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.
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