The mammalian epididymis is more than a highly convoluted tube divided into four regions: initial segment, caput, corpus and cauda. It is a highly segmented structure with each segment expressing its own and overlapping genes, proteins, and signal transduction pathways. Therefore, the epididymis may be viewed as a series of organs placed side by side. In this review we discuss the contributions of septa that divide the epididymis into segments and present hypotheses as to the mechanism by which septa form. The mechanisms of Wolffian duct segmentation are likened to the mechanisms of segmentation of the renal nephron and somites. The renal nephron may provide valuable clues as to how the Wolffian duct is patterned during development, whereas somitogenesis may provide clues as to the timing of the development of each segment. Emphasis is also placed upon how segments are differentially regulated, in support of the idea that the epididymis can be considered a series of multiple organs placed side by side. One region in particular, the initial segment, which consists of 2 or 4 segments in mice and rats, respectively, is unique with respect to its regulation and vascularity compared to other segments; loss of development of these segments leads to male infertility. Different ways of thinking about how the epididymis functions may provide new directions and ideas as to how sperm maturation takes place.

Without a fully developed initial segment, the most proximal region of the epididymis, male infertility results. Therefore, it is important to understand the development and regulation of this crucial region. In addition to distinctively high activity levels of the components of the ERK pathway, which are essential for initialsegment differentiation, the initial segment exhibits high protein and activity levels of phosphatase and tensin homolog (PTEN). To understand the role of PTEN in the regulation of the initial segment, we generated a mouse model with a conditional deletion of Pten from the epithelial cells of the proximal epididymis from postnatal day 17 (P17) onward. Shortly after Pten deletion, hypertrophy of the proximal epididymis became evident. Loss of Pten resulted in activation of the AKT (protein kinase B) pathway components from P28 onward, which in turn gradually suppressed RAF1 proto-oncogene serine/threonine kinase (RAF1)/ERK signaling through the interaction between AKT and RAF1. Consistent with progressive changes in RAF1/ERK signaling, loss of Pten progressively altered cell shape, size, organization, proliferation, and survival in the initial-segment epithelium and resulted in dedifferentiation and extensive epithelial folding. Most importantly, knockout males progressively lost fertility and became infertile from 6 to 12 mo. Spermatozoa from older knockout mice showed a lower percentage of motility and a higher percentage of flagellar angulation compared with controls, suggesting compromised sperm maturation. Therefore, under normal physiological conditions, PTEN suppresses AKT activity to maintain activation of the RAF1/ERK signaling pathway, which in turn maintains normal function of the initial segment and therefore, normal sperm maturation.S permatozoa enter the epididymis from the testes as immotile cells that are incapable of fertilization. During epididymal transit, a complex process called sperm maturation allows for conversion of spermatozoa into motile and fertilization-competent cells. The long, convoluted epididymal duct-which is ∼6 m long in humans and 1 m in mice (1)-provides a luminal fluid microenvironment that is necessary for sperm maturation (2).It is apparent that the most proximal region of the epididymis, the initial segment, plays an important role in sperm maturation. C-ros oncogene 1 (Ros1) knockout male mice that lacked prepubertal differentiation of the epididymal initial segment were healthy but infertile (3). Spermatozoa from Ros1 knockouts showed flagellar angulation, a defect in sperm maturation (4). Therefore, it is important to examine the mechanisms by which the initial segment develops, functions normally, and contributes to normal sperm maturation.The initial segment exhibits high activity levels of the ERK pathway components. The ERK pathway [also known as the RAF (RAF proto-oncogene serine/threonine kinases)/MEK/ERK pathway] is an intracellular protein kinase cascade containing at least MAPK3 and/or MAPK1 (commonly known as ERK1 and ERK2), a MAPKK (com...

The Wolffian duct, the proximal end of the mesonephric duct, undergoes non-branching morphogenesis to achieve an optimal length and size for sperm maturation. It is important to examine the mechanisms by which the developing mouse Wolffian duct elongates and coils for without proper morphogenesis, male infertility will result. Here we show that highly proliferative epithelial cells divide in a random orientation relative to the elongation axis in the developing Wolffian duct. Convergent extension (CE)-like of cell rearrangements is required for elongating the duct while maintaining a relatively unchanged duct diameter. The Wolffian duct epithelium is planar polarized, which is characterized by oriented cell elongation, oriented cell rearrangements, and polarized activity of regulatory light chain of myosin II. Conditional deletion of protein tyrosine kinase 7 (PTK7), a regulator of planar cell polarity (PCP), from mesoderm results in loss of the PCP characteristics in the Wolffian duct epithelium. Although loss of Ptk7 does not alter cell proliferation or division orientation, it affects CE and leads to the duct with significantly shortened length, increased diameter, and reduced coiling, which eventually results in loss of sperm motility, a key component of sperm maturation. In vitro experiments utilizing inhibitors of myosin II results in reduced elongation and coiling, similar to the phenotype of Ptk7 knockout. This data suggest that PTK7 signaling through myosin II regulates PCP, which in turn ensures CE-like of cell rearrangements to drive elongation and coiling of the Wolffian duct. Therefore, PTK7 is essential for Wolffian duct morphogenesis and male fertility.

Without a fully developed and functioning initial segment, the most proximal region of the epididymis, male infertility results. Therefore, it is important to understand the development of the initial segment. During postnatal development of the epididymis, many cellular processes of the initial segment are regulated by lumicrine factors, which are produced by the testis and enter the epididymis with testicular luminal fluid. In this report, we showed that prior to Postnatal Day 15 (P15), the initial segment was lumicrine factor independent in the mouse. However, from P19 onward, lumicrine factors were essential for the proliferation and survival of initial segment epithelial cells. Therefore, P15 to P19 was a critical window that established the dependency of lumicrine factors in the initial segment epithelium. The initial segment-specific kinase activity profile, a marker of initial segment differentiation, was also established during this window. The SFK (SRC proto-oncogene family kinases), ERK pathway (known as the RAF/MEK/ERK pathway) components, and AMPK (AMP-activated protein kinases) pathway components had increased activities from P15 to P19, suggesting that lumicrine factors regulated SFK/ERK/AMPK signaling to initiate differentiation of the initial segment from P15 to P19. Compared with litter mate controls, juvenile Src null mice displayed lower levels of MAPK3/1 (mitogen-activated protein kinase 3/1) activity and a reduced level of differentiation in the initial segment epithelium, a similar phenotype resulting from inhibition of SRC activity within the window of P15 to P19. Therefore, lumicrine factor-dependent SRC activity signaling through MAPK3/1 is important for the initiation of initial segment differentiation during a critical window of development.