Effects of ICI 182780 on estrogen receptor expression, fluid absorption and sperm motility in the epididymis of the bonnet monkey

Abstract

Background: The importance of estrogen in regulation of fluid absorption and sperm maturation in the rodent epididymis has been established from studies on estrogen receptor-alpha knockout mice. However, functional studies on the role of estrogen in primate epididymis have been few. The main objective of this study was therefore to extend these observations and systematically analyze the presence and function of estrogen receptors in modulating the function of the primate epididymis, using the bonnet monkey (Macaca radiata) as a model system.

Methods: A steroidal estrogen receptor (ER) antagonist, ICI 182780 (ICI), was administered to adult male bonnet monkeys via mini-osmotic pumps for a duration of 30 to 180 days. The expression of key estrogen-regulated genes (ER-alpha, Na-K ATPase alpha-1 and Aquaporin-1) was examined at specific time points. Further, the effect of ICI in modulating fluid reabsorption in efferent ductules was monitored, and critical sperm-maturation parameters were also analyzed.

Results: Our studies in the bonnet monkey revealed that both ER-alpha and ER-beta were expressed in all the three regions of the epididymis. We observed an increase in ER-alpha mRNA and protein in the caput of ICI-treated monkeys. Steady state mRNA levels of the water-channel protein, Aquaporin-1, was significantly lower in the caput of ICI-treated monkeys compared to controls, whereas the mRNA levels of Na-K ATPase alpha-1 remained unchanged. In vitro incubation of efferent ductules with ICI resulted in two-fold increase in tubular diameter, indicating affected fluid reabsorption capacity. Furthermore, sperm from ICI-treated monkeys were immotile.

Conclusion: Taken together, our results point to an integral role for estrogen in modulating the functions of the bonnet monkey epididymis. This study also demonstrates possible differences in the epididymal physiology of rodents and non-human primates, and thus underscores the significance of reports such as these, that examine the physiology of non-human primates (as opposed to rodents), in an attempt to understand similar events in the human.

Figure 1

a RT-PCR analyses of ERα (Panel A), AQP-1 (Panel B), Na⁺-K⁺ ATPase-α1 (Panel C) and GAPDH (Panel D) in the bonnet monkey epididymis: RNA isolated from the caput, corpus and cauda regions of the bonnet monkey epididymis was reverse transcribed and the cDNA so obtained was subjected to semi-quantitative PCR in the linear range of amplification with GAPDH amplification as an internal control. Panel E is a graphical representation of this data wherein, the densitometric signal intensities obtained for ERα, AQP-1, Na⁺-K⁺ ATPase-α1 were normalized against that of GAPDH and plotted as percent change of the individual intensities with respect to the caput region. Data represents Mean ± SEM of three independent experiments. **P < 0.05, ***P < 0.001. b Western blot analyses for ERα and ERβ in the bonnet monkey epididymis: 100 μg each of protein lysates obtained from caput, corpus and cauda regions of the bonnet monkey epididymis was electrophoresed on 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with antibody specific to ERα. The blot was stripped and reprobed with an antibody specific to ERβ. Following this, the blot was once again stripped with an antibody specific to actin which served as an internal control for assessing equality of protein loading. Panel F is a graphical representation of this data wherein, the densitometric signal intensities obtained for ERα and ERβ were normalized against that of actin and plotted as percent change of the individual intensities with respect to the caput region. The figure is representative of aleast three independent experiments.

Figure 2

Immunohistochemical localization of ERα and ERβ in the bonnet monkey epididymis: a. ERα staining: Immuno-peroxidase staining for ERα showing intense nuclear staining in caput (Panel A), corpus (Panel B) and cauda (Panel C) regions of the bonnet monkey epididymis. Staining was most intense in the tubular epithelial cells lining the lumen (inset; staining indicated by arrow). Panels D-F show the negative controls for each region, wherein the primary antibody was incubated with the blocking peptide. Panel G shows intense staining in the nuclei of mouse uterus tissue, which served as the positive control. Panel H shows an enlarged epididymal tubule representing the staining pattern in different cell types- while staining was intense in the nuclei of the tubular epithelium, weak staining was observed in the smooth muscle cells surrounding the tubule and that of the vascular ducts ; TE-tubular epithelial cell, PSMC- peri-tubular smooth muscle cell, VSMC- vascular smooth muscle cell. Bar in Panel A = 100 μ b. The caput, corpus and cauda regions of the bonnet monkey epididymis (Panels A-C, respectively) show intense staining for ERβ both in the nuclei of the epithelial cells lining the lumen (inset; staining indicated by arrow), and the surrounding stroma. Panels D-F represent the negative controls for each region wherein the addition of the primary antibody was omitted. Panel G shows intense staining in rat ventral prostate tissue, which served as a positive control. Panel H shows absence of ERβ staining in rat liver tissue, which was used as a negative control. Panel I shows an enlarged epididymal tubule depicting staining in the various cell types, in and around the tubule- considerable staining was also observed in the smooth muscle cells; TE-tubular epithelial cell, PSMC-peri-tubular smooth muscle cell, VSMC- vascular smooth muscle cell. Bar in Panel A = 100 μ.

Figure 3

Serum testosterone levels in the ICI-treated adult male bonnet monkeys: Following vehicle or ICI treatment in monkeys for 30, 60 or 180 days, serum testosterone levels were analyzed by radio-immuno assay. We did not observe any significant changes in serum testosterone levels. Nocturnal surge of testosterone (PM values compared to AM values) also remained unchanged following ICI treatment. Data shown is a Mean ± SEM of three experiments.

Figure 4

a-b RT-PCR analysis for ERα, AQP-1, Na⁺-K⁺ ATPase-α1 and GAPDH in the caput regions of control and 30-day (Fig 4a) and 60-day (Fig 4b) ICI treated monkeys: RNA isolated from the caput region of vehicle-and ICI-treated bonnet monkeys, was reverse transcribed and the cDNA was subjected to semi-quantitative PCR in the linear range of amplification with GAPDH amplification as an internal control. The intensity of ERα expression (Panel A) increased upon ICI treatment compared to that of vehicle-treated controls, with the increase being appreciably greater in the 30-day treatment period. AQP-1 levels were reduced in the ICI-treated group compared to controls (Panel B). There was no discernable change in the Na⁺-K⁺ ATPase-α1 levels between the ICI-treated groups and vehicle controls (Panel C). Panel E is a graphical representation of this data wherein, the densitometric signal intensities obtained for ERα, AQP-1 and Na-K ATPAse-α1 were normalized to that of GAPDH (Panel D). Data represents Mean ± SEM of three experiments. **P < 0.05, ***P < 0.001.

Figure 5

Expression of ERα in the caput region of 30- and 60-day ICI-treated bonnet monkeys: Immunoperoxidase staining for ERα showed striking differences in expression intensities following 30 and 60 days of ICI treatment. Staining as seen previously was nuclear (inset; indicated by arrow). Compared to the 30-day vehicle-treated control caput sample (Panel A), the intensity of ERα staining was unchanged in the caput after 30-day ICI treatment (Panel B). Staining was very intense in the 60-day ICI-treated caput (Panel D) in comparison to the corresponding 60-day vehicle treated control (Panel C). Bar in Panel A = 100 μ.

Figure 6

Change in the luminal diameter of efferent ductules after in-vitro incubation with ICI: Efferent ductules were minced and tissue fragments of 2–3 mm were incubated in M199 medium containing dihydrotestosterone (1 nM), 17β-estradiol (1 nM) with or without ICI (1 μM) for 24 hr. The ends of the tubules were ligated with a sterile catgut and incubated in a fresh medium of the same composition, for further 24 hr. Subsequent to this total incubation period of 48 hr, the ductules were rapidly fixed in Bouin's fluid and embedded in paraffin. Sections were stained in hematoxylin and eosin and diameter of the tubules measured. In absence of ICI, the control (Panel A) shows reduced luminal diameter compared to ICI-incubated efferent ductules (Panel B). Panel C is a graphical representation of the luminal diameters. Values represent Mean ± SEM from three independent experiments. Bar in Panel A = 100 μ; *** P < 0.001.

Figure 7

Effect of ICI treatment on the sperm count in bonnet monkeys: Semen from the monkeys from vehicle- and ICI-treated bonnet monkeys was collected by electro-ejaculation, and sperm count was determined using Neubauer hemacytometer following 1:200 dilution of the semen in saline. As seen from the Panels A-C, sperm count does not change considerably in the ICI-treated groups compared to the control. Data represented is a Mean ± SEM of three independent experiments.