Deletion of NKX3.1 via CRISPR/Cas9 Induces Prostatic Intraepithelial Neoplasia in C57BL/6 Mice

Several techniques have been employed for deletion of the NKX3.1 gene, resulting in developmental defects of the prostate, including alterations in ductal branching morphogenesis and prostatic secretions as well as epithelial hyperplasia and dysplasia. To investigate whether the CRISPR/Cas9-mediated technique can be applied to study prostate carcinogenesis through exon I deletion of NKX3.1 gene, alterations in the prostatic intraepithelial neoplasia (PIN) and their regulatory mechanism were observed in the prostate of NKX3.1 knockout (KO) mice produced by the CRISPR/Cas9-mediated NKX3.1 mutant gene, at the ages of 16 and 24 weeks. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were observed to be increased in only the 24 weeks KO mice, although morphogenesis was constant in all groups. Obvious PIN 1 and 2 lesions were frequently detected in prostate of the 24 weeks KO mice, as compared with the same age wild type (WT) mice. Ki67, a key indicator for PIN, was densely stained in the epithelium of prostate in the 24 weeks KO mice, while the expression of p53 protein was suppressed in the same group. Also, both the 16 and 24 weeks KO mice reveal inhibition of the PI3K/AKT/mTOR pathway in the prostate. However, prostate specific antigen (PSA) levels and Bax/Bcl-2 expressions were decreased in the prostate of 16 weeks KO mice, and were increased in only the 24 weeks KO mice. Taken together, the results of the present study provide additional evidence that CRISPR/Cas9-mediated exon 1 deletion of the NKX3.1 gene successfully induces PIN lesions, along with significant alterations of Ki67 expression, EGFR signaling pathway, and cancer-regulated proteins.

Environmental selection during the last ice age on the mother-to-infant transmission of vitamin D and fatty acids through breast milk

Because of the ubiquitous adaptability of our material culture, some human populations have occupied extreme environments that intensified selection on existing genomic variation. By 32,000 years ago, people were living in Arctic Beringia, and during the Last Glacial Maximum (LGM; 28,000–18,000 y ago), they likely persisted in the Beringian refugium. Such high latitudes provide only very low levels of UV radiation, and can thereby lead to dangerously low levels of biosynthesized vitamin D. The physiological effects of vitamin D deficiency range from reduced dietary absorption of calcium to a compromised immune system and modified adipose tissue function. The ectodysplasin A receptor (EDAR) gene has a range of pleiotropic effects, including sweat gland density, incisor shoveling, and mammary gland ductal branching. The frequency of the human-specificEDAR V370Aallele appears to be uniquely elevated in North and East Asian and New World populations due to a bout of positive selection likely to have occurredcirca20,000 y ago. The dental pleiotropic effects of this allele suggest an even higher occurrence among indigenous people in the Western Hemisphere before European colonization. We hypothesize that selection onEDAR V370Aoccurred in the Beringian refugium because it increases mammary ductal branching, and thereby may amplify the transfer of critical nutrients in vitamin D-deficient conditions to infants via mothers’ milk. This hypothesized selective context forEDAR V370Awas likely intertwined with selection on the fatty acid desaturase (FADS) gene cluster because it is known to modulate lipid profiles transmitted to milk from a vitamin D-rich diet high in omega-3 fatty acids.

Localization Of MMP-2 And MMP-9 In Canine Mammary Gland During Pregnancy

SummaryThe mammary gland is unique in its development because most of its branching occurs in adolescent rather than in prenatal development. During early pregnancy extensive ductal side branching occurs while during the second half, secretory lobuloalveolar units are formed within the mammary gland. As modulators of the extracellular matrix, matrix metalloproteinases (MMPs) are the major enzymes taking part in the development of the gland. The activity of MMP-2 and MMP-9 has mostly been associated with tumor progression, while their participation in the physiological development of the mammary gland is not well characterized. In the present study the cell-specific localization of MMP-2 and MMP-9 in the developing dog mammary gland during pregnancy was investigated. In the early stages, both gelatinases were present, being located mostly in the epithelium of the ducts and less so in the surrounding stroma. After the formation of alveoli, MMP-2 was still present but MMP-9 was absent from the glandular epithelium and the stroma, being present only in the epithelium of the larger ducts. The results show that most likely, both gelatinases take part in ductal branching during early pregnancy, but only MMP-2 is associated with the differentiated stage of lactation.

Normal mammary gland growth and lactation capacity in pregnant relaxin-deficient mice

Pups born to mice with a targeted deletion of relaxin or its receptor (Rxfp1) die within 24 h postpartum. This has been attributed, in part, to abnormal mammary gland development in relaxin-mutant mice (Rln–/–). However, mammary development is normal in relaxin receptor-mutant (Rxfp1–/–) mice. The present study aimed to verify the mammary phenotypes in late pregnant and early lactating Rln–/– mice and to test the hypothesis that relaxin is involved in milk protein synthesis. Comparisons between late pregnant and early lactating wildtype (Rln+/+) and Rln–/– mice showed no differences in lobuloalveolar structure or ductal branching in the mammary gland. Mammary explants from Rln–/– mice also expressed β-casein and α-lactalbumin in response to lactogenic hormones at a similar level to Rln+/+ mice, implying normal milk protein synthesis. Pregnant Rln–/– mice infused with relaxin for 6 days gave birth to live pups without difficulty, and 96% of pups survived beyond 7 days. This is in contrast with the 100% pup mortality in saline-treated Rln–/– mice or 3-day relaxin-treated Rln–/– mice. Pups born to relaxin-treated Rln–/– dams weighed significantly less than Rln+/+ pups but had similar growth rates as their wildtype counterparts. In summary, relaxin is not critical for mammary gland development or β-casein and α-lactalbumin expression in late pregnant mice. In addition, Rln–/– dams did not need to be treated with relaxin postpartum for the pups to survive, suggesting that relaxin has no role in the maintenance of lactation in mice.

Estrogen Receptor-α Signaling in Growth of the Ventral Prostate: Comparison of Neonatal Growth and Postcastration Regrowth

A role for estrogen receptor (ER)-α in branching morphogenesis in the ventral prostate (VP) has previously been demonstrated; in the VP of ERα−/− mice, there are fewer side branches than in wild-type littermates. In the present study, we show that in the postnatal VP, fibroblast growth factor 10 (FGF10) is expressed in wild-type mice but not in ERα−/− mice, and because branching involves proliferation pathways also used in malignant growth, we investigated whether branching during regrowth of the VP after castration involves ERα and FGF10. ERα was not detectable in the prostates of sham-operated or castrated mice but was expressed in the prostatic epithelium between d 3 and 5 after testosterone replacement. Blocking either ERα or ERβ with ICI 182,780 had no detectable effects on epithelial cell proliferation during regrowth by testosterone. The ERα agonist, propylpyrazoletriol, did not induce regrowth by itself, but exposure to propylpyrazoletriol on d 3–5 of testosterone replacement resulted in cyclin D1-positive cells in the ductal epithelium, invasion of FGF10-positive immune cells in the regrowing prostate, and budding 14 d later. Testosterone replacement alone did not induce cyclin D1, FGF10, or bud formation. These results indicate that stimulation of ERα is essential for ductal branching during postnatal prostate growth. During regrowth after castration, there is a window in time when selective stimulation of ERα can also induce ductal branching. The FGF10 for this growth comes from the immune system, not from the prostatic mesenchyme.