Epididymal hypo-osmolality induces abnormal sperm morphology and function in the estrogen receptor alpha knockout mouse

Abstract

Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility.

FIG. 1.

Esr1KO sperm have increased frequency of spontaneous acrosome reactions. A) Sperm from the initial segment, caput, corpus, and cauda were assessed for acrosome status under light microscopy by Coomassie Blue staining. Arrowheads indicate sperm loss of acrosomal contents. B) Wild-type (WT) and heterozygote (HET) mice have a basal level of spontaneous acrosome reactions that remain stable in all regions of the epididymis (3 ± 1%). In comparison, Esr1KO mice have a significantly increased proportion of spontaneous reactions in the initial segment, caput, and corpus. Each bar represents the mean ± SEM. C) Acrosomal status of cauda sperm at 6 and 14 wk of age (two animals per group). The large variability in acrosome status in the Esr1KO cauda epididymis may be associated with an age-dependent defect that progressively worsens and approaches 100% in 14-wk-old mice. Each bar represents the mean ± SD. D) Electron microscopy of WT sperm reveals normal intact plasma membranes surrounding the nucleus (N) and acrosomic vesicle (A). Esr1KO sperm revealing a phenotype of vesiculated, irregular, or broken plasma membranes (arrows) that overlie the acrosomic vesicle (A) and nucleus (N). M, mitochondria in the midpiece. *Denotes significant difference from WT/HET within an epididymal segment (P ≤ 0.01).

FIG. 2.

Esr1KO sperm exhibit flagellar angulation and coiling. A) Light microscopy revealed varying degrees of coiling in Esr1KO sperm. Hairpin loops (loop) were the mildest form observed and were frequently found in control animals. Coils isolated to the tail midpiece region (MC) and coils that encompassed the entire tail length (WC) were more severe. B) Electron microscopy (TEM) of wild-type (WT) sperm within the caput epididymis showing normal nucleus (N) and midpiece region with mitochondria (M). C) TEM of Esr1KO sperm within the caput epididymis showing the coiling phenotype in situ, where sperm were frequently imaged with two or more midpiece or flagellar cross sections enclosed in one membrane. M, mitochondria. D) TEM of WT sperm showing midpiece cross sections with normal classical axonemal architecture. M, mitochondria. E) TEM of Esr1KO sperm showing the coiled phenotype with two or more cross sections of the midpiece with mitochondria (M) and flagellar axonemal complex (F).

FIG. 3.

Sperm tail coiling is more pronounced in the Esr1KO and increases with transit through the epididymis. A) Quantification of sperm tail morphology via light microscopy revealed that control wild-type (WT) and heterozygote (HET) mice have consistently straight-tail morphology when collected from the initial segment, caput, and corpus epididymis (93 ± 3%). In the control cauda, the percentage of straight flagella decreased to 75 ± 4%, reflecting an increase in hairpin loops. B) In comparison, Esr1KO mice had a significantly reduced proportion of straight sperm, relative to the control mice, recovered from the initial segment, caput, corpus, and cauda that reflects an increase in the proportion of both loops and more severe forms of tail coiling (midpiece and whole tail coils described in Fig. 2). Each bar represents the mean ± SEM. *Denotes significant difference from WT/HET within an epididymal segment (P ≤ 0.04).

FIG. 4.

Esr1KO sperm tail coiling is a function of epididymal transit. A) Analysis of adult Esr1KO testes showed an approximate 10% decrease in straight-tail morphology, relative to control wild-type (WT) and heterozygote (HET) mice. However, sperm recovered from young (5-wk-old) Esr1KO testes that had not yet developed the characteristic fluid accumulation in the testes showed no difference in the percentage of straight flagella when compared to control mice (98 ± 1%). B) Nevertheless, sperm recovered from 5-wk-old Esr1KO caput had a small but statistically significant reduction in straight flagella. Each bar represents the mean ± SEM. *Denotes significant difference from corresponding sperm morphology type in WT/HET (P ≤ 0.05).

FIG. 5.

Esr1KO testis exhibits sperm flagellar angulation and coiling only in the lumen. A) Light microscopy of wild-type (WT) seminiferous tubules reveals normal development of germ cells in stages XI and VII. Flagella of elongated spermatids extend straight into the lumen (arrow). B) In Esr1KO testis, spermatids in the stage VII–VIII epithelia appear normal, with straight tails projecting into the lumen. C) When the lumen becomes dilated because of fluid back pressure, the sperm display coiled tails and angulation. D) Higher magnification of the luminal area in C. Sperm with coiled tails (cT) were observed in the lumen.

FIG. 6.

Osmolality of Esr1KO luminal fluids is reduced in comparison to control. Osmolality was measured from luminal fluids collected from the testes and whole caput region (including initial segment) of wild-type (WT), heterozygote (HET), and Esr1KO mice. The testicular fluid of WT and HET mice was 362 ± 3 mmol/kg and increased in the caput to 435 ± 11 and 424 ± 4 mmol/kg, respectively. In comparison, the luminal contents of the Esr1KO were significantly hypo-osmotic in the testes (346 ± 2 mmol/kg) and caput (395 ± 1 mmol/kg) relative to both WT and HET animals. Each point in the histogram represents an individual measurement. The three determinations for Esr1KO caput luminal fluid overlie one another. Bars = mean. *Denotes significant difference from corresponding regions in WT/HET (P ≤ 0.01).

FIG. 7.

Increased tonicity partially rescues the coiling defect of Esr1KO sperm. Tail morphology was assessed after sperm were released into a more wild-type-like tonicity, with 300 mmol/kg being the standard osmolality of sperm collection media. Sperm recovered from the Esr1KO caput responded with an approximately 20% increase in straight-tail morphology when placed into media of 350 and 430 mmol/kg. Although there was a similar trend in sperm recovered from the Esr1KO cauda, the degree of rescue was minimal. Each bar represents the mean ± SEM. *Denotes significant difference from corresponding sperm morphology in 300 mmol/kg (P ≤ 0.05).

FIG. 8.

Decreased osmolality with increased pH produces Esr1KO-like morphological defects in wild-type sperm. Wild-type sperm were recovered from either the caput or cauda and introduced into media of osmolality 300, 260, and 220 mmol/kg, pH 7.8. Morphology was assessed and compared to basal medium (300 mmol/kg, pH 7.4). A) Although the increase in pH alone did not affect tail morphology, decreasing osmolality with increased pH resulted in a decreased proportion of sperm with straight flagellar morphology and increased coiling. B) Cauda sperm exposed to a similar media, with decreasing tonicity and increased pH, led to a decrease of straight flagella. Each bar represents the mean ± SEM. *Denotes significant difference from corresponding sperm morphology in 300 mmol/kg, pH 7.8 (P ≤ 0.03).