Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Dec 18;109(51):21140-5.
doi: 10.1073/pnas.1216189110. Epub 2012 Dec 4.

Transactivating function (AF) 2-mediated AF-1 activity of estrogen receptor α is crucial to maintain male reproductive tract function

Yukitomo Arao  1 , Katherine J Hamilton, Eugenia H Goulding, Kyathanahalli S Janardhan, Edward M Eddy, Kenneth S Korach
Affiliations

Transactivating function (AF) 2-mediated AF-1 activity of estrogen receptor α is crucial to maintain male reproductive tract function

Yukitomo Arao et al. Proc Natl Acad Sci U S A. .

Abstract

Estrogen receptor alpha (ERα) is a ligand-dependent transcription factor containing two transcriptional activation function (AF) domains. AF-1 is in the N terminus of the receptor protein, and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. We recently showed that two point mutations converting leucines 543 and 544 to alanines in helix 12 (AF2ER) minimized estrogen-dependent AF-2 transcriptional activation. A characteristic feature of AF2ER is that the estrogen antagonists ICI182780 and tamoxifen (TAM) act as agonists through intact AF-1, but not through mutated AF-2. Here we report the reproductive phenotype of male AF2ER knock-in (AF2ERKI) mice and demonstrate the involvement of ERα in male fertility. The AF2ERKI male homozygotes are infertile because of seminiferous tubular dysmorphogenesis in the testis, similar to ERα KO males. Sperm counts and motility did not differ at age 6 wk in AF2ERKI and WT mice, but a significant testis defect was observed in adult AF2ERKI male mice. The expression of efferent ductal genes involved in fluid reabsorption was significantly lower in AF2ERKI males. TAM treatment for 3 wk beginning at age 21 d activated AF-2-mutated ERα (AF2ER) and restored expression of efferent ductule genes. At the same time, the TAM treatment reversed AF2ERKI male infertility compared with the vehicle-treated group. These results indicate that the ERα AF-2 mutation results in male infertility, suggesting that the AF-1 is regulated in an AF-2-dependent manner in the male reproductive tract. Activation of ERα AF-1 is capable of rescuing AF2ERKI male infertility.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AF2ERKI homozygote males are infertile. (A) Sperm counts from the cauda epididymis of 6-wk-old and adult WT and AF2ERKI mice. Data are expressed as mean ± SEM. Statistical analysis was done by ANOVA.*P < 0.05; ns, not significant. (B) Motility of sperm from the cauda epididymis of 6-wk-old and adult WT and AF2ERKI mice, determined with a computer-assisted sperm analyzer after a 60-min incubation in M2 medium. Data are expressed as mean ± SEM. Statistical analysis was done by ANOVA. *P < 0.05; ns, not significant. (C) Representative longitudinal sections from adult WT and AF2ERKI testes and epididymis. Arrow indicates efferent duct; T indicates testis; r indicates rete testis. (Scale bars: 3 mm.) (D) Representative ERα expression levels in the efferent ducts and testes of adult WT, AF2ERKI, and αERKO mice. Immunohistochemical studies were performed as described in Materials and Methods. Signals were developed by DAB chromogen, and the slides were counterstained in modified Harris hematoxylin. (Scale bars: 0.3 mm for efferent duct; 0.2 mm for testis). (E) Representative histology of testes from 10-d-old, 20-d-old, 6-wk-old, and adult WT, AF2ERKI, and αERKO mice. Sections were stained with H&E.
Fig. 2.
Fig. 2.
Efferent ductal gene expression in AF2ERKI and αERKO mice. (A) Representative expression levels of efferent duct SLC9A3 and AQP9 in adult WT, AF2ERKI, and αERKO mice. Immunohistochemical studies were performed as described in Materials and Methods. Signals were developed by DAB chromogen. The slides were counterstained in modified Harris hematoxylin. (Scale bars: 0.3 mm.) (B) mRNA levels of Slc9a3, Car2, Aqp1, and Aqp9 in efferent ducts from WT, AF2ERKI, and αERKO mice, quantified by qPCR. mRNA levels are presented as fold change vs. WT. Values are mean ± SEM. Statistical analysis was done by ANOVA. *P < 0.05 against WT. (C) mRNA levels of Slc9a3, Car2, Aqp1, and Aqp9 in vehicle (Veh)- and TAM (Tam)-treated efferent ducts from WT (+/+), AF2ERKI (KI/KI), and αERKO (−/−) mice, quantified by qPCR. mRNA levels are presented as fold change vs. vehicle-treated WT mice. Values are mean ± SEM. Statistical analysis was done by ANOVA. a indicates P < 0.05 against WT (+/+) vehicle-treated mice; b indicates P < 0.05 against AF2ERKI (KI/KI) vehicle-treated mice.
Fig. 3.
Fig. 3.
AF2ERKI homozygote male fertility was recovered by TAM treatment. (A) Sperm counts from the cauda epididymis of WT and AF2ERKI mice after breeding. Data are expressed as mean ± SEM. Statistical analysis was performed using the Mann–Whitney U test between placebo and TAM. ns, not significant. (B) Sperm motility from the cauda epididymis of WT and AF2ERKI mice after 60 min of incubation in M2 medium. Data are expressed as mean ± SEM. Statistical analysis was performed using the Mann–Whitney U test between placebo and TAM. *P < 0.05; ns, not significant. (C) (Top) Representative low-magnification view of sections for placebo- and TAM-treated WT and AF2ERKI testes. The sections were stained with H&E. (Scale bars: 2 mm.) (Middle) Representative histology of testes from placebo- and TAM-treated WT and AF2ERKI. (Scale bars: 0.3 mm.) (Bottom) Representative histology of cauda epididymis from placebo- and TAM-treated WT and AF2ERKI. (Scale bars: 0.6 mm.)
Fig. 4.
Fig. 4.
Expression of efferent ductal genes was recovered by TAM treatment in AF2ERKI mice. (A) mRNA levels of Slc9a3, Car2, Aqp1, and Aqp9 in the placebo- and TAM (Tam)-treated efferent ducts from WT (+/+) and AF2ERKI (KI/KI) mice, quantified by qPCR. mRNA levels are presented as fold change vs. placebo-treated WT. Values are mean ± SEM. Statistical analysis was done by ANOVA. a indicates P < 0.05 against WT (+/+) placebo-treated mice; b indicates P < 0.05 against AF2ERKI (KI/KI) placebo-treated mice. (B) Immunohistochemical results of placebo- and TAM-treated WT and AF2ERKI efferent ducts. Representative results of SLC9A3 and the highest AQP9 expression are shown. Immunohistochemistry was performed as described in Materials and Methods. Signals were developed by DAB chromogen, and the slides were counterstained in modified Harris hematoxylin. (Scale bar: 0.2 mm.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Eddy EM, et al. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology. 1996;137(11):4796–4805. - PubMed
    1. Hess RA, et al. A role for oestrogens in the male reproductive system. Nature. 1997;390(6659):509–512. - PMC - PubMed
    1. Couse JE, Mahato D, Eddy EM, Korach KS. Molecular mechanism of estrogen action in the male: Insights from the estrogen receptor null mice. Reprod Fertil Dev. 2001;13(4):211–219. - PubMed
    1. Hess RA. Estrogen in the adult male reproductive tract: A review. Reprod Biol Endocrinol. 2003;1:52. - PMC - PubMed
    1. Zhou Q, et al. Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proc Natl Acad Sci USA. 2001;98(24):14132–14137. - PMC - PubMed

Publication types

LinkOut - more resources