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. 2001 Nov 20;98(24):14132-7.
doi: 10.1073/pnas.241245898. Epub 2001 Nov 6.

Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function

Q Zhou  1 , L Clarke, R Nie, K Carnes, L W Lai, Y H Lien, A Verkman, D Lubahn, J S Fisher, B S Katzenellenbogen, R A Hess
Affiliations

Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function

Q Zhou et al. Proc Natl Acad Sci U S A. .

Abstract

Estrogen receptor alpha (ER alpha) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na(+)/H(+) exchanger-3 (NHE3) and the rate of (22)Na(+) transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ER alpha knockout (alpha ERKO) efferent ductules. Targeted gene-deficient mice were compared with alpha ERKO, and the NHE3 knockout and CAII-deficient mice showed alpha ERKO-like fluid accumulation, but only the NHE3 knockout and alpha ERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in alpha ERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in alpha ERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in alpha ERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na(+) transport. Finally, these data raise the possibility of targeting ER alpha in developing a contraceptive for the male.

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Figures

Figure 1
Figure 1
Immunohistochemical staining for NHE3, CAII, and AQP-1 in efferent ductules of WT and αERKO mice. (A and B) NHE3 is abundant along the brush border of WT (αERKO littermate) nonciliated cells (arrow) but is absent in αERKO (B). (C and D) CAII staining found in the cytoplasm (arrowhead) and along the apical border of the epithelial cells (arrow) in WT, but CAII in αERKO is reduced in the cytoplasm (arrowhead) and absent along the apical border. (E and F) AQP1 is abundant along the brush border of WT nonciliated cells (arrow), but AQP1 in αERKO staining is absent (arrowhead) in the proximal ductules (prox) or intermittent in the more distal regions (arrow). (Bar = 25 μm.)
Figure 2
Figure 2
Histological comparison of rete testis (A–E), efferent ductule lumen (F–J), and electron microscopy of apical epithelium (K–O) in gene-deficient and WT mice (αERKO). The rete testis (arrow) is small and narrow in WT (A), whereas there are large dilations in αERKO (B), NHE3KO (C), and CAIIdef (D) mice. AQP1KO has a slight dilation of the rete (E). The efferent ductule lumen (L) has a small diameter in WT (F) and AQP1KO (J) mice, but is dilated in αERKO (G), NHE3KO (H), and CAIIdef (I). The lumen in some NHE3KO ductules was 2- to 3-fold greater in diameter than in αERKO. Ultrastructure of apical cytoplasm in nonciliated cells of the efferent ductules reveals a loss or shortening of microvilli (double arrow) and a decreased number of endocytotic vesicles in αERKO (L) compared with WT (K). Microvilli in NHE3KO (M), CAIIdef (N), and AQP1KO mice (O) are taller than WT. [Bar = 25 μm (A–E), 100 μm (F–J), and 10 μm (K–O).]
Figure 3
Figure 3
Alterations in mRNA expression in efferent ductules (ED) of the αERKO (−/−) mice compared with WT (+/+). NHE3 (5.9 kb) is reduced nearly 6-fold in αERKO, whereas CAII (1.7 kb) and AQP1 (3.2 kb) show no change in mRNA expression. All Northern blots were normalized for loading by a final hybridization with the 36B4 probe, which is not responsive to estrogen (24).
Figure 4
Figure 4
The rate of 22Na uptake sensitive to 100 μM EIPA in efferent ductule segments is reduced nearly 4-fold in αERKO mice compared with WT male littermates. Means ± SEM; n = 7 pairs. Significant difference (*) was determined by the paired t test, P < 0.01.
Figure 5
Figure 5
Effects of ICI 182,780 treatment for 8 days in adult males. Immunohistochemical staining for NHE3 (A and B), CAII (C and D), and AQP-1 (E and F) in efferent ductules of control and treated mice. NHE3 is greatly reduced in staining after ICI treatment (arrow), whereas CAII may be reduced slightly (arrow) and AQP1 (arrow) shows no change compared with controls (C and E, respectively). (Bar = 25 μm.)
Figure 6
Figure 6
NHE3 mRNA from efferent ductules is decreased nearly 63% on day 8 after ICI 182,780 treatment. CAII and AQP1 mRNAs show no change.
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