Notch signaling in the epididymal epithelium regulates sperm motility and is transferred at a distance within epididymosomes.

Spermatozoa undergo sequential maturation changes during their transit along the epididymis. These changes are modulated by the epididymal epithelium and require a finely tuned gene expression. The Notch cell signaling pathway is a major regulator of cell fate decisions in several tissues, including the testis. Here, we evaluated the transcription and expression patterns of Notch components (Notch1-3, Dll1, Dll4, and Jagged1) and effectors (Hes1-2 and Hes5) in the adult mouse epididymis, and evaluated the role of Notch signaling in the epididymis through its in vivo blockade following administration of an inhibitor (DAPT). Notch components and effectors were dynamically transcribed and expressed in the epididymis and vas deferens, each segment exhibiting a specific combination of epithelial receptor/ligand/effector expression patterns. Nuclear detection of Notch effectors indicates that Notch signaling was active. Notch components (but not effectors) were identified in the cytoplasmic droplet of spermatozoa, in a dynamic and specific pattern along the epididymis. In addition, Notch components were identified within large and small vesicles in the epididymal lumen. A purified population of these membranous vesicles from different epididymal segments was obtained, and through dot blot analysis, it was confirmed that Notch components were carried within these vesicles in a dynamic pattern along the epididymal lumen. We hypothesize that these vesicles (epididymosomes) allow Notch signaling at distance from epididymal epithelial cells to spermatozoa. DAPT-induced in vivo Notch signaling blockade, although showing a low efficiency, disrupted the expression patterns of Notch components and effectors in the epididymal epithelium and in spermatozoa, and significantly decreased sperm motility, although not affecting male fertility. These results prompt for a regulatory role of Notch signaling in epididymal epithelial function and sperm maturation.

Dynamics of Notch signalling in the mouse oviduct and uterus during the oestrous cycle.

The oviduct and uterus undergo extensive cellular remodelling during the oestrous cycle, requiring finely tuned intercellular communication. Notch is an evolutionarily conserved cell signalling pathway implicated in cell fate decisions in several tissues. In the present study we evaluated the quantitative real-time polymerase chain reaction (real-time qPCR) and expression (immunohistochemistry) patterns of Notch components (Notch1-4, Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged1-2) and effector (hairy/enhancer of split (Hes) 1-2, Hes5 and Notch-Regulated Ankyrin Repeat-Containing Protein (Nrarp)) genes in the mouse oviduct and uterus throughout the oestrous cycle. Notch genes are differentially transcribed and expressed in the mouse oviduct and uterus throughout the oestrous cycle. The correlated transcription levels of Notch components and effector genes, and the nuclear detection of Notch effector proteins, indicate that Notch signalling is active. The correlation between transcription levels of Notch genes and progesterone concentrations, and the association between expression of Notch proteins and progesterone receptor (PR) activation, indicate direct progesterone regulation of Notch signalling. The expression patterns of Notch proteins are spatially and temporally specific, resulting in unique expression combinations of Notch receptor, ligand and effector genes in the oviduct luminal epithelium, uterus luminal and glandular epithelia and uterine stroma throughout the oestrous cycle. Together, the results of the present study imply a regulatory role for Notch signalling in oviduct and uterine cellular remodelling occurring throughout the oestrous cycle.

Differential expression of Notch component and effector genes during ovarian follicle and corpus luteum development during the oestrous cycle.

Ovarian dynamics throughout the female oestrous cycle (EC) are characterised by cyclical follicle and corpus luteum (CL) development. These events are tightly regulated, involving extensive cell-to-cell communication. Notch is an evolutionarily well conserved cell-signalling pathway implicated in cell-fate decisions in several tissues. Here, we evaluated the extra-vascular expression patterns of Notch component and effector genes during follicle and CL development throughout the EC. Five mature CD1 female mice were killed at each EC stage. Blood samples were collected for progesterone measurement, ovaries were processed for immunohistochemistry and expression patterns of Notch components (Notch1, 2 and 3, Jagged1 and Delta-like1 and 4) and effectors (Hes1, Hes2 and Hes5) were characterised. Nuclear detection of Notch effectors indicates that Notch signalling is active in the ovary. Notch components and effectors are differentially expressed during follicle and CL development throughout the EC. The spatial and temporal specific expression patterns are associated with follicle growth, selection and ovulation or atresia and CL development and regression.