Influence of Prenatal Diethylstilbestrol (DES) and dietary phytoestrogens on sperm function and reproductive organ development in male mice

Abstract

Objective: The influence of exogenous estrogens on male reproductive parameters remains controversial. Prenatal estrogen exposure may influence male organ development and dietary phytoestrogens have been shown to exert physiologic estrogen activity, with the possible consequence of endocrine disruption. We sought to determine the effects of both in utero exogenous estrogen exposure and dietary phytoestrogen intake on male reproductive organ development and sperm physiology in mice.Design: Experimental animal study.Materials and Methods: In utero exposure of males was performed on pregnant mice fed corn oil with no DES (control), 0.1μg/kg/day DES (low-dose), and 50μg/kg/day DES (high-dose) on gestation days 11–17. Subsequent male pups (n=36) were then fed Purina 5001 (Diet A, 491mg/g diet phytoestrogens) or Purina 5002 (Diet B, 159 mg/g diet phytoestrogens) until sacrifice on postnatal day 90. Testis and epididymal blotted weights were obtained prior to sperm isolation from the cauda epididymis by swim-out after mincing. Morphology, sperm capacitation and acrosome reaction (AR) was evaluated. Cumulus masses from gonadotropin hyperstimulated females were inseminated with sperm at a concentration of 1 million motile sperm per ml for in vitro fertilization (IVF). Percent fertilization, defined as development to the 2-cell stage, and percent embryo development to blastocyst was recorded for each male. Outcome means were analyzed with ANOVA, as well as 2-way ANOVA to detect interactive effects, with p<0.05 considered significant.Results: In utero high-dose DES resulted in decreased testis and epididymal weight, but was not statistically significant unless combined testis/epididymal weight was analyzed (p=0.023). No significant DES effect was seen on sperm morphology, sperm capacitation or acrosome reaction (all p0.05). Fertilization (p=0.477) and embryo development (p=0.410) with IVF was also not adversely affected by in utero DES exposure. No diet effect was seen on epididymal or testis weights or sperm morphology (all p0.05). A decrease in spontaneous capacitation (p<0.001) and AR (p=0.012) was found in the higher phytoestrogen diet. Higher dietary phytoestrogen content also resulted in increased fertilization rates (p=0.014) but lower development to blastocyst (p=0.007). Two-way ANOVA analysis showed no interaction between DES exposure and dietary phytoestrogen content among measured outcomes.Conclusion: In utero high-dose DES exposure decreased combined epididymal/testis weight but had no influence on sperm function or in vitro fertilizing ability. Higher dietary phytoestrogen intake resulted in desirable differences in sperm capacitation and acrosome reaction, as well as improved rates of fertilization with IVF. No interaction between these two exposures existed. Objective: The influence of exogenous estrogens on male reproductive parameters remains controversial. Prenatal estrogen exposure may influence male organ development and dietary phytoestrogens have been shown to exert physiologic estrogen activity, with the possible consequence of endocrine disruption. We sought to determine the effects of both in utero exogenous estrogen exposure and dietary phytoestrogen intake on male reproductive organ development and sperm physiology in mice. Design: Experimental animal study. Materials and Methods: In utero exposure of males was performed on pregnant mice fed corn oil with no DES (control), 0.1μg/kg/day DES (low-dose), and 50μg/kg/day DES (high-dose) on gestation days 11–17. Subsequent male pups (n=36) were then fed Purina 5001 (Diet A, 491mg/g diet phytoestrogens) or Purina 5002 (Diet B, 159 mg/g diet phytoestrogens) until sacrifice on postnatal day 90. Testis and epididymal blotted weights were obtained prior to sperm isolation from the cauda epididymis by swim-out after mincing. Morphology, sperm capacitation and acrosome reaction (AR) was evaluated. Cumulus masses from gonadotropin hyperstimulated females were inseminated with sperm at a concentration of 1 million motile sperm per ml for in vitro fertilization (IVF). Percent fertilization, defined as development to the 2-cell stage, and percent embryo development to blastocyst was recorded for each male. Outcome means were analyzed with ANOVA, as well as 2-way ANOVA to detect interactive effects, with p<0.05 considered significant. Results: In utero high-dose DES resulted in decreased testis and epididymal weight, but was not statistically significant unless combined testis/epididymal weight was analyzed (p=0.023). No significant DES effect was seen on sperm morphology, sperm capacitation or acrosome reaction (all p0.05). Fertilization (p=0.477) and embryo development (p=0.410) with IVF was also not adversely affected by in utero DES exposure. No diet effect was seen on epididymal or testis weights or sperm morphology (all p0.05). A decrease in spontaneous capacitation (p<0.001) and AR (p=0.012) was found in the higher phytoestrogen diet. Higher dietary phytoestrogen content also resulted in increased fertilization rates (p=0.014) but lower development to blastocyst (p=0.007). Two-way ANOVA analysis showed no interaction between DES exposure and dietary phytoestrogen content among measured outcomes. Conclusion: In utero high-dose DES exposure decreased combined epididymal/testis weight but had no influence on sperm function or in vitro fertilizing ability. Higher dietary phytoestrogen intake resulted in desirable differences in sperm capacitation and acrosome reaction, as well as improved rates of fertilization with IVF. No interaction between these two exposures existed.