosterone in rats) and a dehydrogenase (which catalyses the reverse, inactivating reaction), although 11β-HSD-1 largely functions as a reductase in vivo. In contrast, 11β-HSD-2 acts exclusively as a dehydrogenase to inactivate excess cortisol or corticosterone, which allows aldosterone to bind to the MR in specific tissues. The 11β-HSD-1 and -2 enzymes have been localised in the adult rat (Waddell et al. 2003) and porcine (Sharp et al. 2007, 2009) epididymis. Similarly, the GR (Silva et al. 2010) and MR are present in rat epididymis (Hinton & Keefer, 1985; Pearce et al. 1986; Schultz et al. 1993), and the GR has also been identified in rat epididymal sperm (Kaufmann et al. 1992).

Sodium potassium-adenosine triphosphatase (Na+K+-ATPase) is a ubiquitous membrane protein present in all mammalian cells that plays a critical role in the regulation and maintenance of intracellular ion homeostasis (Fuller & Verity, 1990; Ilio & Hess, 1992; Whorwood & Stewart, 1995; Devarajan & Benz, 2000). Na+K+-ATPase activity is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, where it is important for epithelial sodium transport (Barlet-Bas et al. 1990; Ellis et al. 1987; Schmitt & McDonough, 1988; Fuller & Verity, 1990; Katz, 1990; Kinsella, 1990; Whorwood et al. 1994; Whorwood & Stewart, 1995). The Na+K+-ATPase isoform (α4) has been localised to rat and human sperm, and inhibition of Na+K+-ATPase α4 activity decreased sperm motility, suggesting a role for this enzyme in normal sperm function (Woo et al. 1999, 2000; Wagoner et al. 2005; Hlivko et al. 2006).

Although 11β-HSD-1 and -2, GR, MR and Na+K+-ATPase have been identified in the adult rat epididymis, their presence during epididymal development and differentiation has not been fully examined. Puberty is a period of development when significant changes occur in the reproductive tissues, including the epididymis, and it is likely that glucocorticoids play an active role in epididymal physiology during this crucial time. The objective of the present study, therefore, was to examine the spatial and temporal expression profiles of 11β-HSD-1 and -2, GR, MR and Na+K+-ATPase throughout postnatal development of the rat epididymis.

Materials and methods

Animals

Ethical approval for this study was received from the Edith Cowan University Animal Ethics Committee, and the University of Western Australia Animal Ethics Committee. This study used male Wistar rats in each of the age groups: postnatal day (pnd) 1, 7, 15, 28, 40, 60, 75 and 104 (= 5 per group). When their respective target ages were reached, animals were killed and the epididymides removed.

Tissue collection

All animals were anaesthetised at 09:00 hours on the allocated day with 5% (v/v) isofluorane in a mixture of 0.2 L min−1 oxygen and 0.8 L min−1 nitrous oxide, prior to administration of Lethobarb® (Virbac Australia Pty, Peakhurst, NSW, Australia) 1.0 mL kg−1 body weight, via peritoneal injection.

The right epididymides were removed, trimmed of fat and connective tissue, weighed and immediately placed into Histochoice Tissue Fixative (cat # H2904, Sigma-Aldrich, St Louis, USA) for processing for routine paraffin histology as previously described (Burton et al. 1996). The left epididymides were removed, trimmed of fat and connective tissue, and cut into three regions: the caput, corpus and cauda. Each region was immediately snap-frozen on liquid nitrogen prior to storing at −80 °C for subsequent mRNA analysis.

Immunohistochemistry

Immunohistochemistry was performed using 4-μm sections from five rats per group. The 11β-HSD-1 polyclonal antibody (RAH113) was raised against a synthetic peptide derived from the rat 11β-HSD-1 sequence (Obeyesekere et al. 1998). The 11β-HSD-2 immunopurified polyclonal antibody (RAH23) was raised against a C-terminal peptide derived from the cloned rat 11β-HSD-2 protein (Smith et al. 1997). The 11β-HSD-1 and -2 antibodies were kindly donated by Dr Zygmunt Krozowski. The GR affinity-purified rabbit polyclonal antibody raised against a peptide that mapped to the amino terminus of mouse GRα [GR (M-20), cat # sc-1004], the MR rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1–300 mapping at the amino terminus of MR of human origin [MCR (H-300), cat # sc-11412] and Na+K+-ATPase α1 goat polyclonal IgG (cat # sc-16041) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

To block endogenous peroxidase activity, sections were incubated in 3% hydrogen peroxide, and non-specific staining was further blocked by incubating the tissue sections in 2% (w/v) bovine serum albumin–phosphate-buffered saline–0.2% Triton X-100 for 20 min prior to incubation overnight with the primary antibody. Primary antibody dilutions were as follows: 11β-HSD-1, 1 : 500; 11β-HSD-2, 1 : 2000; GR, 1 : 1000; MR, 1 : 200; Na+K+-ATPase, 1 : 500.

Vectastain-ABC Peroxidation Standard Kits (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidene (DakoCytomation, Botany, NSW, Australia) were used to visualise positive immunoreactivity. A negative control (epididymis) was included with each immunohistochemistry run by omission of the primary antibody.

RT-PCR

Real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) was used to confirm and quantify local mRNA expression of 11β-HSD-1, -2 and GR in the caput and cauda epididymis at pnd 28 (pre-puberty) and pnd 75 (post-puberty). Total RNA was isolated from epididymides using Tri-Reagent (Molecular Resources Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. RNA integrity was assessed by agarose gel electrophoresis. Total RNA (5 μg) was used as a template for cDNA synthesis using M-MLV Reverse Transcriptase RNase H Point Mutant and random hexamer primers (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA, USA).

Analyses of expression levels for 11β-HSD-1 and -2 isoforms and for GR were performed by quantitative PCR on the Rotorgene 6000 (Corbett Industries, Sydney, Australia) using Immolase DNA polymerase (Bioline, Alexandria, Australia). Primers for 11β-HSD-1 and -2 and GR were designed using Primer3 software (MIT/ Whitehead Institute, http://www-genome.wi.mit.edu). Each of the selected primer pairs was positioned to span introns to ensure that no product was amplified from genomic DNA. Primers were used at a concentration of 0.2 μm, SYBR Green (Molecular Probes, Eugene, OR, USA) at 1 : 40 000 of stock, MgCl2 at 3 mm, and 0.5 U of Immolase enzyme per reaction. Cycling conditions included an initial denaturation at 95 °C for 10 min to activate the Immolase enzyme, followed by amplification for 45 cycles of the specific profiles indicated. The resulting amplicons were sequenced to confirm specificity. All samples were standardised against Rpl19 as previously described. Standard curves for each product were generated from gel-extracted (QIAEX II; Qiagen, Melbourne, Australia) PCR products using 10-fold serial dilutions and the Rotorgene 6000 software.

Statistical analysis

Two-way anova using epididymal zone and developmental age as factors were used to analyse variation in relative mRNA levels. Where the F-test reached statistical significance (< 0.05), specific differences were assessed by least significant difference tests. Where interactions were found between factors, two-tailed t-tests were used to compare between different ages within the same zone.

Results

The lumen of the epididymis was not fully developed at pnd 1, 7 and 15, and the pattern of immunoreactivity observed at these ages (see below) was similar throughout the length of the lumen and therefore not analysed for individual regions. At pnd 28, the caput and cauda regions were distinct, and by pnd 40 the four regions were clearly evident and were therefore analysed accordingly. Immature sperm were first observed at pnd 40 in the initial segments (IS) of the epididymides of all animals studied.

For the purpose of describing areas or patterns of localisation, the terms ‘scattered’ and ‘intermittent’ are used interchangeably to describe immunoreactive cells that are randomly spaced and appear only a few times within a given region. The term ‘isolated’ refers to an even lesser number of positive cells. Apical edge is the epithelial edge in contact with the epididymal lumen, and apical region is the epithelial cytoplasm closest to the lumen.

11β-HSD-1

A descriptive overview of the developmental pattern of 11β-HSD-1 localisation at all ages examined is provided in Fig. 1. Intensity of immunostaining and the types of cells observed are shown in Table 1. From pnd 28 to 75 there was strong to intense 11β-HSD-1 reactivity at the apical edge of epithelium in the caput, corpus and cauda, which became weaker by pnd 104. Strong reactivity was detected at the apical edge in the IS at pnd 1, 7, 15 and 28, but this became more evenly distributed throughout the apical half of epithelium at pnd 40, 60, 75 and 104. At pnd 60 and 75 a striped pattern of reactivity appeared in the caput and corpus as a result of scattered unreactive clear, apical, narrow and principal cells. From pnd 40, isolated unreactive basal cells were also observed in the IS, caput and corpus. At pnd 104, epithelial reactivity was very weak. Scattered principal cell nuclei were strongly reactive in the IS at pnd 15, 28, 40 and 60, and in the caput at pnd 40. At pnd 75 their reactivity was weak, and at pnd 104 undetectable. Isolated interstitial cells were intensely reactive for 11β-HSD-1 in all regions at all ages.

Figure 1.

 Overview of the immunolocalisation of 11β-HSD-1 in the postnatal developing rat epididymis [original magnification 1000 ×; negative control (epididymis) 400 ×]. pnd, postnatal day.

Table 1.   The postnatal developmental immunolocalisation of 11β-HSD-1.
pnd ISCaputCorpusCauda
  1. A, apical cell; AE, apical edge; B, basal cell; C, clear cell; EC, epithelial cytoplasm; Ep, epithelial cell; IC, interstitial cell; IS, initial segments; N, narrow cell; P, principal cell; Pn, principal cell nucleus; pnd, postnatal days; scat, scattered; SM, smooth muscle cells.

  2. Shaded regions were undeveloped or developing at that age. Striped, pattern of epithelial staining caused by alternating reactive and unreactive cells. ++++, intense; +++, strong; ++, moderate; +, weak; ND, not detected.

1Ep++ (AE)   
IC++++   
7Ep+++ (AE)   
IC+++   
15Ep+++ (AE)   
IC+++   
28Pn++++ (scat)++++ ND
BND++++ ND
C++++ ND
A/N++++ ND
EC++++ ++
AE++ND ++++
SMNDND ND
IC++ND ++++
40Pn+++ (scat)+++ (scat)NDND
BNDNDNDND
CNDNDNDND
A/NND++NDND
EC+++++
AEND++++++++++++
SMNDNDNDND
ICND+++NDND
60Pn+++ (scat)NDNDND
BNDNDNDND
CNDNDNDND
A/NNDNDNDND
EC++StripedStripedStriped
AEND++++++++++
SMNDNDNDND
IC+++++++++
75Pn+ (scat)NDNDND
BNDNDNDND
CNDNDNDND
A/NNDNDNDND
EC++StripedStripedStriped
AEND+++++++++
SMNDNDNDND
IC++++++++
104PNDNDNDND
BNDNDNDND
CNDNDNDND
A/NNDNDNDND
EC+++++
AEND+++++++ND
SMNDNDNDND
ICNDNDND++++

11β-HSD-2

Figure 2 illustrates a descriptive overview of the developmental pattern of 11β-HSD-2 localisation. Intensity of immunostaining and the types of cells observed are shown in Table 2. In contrast to 11β-HSD-1 reactivity, that for 11β-HSD-2 was more discrete and first became apparent in epithelial cells at pnd 15. In the IS and caput there was a distinctive pattern of strong to intense 11β-HSD-2 reactivity in scattered basal, narrow, clear and apical cells from pnd 28. Reactivity was differentially localised at the outer membrane, nucleus and/or the apical edge and stereocilia of the cells, depending on the region and cell type. In the cauda, 11β-HSD-2 reactivity in the epithelium was notably different. At pnd 28 there was weak reactivity at the apical edge, intense reactivity in stereocilia around the entire epithelium and a large quantity of strongly reactive luminal debris. At pnd 40, reactivity was weak at the apical edge, absent in stereocilia and strong in a few reactive luminal round cells. At pnd 60, 75 and 104, 11β-HSD-2 reactivity in the cauda epithelium was weak and disappeared from distal sections.

Figure 2.

 Overview of the immunolocalisation of 11β-HSD-2 in the postnatal developing rat epididymis [original magnification 1000 ×; negative control (epididymis) 400 ×]. pnd, postnatal day.

Table 2.   The postnatal developmental immunolocalisation of 11β-HSD-2.
pnd ISCaputCorpusCauda
  1. A, apical cell; AE, apical edge; B, basal cell; C, clear cell; EC, epithelial cytoplasm; Ep, epithelial cell; IC, interstitial cell; IS, initial segments; N, narrow cell; P, principal cell; pnd, postnatal days; R, round cell in lumen; scat, scattered; SM, smooth muscle cell.

  2. Shaded regions were undeveloped or developing at that age. Reactive cells were in a scattered arrangement throughout. ++++, intense; +++, strong; ++, moderate; +, weak; ND, not detected.

1EpND   
ICND   
7EpND   
ICND   
15Ep+++   
ICND   
28P++++ND ND
B++++++++ ND
C+++++++ ND
A++++++++ ND
N++++++++ ND
ECNDND +
SMNDND ND
ICNDND ND
RNDND ++++
40PNDNDNDND
B++++++++++ND
C++++++++++++ND
A++++++++++ND
N+++++++NDND
ECNDNDNDND
SMNDNDNDND
ICNDNDNDND
RNDNDND++++
60PNDNDNDND
B+++++++++++ND
C++++++++ND
A++++++++++ND
N++++++++NDND
ECNDNDND+ AE(scat)
SMNDNDNDND
ICNDNDNDND
75PNDNDNDND
B+++++++++++ND
C+++++++++ND
A+++++++++++ND
N+++++++++ND
ECNDNDND+AE(scat)
SMNDNDNDND
ICNDNDNDND
104PNDNDNDND
B+++++++++ND
C+++++++++ND
A++++++++++ND
N++++++NDND
ECNDNDND+AE(scat)
SMNDNDNDND
ICNDNDNDND

GR

At all ages examined where reactivity was present, the GR was largely localised in the nuclei of interstitial, basal, apical, narrow, principal and smooth muscle cells (Fig. 3). Intensity of immunostaining and the types of cells observed are shown in Table 3. Reactivity in the epithelial cytoplasm was moderate at pnd 28 and 40, and then was weak or very weak at all later ages. At pnd 1 and at all later ages examined, interstitial cells were intensely reactive for the GR. From pnd 28 onwards GR reactivity in basal cells was intense. At pnd 7, 15 and 28, smooth muscle cells were intensely reactive; however, at later ages were only reactive in the IS. Apical cells were intensely reactive in the caput at pnd 28, and from pnd 40 were only moderately reactive in the IS and caput. At pnd 40 narrow cells were intensely reactive in the caput and principal cells were reactive in all regions. From pnd 60 and at later ages, GR reactivity in principal cells was only observed in the corpus and cauda. GR reactivity was largely absent from clear cells in the caput and corpus at all ages.

Figure 3.

 Overview of the immunolocalisation of the GR in the postnatal developing rat epididymis [original magnification 1000 ×; negative control (epididymis) 400 ×]. pnd, postnatal day.

Table 3.   The postnatal developmental immunolocalisation of the GR.
pnd ISCaputCorpusCauda
  1. A, apical cell; B, basal cell; C, clear cell; EC, epithelial cytoplasm; Ep, epithelial cell; IC, interstitial cell; IS, initial segments; N, narrow cell; P, principal cell; pnd, postnatal days; SM, smooth muscle cell.

  2. Shaded regions were undeveloped or developing at that age. ++++, intense; +++, strong; ++, moderate; +, weak; ±, variable; ND, not detected.

1Ep±   
IC+++   
7Ep±   
SM++++   
IC++++   
15Ep+++   
SM++++   
IC++++   
28P±+++ ±
B++++++++ ++++
CNDND ±
A/N++++++++ ND
EC++ND ++
SM++++++++ ±
IC++++++++ ++++
40P±±++++±
B+++++++++++++++
C±NDNDND
A/N++++NDND
EC+++±+++
SM++++±±ND
IC++++++++++++++++
60PNDND±±
B+++++++++++++++
CNDNDNDND
A/N++++NDND
EC+±±+
SM++NDND++
IC++++++++++++++++
75PNDND±±
B++++++++++++ND
CNDNDNDND
A/N+++NDNDND
EC++++
SM++++±±ND
IC++++++++++++++++
104PNDND±±
B++++++++++++++++
CNDNDNDND
A/N++NDNDND
EC++±±
SM±±NDND
IC++++++++++++++++

MR

MR reactivity was predominantly in the epithelial cytoplasm and scattered interstitial cells at all ages examined (Fig. 4). Intensity of immunostaining and the types of cells observed are shown in Table 4. At pnd 1, 7 and 15, reactivity was weak, but had increased to moderate in the epithelial apical region by pnd 28. At pnd 40, MR reactivity in the IS epithelial apical region was weak; however, there was stronger MR reactivity at the basement membrane in the caput and corpus. At pnd 40 the cauda had intense MR reactivity at the apical edge of scattered epithelial cells. At pnd 60, 75 and 104, the apical half of epithelium in the IS was moderately reactive. Weak MR reactivity was observed in the epithelial cytoplasm in the caput, corpus and cauda at these later ages.

Figure 4.

 Overview of the immunolocalisation of the MR in the postnatal developing rat epididymis. [original magnification 1000 ×; negative control (epididymis) 400 ×]. pnd, postnatal day.

Table 4.   The postnatal developmental immunolocalisation of the MR.
pnd ISCaputCorpusCauda
  1. A, apical cell; AE, apical edge; B, basal cell; BM, basement membrane; C, clear cell; EC, epithelial cytoplasm; Ep, epithelial cell; IC, interstitial cell; IS, initial segments; N, narrow cell; P, principal cell; pnd, postnatal days; SM, smooth muscle cells.

  2. Shaded regions were undeveloped or developing at that age. ++++, intense; +++, strong; ++, moderate; +, weak; ND, not detected.

1Ep+   
ICND   
7Ep+   
ICND   
15Ep+   
ICND   
28Ep++++ +
IC++++++ ND
40PNDNDNDND
BNDNDNDND
CNDNDNDND
ANDNDNDND
NNDNDNDND
EC+++(BM)++ (AE)
SMNDNDNDND
IC++++++++++
60PNDNDNDND
BNDNDNDND
CNDNDNDND
ANDNDNDND
NNDNDNDND
EC++++(AE)++
SMNDNDNDND
IC+++++++++++++
75PNDNDNDND
BNDNDNDND
CNDNDNDND
ANDNDNDND
NNDNDNDND
EC+++++
SMNDNDNDND
IC++++++++++
104PNDNDNDND
BNDNDNDND
CNDNDNDND
ANDNDNDND
NNDNDNDND
EC++++ND
SMNDNDNDND
IC+++++++++++

Na+K+-ATPase

The developmental pattern of Na+K+-ATPase localisation is presented in Fig. 5. Intensity of immunostaining and the types of cells observed are shown in Table 5. Weak Na+K+-ATPase reactivity was first observed at pnd 15 in the epithelial cytoplasm. Na+K+-ATPase at all subsequent ages examined was largely evident in epithelial cytoplasm and sporadically in epithelial nuclei. At pnd 28 there was weak Na+K+-ATPase reactivity in the epithelial cytoplasm of the caput and strong reactivity at the apical edge in the cauda. Strong to intense Na+K+-ATPase reactivity was also observed at the apical edge in the distal cauda at all later ages. From pnd 40, and at all later ages, the epithelial pattern of Na+K+-ATPase reactivity appeared as striped in the caput, corpus and proximal cauda, a result of interspersed, scattered, reactive principal cells and unreactive clear cells. Isolated basal, apical and narrow cells in the IS and caput were moderately to intensely reactive. In the IS, Na+K+-ATPase reactivity at pnd 40 and 60 was observed in scattered principal cell nuclei. There was strong to intense reactivity in isolated interstitial cells in all regions.

Figure 5.

 Overview of the immunolocalisation of Na+K+-ATPase in the postnatal developing rat epididymis [original magnification 1000 ×; negative control (epididymis) 400 ×]. pnd, postnatal day.

Table 5.   The postnatal developmental immunolocalisation of Na+K+ -ATPase.
pnd ISCaputCorpusCauda
  1. A, apical cell; AE, apical edge; B, basal cell; C, clear cell; EC, epithelial cytoplasm; Ep, epithelium; IC, interstitial cell; n, nucleus; N, narrow cell; P, principal cell; SM, smooth muscle cell.

  2. Shaded regions were undeveloped or developing at that age. Striped, pattern of epithelial staining caused by alternating reactive and unreactive cells. ++++, intense; +++, strong; ++, moderate; +, weak; ±, variable; ND, not detected.

1EpND   
ICND   
7EpND   
ICND   
15Ep+   
ICND   
28EC++ +
AENDND +++
IC+++ND +
40P+++ (n)±±±
B++++NDNDND
C++NDNDND
A++NDNDND
NNDNDNDND
EC+StripedStriped++
AENDNDND++++
SMNDNDNDND
IC++++++++++++
60P+ (n)±±+
BNDNDNDND
CNDNDNDND
ANDNDNDND
NND+++NDND
EC+StripedStriped±
AENDNDND+++
SMNDNDNDND
IC++++++++++++
75P+±±+
BNDNDNDND
CNDND±ND
A++++NDNDND
N+++NDNDND
EC+StripedStriped±
AENDNDND+++
SMNDNDNDND
IC++++++++++++
104P±±±+
BND++++NDND
CNDNDNDND
ANDNDNDND
NNDNDNDND
ECStripedStripedStriped±
AENDNDND+++
SMNDNDNDND
IC++++++++++++

11β-HSD-1 mRNA levels of expression in the caput and cauda

There was detectable 11β-HSD-1 mRNA expression at pnd 28 and pnd 75 in both the caput and cauda (Fig. 6). 11β-HSD-1 mRNA expression tended to be higher at pnd 75 compared with pnd 28 (overall age effect = 0.0055; two-way anova). There were no differences in expression of 11β-HSD-1 mRNA in the cauda at pnd 28 and pnd 75 (= 0.1434; two-tailed t-test), and no difference between the caput and cauda at pnd 28 (= 0.6684) or pnd 75 (= 0.3289; two-tailed t-tests).

Figure 6.

 Relative 11β-HSD-1 mRNA levels in the caput and cauda in pre- and post-pubertal animals. Values are the mean ± SEM (= 4–5 per group).

11β-HSD-2 mRNA levels of expression in the caput and cauda

There was 11β-HSD-2 mRNA expression detected at pnd 28 and pnd 75 in both the caput and cauda (Fig. 7), with higher 11β-HSD-2 mRNA expression detected at pnd 28 compared with pnd 75 (= 0.004; two-way anova), and a significant interaction between animal age and epididymal zone (= 0.032; two-way anova). Specifically, the caput had higher 11β-HSD-2 expression at pnd 28 compared with pnd 75 (< 0.01; two-tailed t-test). 11β-HSD-2 mRNA expression was also higher in the caput than the cauda at day 28 (< 0.05, two-tailed t-test), but there was no age difference in expression levels of 11β-HSD-2 mRNA in the cauda (= 0.2749; two-tailed t-test).

Figure 7.

 Relative 11β-HSD-2 mRNA levels in the caput and cauda in pre- and post-pubertal animals. Values are the mean ± SEM (= 5 per group). *< 0.01 compared with caput levels at Day 28 (two-tailed t-test), #< 0.05 compared with caput levels at Day 28 (two-tailed t-test).

GR mRNA levels of expression in the caput and cauda

Expression of GR mRNA was detected in the caput and cauda at pnd 28 and pnd 75 (Fig. 8), but there was no variation in expression due to either age (= 0.222; two-way anova) or epididymal zone (= 0.92; two-way anova).

Figure 8.

 Relative glucocorticoid receptor (GR) mRNA levels in the caput and cauda in pre- and post-pubertal animals. Values are the mean ± SEM (= 3–5 animals per group).

Discussion

The aim of this study was to examine the postnatal development of 11β-HSD-1 and -2, GR, MR and Na+K+-ATPase in the rat epididymis. 11β-HSD-1, GR and MR were observed at pnd 1, whereas 11β-HSD-2 and Na+K+-ATPase were first detected in the epithelium at pnd 15, increasing in intensity by pnd 28 when the epithelium remained intensely reactive for 11β-HSD-1, the GR and MR. The cell-, region- and age-specific patterns of 11β-HSD-1 and -2 localisation in the epididymal epithelium suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but the cell-specific expression of these enzymes still provides a capacity for intricate local control of glucocorticoid exposure across development.

Epididymal 11β-HSD-1 immunolocalisation was observed from pnd 1 with moderate to strong reactivity. Cytoplasmic immunostaining was present in epithelial cells at all ages examined, with sections of the apical edge in all regions showing intense reactivity. Previous reports indicate that 11β-HSD-1 is localised to the nuclear envelope as well as in the endoplasmic reticulum (Monder & White, 1993; Brereton et al. 2001), and the intense nuclear localisation in a small population of scattered principal cells at pnd 28, 40 and 60 in the present study is consistent with this. Epithelial reactivity at pnd 40, 60 and 75 in the caput, corpus and cauda appeared ‘striped’ in some segments, largely due to scattered clear cells lacking 11β-HSD-1 reactivity. There were also intermittent basal, apical, narrow and principal cells devoid of staining for 11β-HSD-1, a pattern of reactivity that highlights the complexity of glucocorticoid regulation in this epithelium. This is consistent with previous observations that 11β-HSD-1 was undetectable in clear cells but was localised to the apical cytoplasm of principal cells of the caput region of the adult rat epididymis (Waddell et al. 2003).

The role of 11β-HSD-1 in reactivating glucocorticoids is well recognised (Seckl, 2001), and the strong presence of 11β-HSD-1 reactivity from pnd 1 demonstrated in this study coinciding with strong GR reactivity suggests a crucial role for glucocorticoids in cellular differentiation and growth of the epididymal epithelium from very early in postnatal development. Indeed, it has been reported that from pnd 16 to pnd 44 narrow, basal, principal and halo cells appear for the first time (Sun & Flickinger, 1979). In the caput, 11β-HSD-1 mRNA expression increased from pre- to post-puberty, an effect that would be expected to increase local concentrations of active glucocorticoid within the epididymal lumen. Accordingly, we previously reported that 11-oxoreductase bioactivity does predominate in the adult caput (Waddell et al. 2003). Because sperm maturation takes place in this epididymal region (Brooks, 1983; Robaire & Hermo, 1988), we propose that increases in 11β-HSD-1 expression after puberty may promote sperm maturation via increased glucocorticoid levels.

In contrast, caput expression of 11β-HSD-2 mRNA levels fell significantly from pre- to post-puberty, raising the possibility that 11β-HSD-2 acts to protect the epididymal fluid environment from glucocorticoid excess. High levels of glucocorticoids may be damaging to differentiating epididymal epithelial cells that have vital functions in maintaining optimal conditions for sperm maturation in the mature epididymis. The 11β-HSD-2 enzyme may also protect the MR from excess glucocorticoids, so that aldosterone can bind to the MR and maintain optimal ion and fluid transport in the caput. It is well recognised that fluid reabsorption largely takes place in the initial segment and caput regions. Previous research demonstrated that 11β-dehydrogenase activity was less than 11β-oxoreductase activity in the adult rat epididymis (Waddell et al. 2003); however, 11β-HSD-2 bioactivity has not previously been measured at pre-puberty. The strong localisation of 11β-HSD-2 in scattered basal, narrow, clear and apical cells of the epididymal epithelium suggests an intricate and highly specialised regulation of glucocorticoid levels in the epididymis, indicating that a finely balanced level of glucocorticoids is required for normal epididymal function and sperm maturation.

For the first time, intense GR reactivity has been demonstrated in epididymal interstitial cells at pnd 1, 7 and 15. The present study has also demonstrated strong to intense reactivity for the GR in basal and smooth muscle cells, and a small number of principal, apical and narrow cells at pnd 28, 40, 60, 75 and 104. Overall, the GR was largely localised to the nuclei of basal and interstitial cells, suggesting GR was activated by glucocorticoids in these cells. The GR was also localised in the epithelial cytoplasm, although reactivity was only moderate at pnd 28 and 40, and became weaker from pnd 60, suggesting that GR are not activated in these cells at these developmental stages. The GR localisation demonstrated here is partly in accordance with previous research that reported the GR to be present only in basal cells and fibroblasts in the adult rat epididymis (Schultz et al. 1993; Silva et al. 2010). The intense localisation demonstrated in the present study at pnd 1, 7 and 15 suggests a significant role for glucocorticoids in epididymal tissue at these ages, most likely in the differentiation of the epididymal tubule and of individual cell types within the epithelium. Indeed, pnd 16–44 is recognised as a time of cellular differentiation in the rat epididymis (Sun & Flickinger, 1979), and glucocorticoids are involved in cellular induction and differentiation in many other tissues (Chrousos & Gold, 1992). The localisation demonstrated here is therefore suggestive of a similar role in the epididymis.

The relative levels of GR mRNA expression were the same in the caput and cauda at pre-and post-puberty, suggesting that glucocorticoids are important for epididymal epithelial functions at both pre- and post-puberty in both regions.

Mineralocorticoid receptor was present in the epididymal epithelial cytoplasm from pnd 1. At later ages examined there were sections of stronger reactivity along the basement membrane in the caput and intense localisation at the apical edge of scattered epithelial cells in the cauda. The MR was absent from scattered clear cells in the corpus and cauda from pnd 60, similar to the pattern of localisation demonstrated for 11β-HSD-1. Localisation appeared to be maximal at pnd 28, 40 and 60, and largely confined to the IS and caput regions. Previous studies have reported MR binding to clear cells in the adult rat epididymis (Hinton & Keefer, 1985; Pearce et al. 1986).

Na+K+-ATPase immunoreactivity was first observed at pnd 15 in the caput, and then at pnd 28 in both the caput and cauda. Strong reactivity was present at the epithelial apical edge of the cauda from pnd 28 and at all later ages examined, similar to the pattern of localisation demonstrated in that region for the MR and 11β-HSD-1. The striped pattern of Na+K+-ATPase reactivity demonstrated in the epithelium of the caput and corpus regions was largely a result of the differential staining intensity of principal cells, unlike the striped epithelium shown in the 11β-HSD-1 localisation that was caused by the absence of that enzyme in scattered clear cells. This pattern of Na+K+-ATPase localisation is in contrast to a previous study, which reported that staining was basolateral, absent from the apical region and less intense in more distal regions of the epididymis (Byers & Graham, 1990).

Na+K+-ATPase activity is known to be regulated by both mineralocorticoids and glucocorticoids in the kidney and colon where it is important for epithelial sodium transport (llis et al. 1987; Schmitt & McDonough, 1988; Barlet-Bas et al. 1990; Fuller & Verity, 1990; Katz, 1990; Kinsella, 1990; Whorwood et al. 1994; Whorwood & Stewart, 1995). The pattern of localisation observed in the present study suggests a role consistent with sodium and water regulation in the IS, caput and corpus across the basement membrane, and in the cauda, across the apical plasma membrane. The apical localisation of Na+K+-ATPase in the cauda is consistent with the sperm storage function of that region, where ion and fluid transport mechanisms are vital to maintaining optimal conditions in the luminal fluid environment. Na+K+-ATPase is also essential for maintaining the integrity of the intricate network of tight junctions in the epididymal epithelium, and its intense localisation at the apical edge in the cauda from pnd 28 in the present study is consistent with that function. Furthermore, Na+K+-ATPase has been shown to be sensitive to oxidative stress in tissues including heart and kidney (Dobrota et al. 1999; Rodrigo et al. 2002).

In conclusion, the cell-, region- and age-specific patterns of 11β-HSD-1 and -2 reactivity demonstrated in the epididymal epithelium in the present study strongly indicate that glucocorticoids may have specialised roles in different regions and cell types of the rat epididymis that vary according to the postnatal developmental stage. It is also proposed that the strong peak in reactivity demonstrated at pnd 28 for 11β-HSD-1 and -2, GR, MR and Na+K+-ATPase, coinciding with the differentiation of basal and principal cells and the formation of tight junctions in the epithelium, also parallels the maturation of the HPA axis and indicates that glucocorticoids are especially crucial in mediating developmental and protective mechanisms in the epididymal epithelium at that time. Furthermore, it is suggested that glucocorticoids are essential for preparing and maintaining the epididymal epithelium for the arrival of immature sperm from the testes around the time of puberty.

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