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. 2016 Jul;157(7):2909-19.
doi: 10.1210/en.2016-1085. Epub 2016 May 4.

Membrane-Localized Estrogen Receptor 1 Is Required for Normal Male Reproductive Development and Function in Mice

Manjunatha K Nanjappa  1 , Rex A Hess  1 , Theresa I Medrano  1 , Seth H Locker  1 , Ellis R Levin  1 , Paul S Cooke  1
Affiliations

Membrane-Localized Estrogen Receptor 1 Is Required for Normal Male Reproductive Development and Function in Mice

Manjunatha K Nanjappa et al. Endocrinology. 2016 Jul.

Abstract

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17β-estradiol (E2). Male Esr1 knockout (Esr1KO) mice are infertile due to efferent ductule and epididymal abnormalities. The majority of ESR1 is nuclear/cytoplasmic; however, a small fraction is palmitoylated at cysteine 451 in mice and localized to cell membranes, in which it mediates rapid E2 actions. This study used an Esr1 knock-in mouse containing an altered palmitoylation site (C451A) in ESR1 that prevented cell membrane localization, although nuclear ESR1 was expressed. These nuclear-only estrogen receptor 1 (NOER) mice were used to determine the roles of membrane ESR1 in males. Epididymal sperm motility was reduced 85% in 8-month-old NOER mice compared with wild-type controls. The NOER mice had decreased epididymal sperm viability and greater than 95% of sperm had abnormalities, including coiled midpieces and tails, absent heads, and folded tails; this was comparable to 4-month Esr1KO males. At 8 months, daily sperm production in NOER males was reduced 62% compared with controls. The NOER mice had histological changes in the rete testes, efferent ductules, and seminiferous tubules that were comparable with those previously observed in Esr1KO males. Serum T was increased in NOER males, but FSH, LH, and E2 were unchanged. Critically, NOER males were initially subfertile, becoming infertile with advancing age. These findings identify a previously unknown role for membrane ESR1 in the development of normal sperm and providing an adequate environment for spermatogenesis.

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Figures

Figure 1.
Figure 1.. Reproductive organ weights in WT, NOER, and Esr1KO mice at 4 and 8 months of age.
Paired testes weights (A) were increased in NOER males and decreased in Esr1KO mice vs WT at 4 months of age, but NOER testes weights were not different from WT at 8 months of age. Weights of the seminal vesicle and coagulating glands in NOER and Esr1KO mice (B) were significantly increased compared with WT at 4, but not 8, months of age. For both WT and NOER, n = 8 at 8 months, and n = 4–5 for all genotypes at 4 months. **, P < .01; ***, P < .001. ns, not significantly different
Figure 2.
Figure 2.. Testicular abnormalities in NOER mice.
In transverse sections, rete testis (RT) in 4-month-old NOER (B) testes was highly dilated compared with 4-month-old WTs (A). The RT enlargement in NOER testes did not increase between 4 and 8 months of age (not shown), and RT size in NOER mice was similar to that seen in 8-month-old Esr1KO mice (C). Luminal diameters of seminiferous tubules in NOER (B, E, and G) and Esr1KO (C) testes also appeared increased compared with WT (A, D, and F). Seminiferous tubules of WT testis at 4 months (D and F) contain complete layers of germ cells showing normal spermatogenesis and fairly consistent tubular size. In contrast, 4-month NOER testes (E and G) contained increased numbers of abnormal seminiferous tubules (ab) that were atrophic or showed degenerative changes in spermatogenesis, sometimes accompanied by vacuolation. Some dilated seminiferous tubules (arrows) were also seen in NOER testes. All sections stained with Masson's trichrome. Bars, 1 mm (A–C), 100 μm (D and E), and 50 μm (F and G).
Figure 3.
Figure 3.. Changes in testis, rete testis (RT), and proximal efferent ductules (PED) in NOER mice.
Sagittal sections of the 4-month NOER testis (A) revealed that atrophic changes were seen preferentially in the caudal pole of the testis. The RT area (B) was increased more than 10-fold in 4-month-old NOER vs WT testes. Luminal diameter (C) and epithelial height (D) were increased and decreased, respectively, in the PED of NOER testes. Increased PED luminal diameter is seen in panel A) as well. Bar, 500 μm in panel A (n = 4 and 5 for WT and NOER, respectively). **, P < .01; ***, P < .001; ****, P < .0001. DED, distal efferent duct.
Figure 4.
Figure 4.. Efferent ductule and epididymal histology is altered in NOER mice.
In the proximal efferent ductules (PED) of 4-month-old WT mice (A), luminal diameter (arrow) is less than in NOER PED (B). The NOER epithelial height is decreased (B, inset) compared with WT (A, inset). In the distal efferent duct (DED), luminal diameter and epithelial height of WT (C) and NOER (D) mice are comparable, although NOER ducts shows larger variation. The initial segment of the epididymis (IS) in WT mice (C and E) contains a distinctly tall epithelium (double headed arrow in E) with numerous principal cells (P) containing basal nuclei and narrow cells (N) with nuclei in the apical cytoplasm. The NOER IS epithelial height (D and F) is reduced compared with WT (G). Bars, 50 μm (A and B), 10 μm (A and B insets), 100 μm (C and D) and 20 μm (E and F). ci, cilia. All sections were stained with Masson's trichrome.
Figure 5.
Figure 5.. Increased abnormalities in cauda epididymal sperm from NOER mice.
Sperm from both 8-month-old NOER and 4-month-old Esr1KO males had an increased incidence of structural defects (A). A normal WT sperm is shown in panel B, with major regions (head, midpiece, and principal piece) identified by arrows. Various structural abnormalities were seen in NOER sperm, including coiled midpieces that sometimes involved the head (C and D), folded midpieces, which sometimes involved the head (E–G), and headless tails (H–J), which also sometimes involved folding or coiling of the tail. Similar abnormalities occur in Esr1KO sperm. All images are the same magnification, and magnification bar is 10 μm. **, P < .01; ***, P < .001; ****, P < .0001
Figure 6.
Figure 6.. Sperm motility, progressive motility, and viability in 8-month-old WT and NOER and 4-month-old Esr1KO males.
The NOER mice showed decreased sperm motility and viability compared with WT mice. Spermatogenesis is almost completely lost by 8 months in Esr1KO males, and thus, these were not used for comparison purposes (n = 8 for both WT and NOER at 8 months of age and n = 5 for 4-month-old Esr1KO males). *, P < .05; ****, P < .0001.
Figure 7.
Figure 7.. Daily sperm production (DSP) and efficiency in WT, NOER, and Esr1KO mice at 4 and 8 months.
At 4 months, the DSP was decreased in Esr1KO, but not NOER, mice (A). By 8 months, the DSP was reduced 62% in NOER vs WT. Spermatogenesis is essentially totally lost by 8 months in Esr1KO males, and thus, these could not be compared with 8-month NOERs. Efficiency of sperm production was also significantly reduced by 8 months in NOER males (B). The number for all genotypes and ages are as in Figure 1. ****, P < .0001. ns, not significantly different at P < .05.
Figure 8.
Figure 8.. Serum hormone concentrations in 8-month WT and NOER mice (n = 8 for both genotypes), and data are shown as mean ± SEM.
Log-transformed serum T concentrations (A) were greater in NOER mice compared with WT. Serum LH (B), E2 (C), and FSH (D) concentrations did not differ in WT and NOER mice.
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