OXTR overexpression leads to abnormal mammary gland development in mice

in Journal of Endocrinology
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Oxytocin receptor (OXTR) is a G-protein-coupled receptor and known for regulation of maternal and social behaviors. Null mutation (Oxtr−/−) leads to defects in lactation due to impaired milk ejection and maternal nurturing. Overexpression of OXTR has never been studied. To define the functions of OXTR overexpression, a transgenic mouse model that overexpresses mouse Oxtr under β-actin promoter was developed (++ Oxtr). ++ Oxtr mice displayed advanced development and maturation of mammary gland, including ductal distention, enhanced secretory differentiation and early milk production at non-pregnancy and early pregnancy. However, ++ Oxtr dams failed to produce adequate amount of milk and led to lethality of newborns due to early involution of mammary gland in lactation. Mammary gland transplantation results indicated the abnormal mammary gland development was mainly from hormonal changes in ++ Oxtr mice but not from OXTR overexpression in mammary gland. Elevated OXTR expression increased prolactin-induced phosphorylation and nuclear localization of STAT5 (p-STAT5), and decreased progesterone level, leading to early milk production in non-pregnant and early pregnant females, whereas low prolactin and STAT5 activation in lactation led to insufficient milk production. Progesterone treatment reversed the OXTR-induced accelerated mammary gland development by inhibition of prolactin/p-STAT5 pathway. Prolactin administration rescued lactation deficiency through STAT5 activation. Progesterone plays a negative role in OXTR-regulated prolactin/p-STAT5 pathways. The study provides evidence that OXTR overexpression induces abnormal mammary gland development through progesterone and prolactin-regulated p-STAT5 pathway.

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  • Supplementary Fig. 1 Phenotypic analysis of ++Oxtr mice. (A) H&E staining of paraffin longitudinal sections from WT and ++Oxtr mammary glands (3rd pair) at pregnancy (P3.5, P9.5) and lactation (L4, L14 L18). Scale bar: 300um. Original magnifications: x4. (B) Representative pup size images and body weight of pups (n=9) nursed by CD1 mothers (n=3) and forested ++Oxtr mothers (n=3). (C) Quantitative RT-PCR analysis of involution related gene expression of L1 and L18 WT and ++Oxtr mammary glands, n=6 for each time point. Data are represented as mean ± SEM. **p <0.01; ***p <0.001, calculated using two-tailed unpaired t test.
  • Supplementary Fig. 2 Hormonal related pathway in ++Oxtr mice. (A) Serum estradiol level measurements. Serum samples were collected from virgin WT and ++Oxtr mice at 12 weeks, n=5. (B) Quantitative RT-PCR analysis of Stat5a and Stat5b expression at different developmental stages, n=4 for each time point. Gene expression levels were normalized to 18S ribosomal RNA. Data are represented as mean ± SEM, calculated using two-tailed unpaired t test.
  • Supplementary Fig. 3 Phenotypic analysis of ++Oxtr mice with hormone treatment. ++Oxtr mice were treated with P4 or PRL for 3 days at late pregnancy (P18.5 and P19.5) and L1. (A) Immunochemistry analysis of p-STAT5 in L1 mammary gland (3rd pair) after hormonal treatment. Nuclei were stained blue with hematoxylin. Scale bar: 100um. Original magnifications: x20. (B) Survival analysis of pups (n=20) from WT dams (n=3), pups (n=19) from ++Oxtr dams (n=3), pups (n=15) from P4-treated ++Oxtr dams (n=3) and pups (n=19) from PRL-treated ++Oxtr dams (n=3) within 24h of birth. Data are represented as mean ± SEM. p <0.0001, calculated using Log-rank (Mantel-Cox) test.
  • Supplementary Table 1. Primer sequences used for real time PCR

 

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    Overexpression of OXTR and localization during mammary gland development. Total RNA and protein were isolated from mammary gland (4rd pair), ovary, uterus, brain, heart, kidney and white adipose tissue (WAT) of WT and ++Oxtr littermates at 12 weeks. (A) Oxtr mRNA expression in female reproductive system by RT-PCR, n = 4 for each tissue. (B) OXTR expression in female reproductive system by Immunoblotting. GAPDH is served as a loading control. Protein quantifications using Image J, n = 4 for each tissue. (C) Oxtr mRNA expression in brain, heart, kidney and WAT by RT-PCR, n = 3 for each tissue. Mammary glands (4rd pair) were harvested from ++Oxtr and WT littermates at 8 and 12 weeks (8 W and 12 W), pregnancy (P3.5 and P9.5) and lactation (L1, L4 and L18). (D) RT-PCR analysis of Oxtr mRNA expression in mammary glands during development, n = 3 for each time point. (E) Immunoblotting analysis of OXTR in mammary gland. Protein quantifications using Image J, n = 4 for each time point. (F) Immunochemistry staining of OXTR in mammary sections (5 µm) and quantifications of immunostaining using Image Pro Plus. Nuclei were stained blue with hematoxylin. Scale bar: 100 µm. Original magnifications: ×20, n = 3 for each time point. Data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, calculated using two-tailed unpaired t test.

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    OXTR overexpression accelerates mammary gland development and milk production at non-pregnancy and pregnancy. Mammary glands (4rd pair) of ++Oxtr and WT mice at 3, 8 and 12 weeks (3, 8, 12 W), pregnancy (P3.5, P9.5) were harvested. (A) Whole-mount staining of mammary gland at non-pregnancy (3, 8 and 12 W). Scale bar: 1 mm. Original magnifications: ×1.25. (B) Whole-mount staining of mammary gland at P3.5 and P9.5. Scale bar: 500 µm. Original magnifications: ×4. (C) Mammary duct caliber (mm) of 12 W, P3.5 and P9.5 WT and ++Oxtr mice. Quantifications from whole-mount staining image using CAD software, n = 5 for each time point. (D) H&E staining of mammary gland from 12 W, P3.5 and P9.5 WT and ++Oxtr mice, duct full of proteinaceous material (arrows). Scale bar: 200 µm. Original magnifications: ×10. (E) Macroscopic images of mammary gland (3rd pair), duct full of milk (arrows). Scale bar: 1 cm. (F) Gene expression of major milk protein Csn2 and Wap during development (12 W, P3.5 and P9.5), n = 3 for each time point. Data are represented as mean ± s.e.m. ***P < 0.001, calculated using two-tailed unpaired t test.

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    Impaired lobuloalveologenesis and early involution results in lactation failure of ++Oxtr mice. (A) Survival analysis of newborn pups (n = 51) from WT dams (n = 7) and newborn pups (n = 50) from ++Oxtr dams (n = 9). (B) Milk yield by daily litter weight gain of newborn pups after embryos transferring (ET). 5 pups per litter were nursed by WT or ++Oxtr dams (n = 4). (C) Average body weight of pups. 5 pups per litter were nursed by WT or ++Oxtr dams (n = 4). (D) Survival analysis of pups (n = 37) nursed by WT dams (n = 5) and pups (n = 34) nursed by ++Oxtr dams (n = 5) by cross-fostering experiments. (E) RT-PCR analysis of Csn2 and Wap mRNA in ++Oxtr and WT mammary glands at P9.5, L1, L4, L18, n = 6 for each time point. (F) Whole-mount staining of mammary gland and quantification of lobuloalveolar size in L4 and L14. Scale bar: 500 µm. Original magnifications: ×4, n = 4 for each time point. (G) H&E staining of mammary gland in L1 and L18. Scale bar: 200 µm. Original magnifications: ×10. (H) RT-PCR analysis of mammary gland involution-related gene expressions in L1 and L18, n = 6 for each time point. Mammary glands (4rd pair) were harvested from ++Oxtr and WT mice in lactation (L1, L4, L14 and L18). Data are represented as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001, calculated using two-tailed unpaired t test and Log-rank (Mantel-Cox) test.

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    Mammary gland cross transplantation. The transplanted mammary (4rd pair) fat pads were harvested 8 weeks post transplantation from WT or ++Oxtr mice. (A) Whole-mount staining of transplanted mammary gland. Scale bar: 500 µm. Original magnifications: ×4. (B) Outgrowth coverage of the repopulated fat pad (percentage of duct and alveolar staining area) using Image Pro Plus, n = 3. (C) Average duct calibers, n = 3. Data are represented as mean ± s.e.m. *P < 0.05; **P < 0.01, calculated with one-way ANOVA.

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    Progesterone/RANKL and prolactin/p-STAT5 are altered during mammary gland development in ++Oxtr mice. (A) Serum progesterone levels in WT and ++Oxtr mice at various stages, n = 5 for each time point. (B) Serum prolactin levels in WT and ++Oxtr mice at various stages, n = 5 for each time point. (C) Immunoblotting analysis of p-STAT5 and RANKL in developing mammary gland from ++Oxtr and WT mice. GAPDH is served as a loading control, n = 4 for each time point. (D) Immunochemistry staining of p-STAT5 in developing mammary gland. The enlarged images at the lower left corner show the state of p-STAT5. Nuclei were stained blue with hematoxylin. Scale bar: 100 µm. Original magnifications: ×20. (E) Quantifications of immunostaining using Image Pro Plus, n = 4 for each time point. The mammary glands (4rd pair) were harvested from ++Oxtr and WT littermates at 12 W, P3.5, P9.5, L1 and L18. Data are represented as mean ± s.e.m. *P < 0.05; **P < 0.01, ***P < 0.001, calculated using two-tailed unpaired t test.

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    Progesterone can reverse the PRL/p-STAT5 effect at non-pregnancy. Eleven-weeks WT and ++Oxtr mice were treated with a vehicle or 300 µg (3 mg/mL) P4 for 7 days. The mammary glands (4rd pair) were harvested from ++Oxtr, hormone-treated ++Oxtr and WT littermates at 12 W. (A) Serum prolactin levels of virgin WT and ++Oxtr mice after P4 treatment, n = 3. (B) RT-PCR analysis of Pgr expression in response to hormonal treatment, n = 3. (C) RT-PCR analysis of Oxtr expression after P4 treatment, n = 3. (D) Immunoblotting analysis of OXTR, p-STAT5 and RANKL in virgin mammary gland after P4 treatment. (E) Whole-mount staining of mammary glands in response to hormonal treatment. Scale bar: 500 µm. Original magnifications: ×4. (F) Image of mammary gland (3rd pair) morphology in response to P4 treatment. Scale bar: 1 cm. (G) Quantification of mammary duct caliber (mm) in response to P4 treatment, n = 3. (H) RT-PCR analysis of major milk protein gene expression in mammary glands in response to P4 treatment, n = 3. Data are represented as mean ± s.e.m. **P < 0.01; ***P < 0.001, calculated with one-way ANOVA.

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    Progesterone can exacerbate but prolactin can rescue STAT5 pathway at late pregnancy and lactation. ++Oxtr mice were treated with 300 µg (3 mg/mL) P4 or 50 ng/g PRL for 3 days at late pregnancy (P18.5, P19.5) and L1. The mammary glands (4rd pair) were harvested from ++Oxtr, hormone-treated ++Oxtr and WT littermates in lactation (L1). (A) RT-PCR analysis of Oxtr mRNA expression at L1 in response to hormonal treatment, n = 3. (B) RT-PCR analysis of Prlr mRNA expression at L1 in response to hormonal treatment, n = 3. (C) Immunoblotting analysis of OXTR, p-STAT5 and RANKL in L1 mammary gland after hormonal treatment. (D) Whole-mount staining of mammary glands in response to hormonal treatment. Scale bar: 200 µm. Original magnifications: ×10. (E) Quantification of alveolar size (mm2), n = 3. (F) RT-PCR analysis of major milk protein gene expression in mammary glands in response to hormonal treatment, n = 3. Data are represented as mean ± s.e.m., calculated with one-way ANOVA.

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    Role model of OXTR in mammary gland development. OXTR regulates mammary gland development through progesterone/RANKL axis and PRL/pSTAT5 axis. OXTR regulates PRL/p-STAT5 pathway. Activated STAT5 (p-STAT5) translocates to nucleus to regulate the transcription of Csn2 and Wap. The expression of milk protein Wap and Csn2 marks the mammary epithelium differentiation and maturation. OXTR overexpression inhibits progesterone level. Progesterone/RANKL pathway mediates mammary epithelium proliferation but inhibits PRL/p-STAT5-induced differentiation. RANKL is regulated by both prolactin and progesterone, but prolactin-induced RANKL is prevented in lactation. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0356.

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