Bingfang Xu

p-MAPK1/3 and DUSP6 Regulate Epididymal Cell Proliferation and Survival in a Region-Specific Manner in Mice

A fully developed, functional epididymis is important for male fertility. In particular, it is apparent that without the most proximal region, the initial segment (IS), infertility results. Therefore, it is important to understand the development and regulation of this crucial epididymal region. We have previously shown that many functions of the IS are regulated by luminal fluid factors/lumicrine factors from the testis. This study provides evidence that lumicrine factors activated the ERK pathway only in epithelial cells of the IS from Postnatal Day (P) 14 to P19 and sustained this activation into adulthood. The activated ERK pathway promoted cell proliferation and differentiation in the developing IS, although in the adult, its role was switched to maintain cell survival. To understand further the regulation of cell proliferation in the IS, we examined the role of DUSP6, an MAPK1/3 (ERK1/2) preferred phosphatase that is also regulated by lumicrine factors in the IS. Utilizing Dusp6−/− mice, our studies, surprisingly, revealed that Dusp6 was a major regulator of cell proliferation in the caput and corpus regions, whereas components of the ERK pathway, together with PTEN and SRC, were the major regulators of cell proliferation in the IS. We hypothesize that region-specific regulation of cell proliferation is caused by differences in the balance of activities between pro- and antiproliferation signaling pathway components for each epididymal region. An understanding of the mechanisms of cell proliferation may provide clues as to why the epididymis rarely succumbs to cancer.

How Do You Get Six Meters of Epididymis Inside a Human Scrotum

It is very clear that the epididymis plays a crucial role in the maturation of spermatozoa, and without a fully developed and functional epididymis, male infertility will result. We are especially interested in understanding the mechanisms that regulate the development of this important organ because disruptions to epididymal function will also arise as a consequence of abnormal development. Very little is known either of the process of epididymal development or the nature and causes of congenital defects that lead to male infertility. A major event during Wolffian/epididymal duct embryonic development is elongation and coiling and this short review outlines potential mechanisms by which these events occur. It is hypothesized that elongation is the result of cell proliferation coupled with directed cell rearrangements, the later regulated by the planar cell polarity signaling pathway. Coiling proceeds in a proximal to distal manner, with three-dimensional coiling beginning approximately embryonic day 16.5 to 18.5 in the mouse. The exact mechanisms of coiling are not known but we hypothesize that it involves an interaction between the Wolffian duct epithelium and the surrounding mesenchyme cells, such that the extracellular matrix is remodeled to allow coiling and growth of the duct. Cell proliferation in the Wolffian duct appears to be dependent on the presence of androgens and mesenchymal factors during embryonic development, but lumicrine factors play an additional role during postnatal development.

TSKS concentrates in spermatid centrioles during flagellogenesis.

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

Testicular Lumicrine Factors Regulate ERK, STAT and NFKB Pathways in the Initial Segment of the Rat Epididymis to Prevent Apoptosis

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis.

Gene expression in the efferent ducts, epididymis, and vas deferens during embryonic development of the mouse

The tissues of the male reproductive tract are characterized by distinct morphologies, from highly coiled to un‐coiled. Global gene expression profiles of efferent ducts, epididymis, and vas deferens were generated from embryonic day 14.5 to postnatal day 1 as tissue‐specific morphologies emerge. Expression of homeobox genes, potential mediators of tissue‐specific morphological development, was assessed. Twenty homeobox genes were identified as either tissue‐enriched, developmentally regulated, or both. Additionally, ontology analysis demonstrated cell adhesion to be highly regulated along the length of the reproductive tract. Regulators of cell adhesion with variable expression between the three tissues were identified including Alcam, various cadherins, and multiple integrins. Immunofluorescence localization of the cell adhesion regulators POSTN and CDH2 demonstrated cell adhesion in the epithelium and mesenchyme of the epididymis may change throughout development. These results suggest cell adhesion may be modulated in a tissue‐specific manner, playing an important role in establishing each tissues final morphology. Developmental Dynamics 239:2479–2491, 2010.

PTEN signaling through RAF1 proto-oncogene serine/threonine kinase (RAF1)/ERK in the epididymis is essential for male fertility

Significance Without a fully developed and functioning initial segment, the most proximal region of the epididymis, spermatozoa do not undergo maturation, resulting in male infertility. The tumor suppressor phosphatase and tensin homolog (PTEN) was deleted from the initial segment, which resulted in changes in the activity of downstream signaling components that led to epithelial dedifferentiation and male infertility. Spermatozoa were normal upon leaving the testis, but as they progressed through the duct of the dedifferentiated initial segment, they developed angled flagella and a decrease in motility. These changes prevented spermatozoa from reaching and fertilizing an egg in the female reproductive tract. PTEN plays an essential role in maintaining normal initial-segment differentiation and function and, therefore, male fertility. 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 initial-segment 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.

Is the Epididymis a Series of Organs Placed Side By Side

ABSTRACT 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.

Understanding normal and abnormal development of the Wolffian/epididymal duct by using transgenic mice.

The development of the Wolffian/epididymal duct is crucial for proper function and, therefore, male fertility. The development of the epididymis is complex; the initial stages form as a transient embryonic kidney; then the mesonephros is formed, which in turn undergoes extensive morphogenesis under the influence of androgens and growth factors. Thus, understanding of its full development requires a wide and multidisciplinary view. This review focuses on mouse models that display abnormalities of the Wolffian duct and mesonephric development, the importance of these mouse models toward understanding male reproductive tract development, and how these models contribute to our understanding of clinical abnormalities in humans such as congenital anomalies of the kidney and urinary tract (CAKUT).

The Role of Fibroblast Growth Factor Receptor Substrate 2 (FRS2) in the Regulation of Two Activity Levels of the Components of the Extracellular Signal-Regulated Kinase (ERK) Pathway in the Mouse Epididymis

ABSTRACT The components of the extracellular signal-regulated kinase (ERK) pathway are involved in the regulation of epididymal cellular processes. Interestingly, our previous studies showed that there are two different activity levels of the ERK pathway components in the epididymal epithelium: a basal level in most regions and a higher level in the differentiated initial segment (IS). In this study we analyzed the role of fibroblast growth factor receptor substrate 2 (FRS2) in the regulation of these two levels. Two mouse models were generated. In the first model, Frs2 was deleted from epithelial cells of most epididymal regions except for the IS from the embryonic period onward. Loss of Frs2 dampened the basal activity level of the ERK pathway components, which resulted in an increase in apoptosis along the epididymal duct. This was observed during the period when FRS2 expression level was highest in wild-type epididymides. In the second model, Frs2 was deleted from the proximal epididymal epithelium from Postnatal Day 17 onward. Most of the epididymides in this model exhibited normal morphology. Loss of Frs2 in these epididymides did not affect the high activity level of the ERK pathway components in the IS. However, a subgroup of epididymides in the second model showed increased apoptosis which resulted in an abnormally shaped proximal region or development of granulomas. Therefore, data from these two models showed that FRS2 played different roles in the regulation of two activity levels of the ERK pathway components in the epididymis.

Protein tyrosine kinase 7 is essential for tubular morphogenesis of the Wolffian duct.

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.