A Role for Androgens in Epithelial Proliferation and Formation of Glands in the Mouse Uterus

Abstract

The endometrium consists of stromal and epithelial compartments (luminal and glandular) with distinct functions in the regulation of uterine homeostasis. Ovarian sex steroids, namely 17β-estradiol and progesterone, play essential roles in modulating uterine cell proliferation, stromal-epithelial cross-talk and differentiation in preparation for pregnancy. The effect of androgens on uterine function remains poorly understood. The current study investigated the effect of the non-aromatizable androgen dihydrotestosterone (DHT) on mouse endometrial function. Ovx female mice were given a single sc injection (short treatment) or 7 daily injections (long treatment) of vehicle alone (5% ethanol, 0.4% methylcellulose) or vehicle with the addition of 0.2 mg DHT (n=8/group) and a single injection of bromodeoxyuridine 2 hours prior to tissue recovery. Treatment with DHT increased uterine weight, the area of the endometrial compartment and immunoexpression of the androgen receptor in the luminal and glandular epithelium. Treatment-dependent proliferation of epithelial cells was identified by immunostaining for MKi67 and bromodeoxyuridine. Real-time PCR identified significant DHT-dependent changes in the concentrations of mRNAs encoded by genes implicated in the regulation of the cell cycle (Wee1, Ccnd1, Rb1) and stromal-epithelial interactions (Wnt4, Wnt5a, Wnt7a, Cdh1, Vcl, Igf1, Prl8, Prlr) as well as a striking effect on the number of endometrial glands. This study has revealed a novel role for androgens in regulating uterine function with an effect on the glandular compartment of the endometrium. This previously unrecognized role for androgens has implications for our understanding of the role of androgens in regulation of endometrial function and fertility in women.

Figure 1.

Treatment with DHT induces a trophic effect in the mouse uterus. Mice were ovx and treated with VC or DHT as described in Materials and Methods. A, At 24 h there was a modest but significant increase in uterine wet weight with DHT treatment whereas after 7 d of treatment the same effect was more pronounced. B–D, Similar effects for a time-dependent increase with DHT treatment were observed in total uterine surface area (B), endometrial area (C), and myometrial area (D). E, Treatment with DHT resulted in a significant time-dependent decrease in uterine cell density at 24 h and 7 d. F, Representative images of hematoxylin- and eosin-stained mouse uterine cross-sections after steroid treatments. All error bars represent SEM, and Student's unpaired t test was used for statistical analysis comparing VC- and DHT-treated samples of the respective time point (n = 4–8 per treatment group). *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001. Scale bar: 500 μm.

Figure 2.

Treatment with DHT results in a marked increase of AR immunoreactivity in uterine epithelial cells accompanied by a down-regulation of Ar mRNA. A and C, Expression of AR was restricted to the stromal compartment and the myometrial smooth muscle cells of the vehicle groups at both 24 h (A) and 7 d (C) with glandular (black arrowheads) and luminal epithelial AR levels being low to undetectable. B and D, Treatment with DHT resulted in intense AR immunostaining in the stroma accompanied by expression of AR in the luminal and glandular epithelium after 24 h (B, red arrowheads), with the glandular epithelium retaining AR expression after 7 d of DHT treatment (D). E, Treatment with DHT for 7 d resulted in a significant down-regulation of Ar mRNA in the mouse uterus. All error bars represent SEM (n = 8 per treatment group). **, P < .01. Scale bars: 100 μm. Neg: negative control.

Figure 3.

Short-term treatment with DHT increases MKi67 expression in uterine epithelial cells. A and C, VC-treated groups displayed nuclear immunostaining for MKi67 (in red staining) in a small proportion of epithelial cells (arrowheads indicate MKi67-positive epithelial cells) at both 24 h (A) and 7 d (C). B, Treatment with DHT for 24 h induced marked expression of MKi67 in the luminal and glandular epithelium, whereas after 7 d MKi67 staining did not differ from VC-treated groups (D). E, Real-time PCR revealed a significant increase in Mki67 mRNA levels after DHT treatment at 24 h, with no difference observed at 7 d between vehicle and DHT. All error bars represent SEM (n = 8 per treatment group). ****, P < .0001. Scale bars: 200 μm. L, lumen; Neg, negative control.

Figure 4.

DHT stimulates transient epithelial cell proliferation in both glandular and luminal compartments. A and B, With VC treatment, BrdU was detected in very few epithelial cells after 24 h, whereas treatment with DHT induced pronounced luminal and glandular epithelial nuclear BrdU incorporation. C, Quantification of BrdU-positive cells revealed a significant increase of luminal (LE) and glandular (GE) epithelial proliferation with DHT after 24 h of treatment. D–F, After 7 d of treatment, proliferation index showed no significant difference between VC and DHT groups. All error bars represent SEM (n = 8 per treatment group), ***, P < .001; ****, P < .0001. Scale bars: 200 μm. Neg: negative control.

Figure 5.

Treatment with DHT has time-dependent effect on concentrations of mRNAs encoded by genes implicated in regulation of the cell cycle and stromal-epithelial cross-talk. Real-time PCR analysis of whole-tissue uterine homogenates identified altered expression of cell-cycle regulators and proliferation markers (Wee1 [A], Ccnd1 [B], Rb1 [C]) and genes involved in stromal-epithelial interactions and epithelial growth factors (Wnt4 [D], Wnt5α [E], Wnt7α [F], Cdh1 [G], Vcl [H], Igf1 [I], Prl8 [J], Prlr [K], Foxa2 [L], Fgf8 [M], Hgf [N]). For all RT-PCR analyses, n = 8 per treatment group per time point were used and all data were normalized to the housekeeping gene 18s. All error bars represent SEM, *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001.

Figure 6.

Androgen treatment stimulates the glandular compartment of the mouse uterus. Immunohistochemistry for the gland-specific transcription factor FOXA2 revealed more glands after DHT treatment (B) compared with VC (A) at 7 d. Quantification of the glands (C) demonstrated a significant increase in the total number of glands per cross-section after DHT treatment compared with vehicle. All error bars represent SEM (n = 8 per treatment group). ****, P < .0001. Scale bars: 200 μm. Neg: negative control.