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. 2024 Jan 16;165(3):bqad180.
doi: 10.1210/endocr/bqad180.

Role for Nongenomic Estrogen Signaling in Male Fertility

Affiliations

Role for Nongenomic Estrogen Signaling in Male Fertility

Jones B Graceli et al. Endocrinology. .

Abstract

Estrogen actions are mediated by both nuclear (n) and membrane (m) localized estrogen receptor 1 (ESR1). Male Esr1 knockout (Esr1KO) mice lacking functional Esr1 are infertile, with reproductive tract abnormalities. Male mice expressing nESR1 but lacking mESR1 (nuclear-only estrogen receptor 1 mice) are progressively infertile due to testicular, rete testis, and efferent ductule abnormalities similar to Esr1KO males, indicating a role for mESR1 in male reproduction. The H2NES mouse expresses only mESR1 but lacks nESR1. The goal of this study was to identify the functions of mESR1 alone in mice where nESR1 was absent. Breeding trials showed that H2NES males are fertile, with decreased litter numbers but normal pup numbers/litter. In contrast to Esr1KO mice, H2NES testicular, and epididymal weights were not reduced, and seminiferous tubule abnormalities were less pronounced. However, Esr1KO and H2NES males both had decreased sperm motility and a high incidence of abnormal sperm morphology. Seminiferous tubule and rete testis dilation and decreased efferent ductule epithelial height characteristic of Esr1KO males were reduced in H2NES. Consistent with this, expression of genes involved in fluid transport and ion movement that were reduced in Esr1KO (Aqp1, Car2, Car14, Cftr) were partially or fully restored to wild-type levels in H2NES. In summary, in contrast to Esr1KO males, H2NES males are fertile and have reduced phenotypic and functional abnormalities in the testis and efferent ductules. Thus, mESR1 alone, in the absence of nESR1, can partially regulate male reproductive tract structure and function, emphasizing its importance for overall estrogen action.

Keywords: ESR1; efferent ductules; epididymis; estrogen; steroid receptors.

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Figures

Figure 1.
Figure 1.
Expression of ESR1 in testis (A-D) and epididymis (E-H) of WT (A, C, E, G) and H2NES (B, D, F, H) mice. Regions outlined by boxes in A, B, E, and F are shown at higher magnifications in C, D, G, and H, respectively. Bar in all panels = 50 µm. (I) Western blot showing ESR1 in individual samples of the testis and epididymis of WT and H2NES mice. To, total protein; Nu, nuclear protein. Quantitation of the Western blot (J) revealed that although total and nuclear protein were closely comparable in WT testis and epididymis, nuclear protein was reduced by 63% and 90% compared to total protein in the H2NES testis and epididymis, respectively.
Figure 2.
Figure 2.
Fertility assessment in WT, H2NES, and Esr1KO mice. (A) Breeding protocol. One male was housed with 2 or 3 proven breeder females for 30 consecutive days or until pregnancy occurred. The number whitin dark boxes represent the size of the litter in a successful pregnancy, and boxes marked with (M) represent matings that did not result in pregnancy. (B) Number of pups/litter. ***P < .001; n = 10.
Figure 3.
Figure 3.
Sperm analysis in WT, H2NES, and Esr1KO males. (A) Daily sperm production. (B) Sperm concentration in the caudal epididymis. (C) Motility of sperm in the caudal epididymis. (D) Forward motility of sperm. (E) Incidence of sperm abnormalities. (F) Representative images comparing normal sperm and sperm with morphological abnormalities. Bar = 20 µm. *P < .05; ***P < .001; n = 4-5 group.
Figure 4.
Figure 4.
Reproductive organ weights, histology, and morphometrics in WT, H2NES, and Esr1KO males. Weights of the testes (A) and epididymis (B) were reduced in Esr1KO but not H2NES mice compared to WT. (C) In sagittal sections, rete testis (asterisk, RT) in 4-month-old H2NES and Esr1KO testes was highly dilated compared with WT controls. (D, E) Seminiferous tubule lumens in H2NES and Esr1KO males were enlarged compared with WT mice. (F) Frequency of histological abnormalities was increased in both H2NES and Esr1KO testes. (G) Epithelial height of efferent ducts is reduced in H2NES and Esr1KO compared with WT mice. For tubule luminal diameter, % of abnormal tubules, and efferent ductule epithelial height, abnormalities in the Esr1KO mice were significantly more pronounced than for H2NES mice, indicating an mESR1 effect for all of these parameters in the H2NES. *P < .05, **P < .01; ***P < .001; n = 3-4.
Figure 5.
Figure 5.
Dysregulation of transcripts associated with water and ion transport and pH regulation in H2NES and Esr1KO males. RT-qPCR for key genes associated with water transport (Aqp1, Aqp4, Aqp9), ion transport, and pH regulation (Car2, Car14, Cftr, Slc9a3, Slc26a3) in WT, H2NES and Esr1KO epididymis and efferent ductules. Gene expression was normalized to an internal housekeeping control (Gapdh). (A) Efferent ductules. (B) Epididymis. Bars are mean ± SEM (n = 3). *P < .05, **P < .01. (C) Summary of gene expression differences in H2NES and Esr1KO compared to WT mice. Upregulated genes are shown in green boxes (↑) and downregulated genes are shown in red boxes (↓). Trends in unchanged genes in gray boxes are indicated by arrows.
Figure 6.
Figure 6.
Morphometric and immunohistochemical assessment of efferent ductules in WT, H2NES, and Esr1KO mice. (A-F) AQP1 immunohistochemistry in distal efferent ducts of WT (A, D), H2NES (B, E), and Esr1KO (C, F) mice. Regions outlined by boxes in A, B, and C are shown at higher magnifications in D, E, and F, respectively. Higher power views of the regions outlined by boxes in D, E, and F are shown as insets in the bottom right-hand corner of those images. Efferent ductule epithelial thickness is highlighted by double-headed arrows in insets in D, E, and F to illustrate differences in epithelial height in WT, H2NES, and Esr1KO mice. DED, distal efferent ductules; IS, initial segment of epididymis. Bar size in all panels is indicated in the figure. (G) Western blot and quantitation (H) of AQP1 expression in efferent ducts from WT, H2NES, and Esr1KO mice. *P < .05; n = 3-4 for all groups).

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