Brief exposure to progesterone during a critical neonatal window prevents uterine gland formation in mice

Abstract

Uterine gland development (adenogenesis) in mice begins on Postnatal Day (PND) 5 and is completed in adulthood. Adenogenesis depends on estrogen receptor 1, and progesterone (P4) inhibits mitogenic effects of estrogen on uterine epithelium. This progestin-induced effect has been used to inhibit uterine gland development; progestin treatment of ewes for 8 wk from birth has produced infertile adults lacking uterine glands. The goals of the present study were to determine if a window of susceptibility to P4-mediated inhibition of uterine gland development exists in mice and whether early P4 treatment abolishes adenogenesis and fertility. Mice were injected daily with P4 (40 μg/g) or vehicle during various postnatal windows. Adenogenesis, cell proliferation, and expression of key morphoregulatory transcripts and proteins were examined in uteri at PNDs 10 and 20. Additionally, adenogenesis was assessed in isolated uterine epithelium. Treatment during PNDs 3-9, 5-9, or 3-7 abolished adenogenesis at PND 10, whereas treatments during PNDs 3-5 and 7-9 did not. Critically, mice treated during PNDs 3-9 lacked glands in adulthood, indicating that adenogenesis did not resume after this treatment. However, glands were present by PND 20 and later following treatment during PNDs 5-9 or 3-7, whereas treatment during PNDs 10-16 produced partial inhibition of adenogenesis at PND 20 and later. Epithelial proliferation at PND 10 was low following P4 treatment (PNDs 3-9) but exceeded that in controls at PND 20, indicating a rebound of epithelial proliferation following treatment. Messenger RNA for Wnt, Fzd, and Hox genes was altered by neonatal P4 treatment. All groups cycled during adulthood. Mice treated with P4 during PNDs 3-9, but not during other developmental windows, showed minimal fertility in adulthood. In summary, brief P4 treatment (7 days) during a critical neonatal window (PNDs 3-9) transiently inhibited epithelial proliferation but totally and permanently blocked adenogenesis and adult fertility. This resulted in permanent loss of uterine glands and, essentially, total infertility during adulthood. The narrow window for inhibition of adenogenesis identified here may have implications for development of this methodology as a contraceptive strategy for animals.

FIG. 1.

Uterine adenogenesis in C57BL/6 control mice and mice exposed to P4 (40 mg/kg) during PNDs 3–9. A) Adenogenesis begins on PND 5, as shown by the absence (top) and presence (bottom; arrows) of uterine glands in isolated epithelium from a 5- and 6-day-old animal, respectively. B–D) Both gland number and size (arrows) are increased in PND 10 uterine epithelium isolated from control mice (B) compared to PND 6. Treatment with P4 during PNDs 3–7 (C) or 3–9 (D) resulted in total absence of glands in epithelium at PND 10. Bar = 100 μm.

FIG. 2.

Adenogenesis in 20-day-old control mice and mice exposed to P4 postnatally. Adenogenesis in controls (arrows) was more extensive at PND 20 (A) than at PND 10 (compare with Fig. 1B). By PND 20, mice treated with P4 during PNDs 3–7 (B) showed gland development (arrows) despite lacking glands at PND 10 (compare with Fig. 1D). In contrast, at PND 20, uterine epithelium from mice treated with P4 during PNDs 3–9 still lacked any gland development (C). Note that the uterine epithelial folds indicated by arrowheads in C are not glands. When mice were treated with P4 during PNDs 10–16, by PND 20 glands were apparent (D), although less extensive than in controls (A), and gland number appeared reduced compared to controls at PND 10. Bar = 100 μm.

FIG. 3.

Effects of neonatal P4 treatment on uterine cell proliferation as measured by immunohistochemical staining of MKI67. At PND 10, extensive proliferation was seen in both epithelium (E) and stroma (S) of control uteri (A). In contrast, uterine epithelial proliferation in 10-day-old mice treated with P4 during PNDs 3–9 was dramatically reduced (B), although stromal (S) proliferation remained robust. By PND 20 in control uteri, both epithelial and stromal proliferation (C) was reduced compared to PND 10. In contrast, 20-day-old uterine epithelium in mice that received P4 during PNDs 3–9 showed robust epithelial proliferation (D) exceeding that of controls. Bars = 100 μm. Quantitation of uterine epithelial and stromal labeling index in the groups mentioned above indicates that epithelial proliferation in mice treated during PNDs 3–9 with P4 is sharply reduced compared to controls at PND 10 but significantly exceeds controls by PND 20 (E). Stromal labeling index was not significantly different than control in P4 (P)-treated mice at PNDs 10 or PND 20, despite a trend toward an increase in the former. *P < 0.05 vs. control. Data are shown as mean ± SEM.

FIG. 4.

Immunohistochemical staining of HAND2 in the 5-day-old neonatal mouse uterus. Strong staining was seen in stroma (S) cells, whereas both epithelium (E) and myometrium (M) were unstained. Bar = 100 μm.

FIG. 5.

Spatial localization of Wnt gene expression was examined by in situ hybridization analysis in uteri from control mice (CO) and mice that received P4 (P) injections daily during PNDs 3–9 (n = 5 per treatment per day). In each panel portion, representative photomicrographs of in situ hybridization results are shown with light-field and dark-field illumination, respectively. GE, glandular epithelium; LE, luminal epithelium; M, myometrium; S, stroma. Bars = 100 μm.

FIG. 6.

Spatial localization of Fzd and Hox gene expression was examined by in situ hybridization analysis in uteri from control mice (CO) and mice that received P4 (P) injections daily during PNDs 3–9 (n = 5 per treatment per day). In each panel portion, representative photomicrographs of in situ hybridization results are shown with light-field and dark-field illumination, respectively. GE, glandular epithelium; LE, luminal epithelium; M, myometrium; S, stroma. Bars = 100 μm.

FIG. 7.

Wnt, Fzd, and Hox gene expression was quantitated by real-time RT-PCR analysis in uteri from control (CO) mice and mice that received P4 (P) injections daily during PNDs 3–9. *P < 0.05 vs. CO.

FIG. 8.

Adult uteri lack glands following neonatal P4 treatment during PNDs 3–9. A) Isolated uterine epithelium from an adult mouse that was treated during PNDs 3–9 with P4. This epithelium showed an absence of glands, although folds of uterine epithelium were still present. In contrast, adult uterine epithelium normally showed extensive glands that covered the epithelial surface (inset). B and C) Histological sections of adult control uteri (B) showed extensive glands (arrows), whereas uteri exposed to P4 during PNDs 3–9 (C) lacked glands in adulthood, corroborating observations in isolated epithelial whole mounts (A). E, epithelium; S, stroma. Bar = 200 μm (A) and 100 μm (B and C).