Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis, converting putrescine into N-methylputrescine. A variety of chemical, environmental, and developmental cues have been implicated in its regulation. Here we have examined the differential expression of beta-glucuronidase (GUS) transgenes under the control of the transcriptional regulatory sequences of four distinct members of the NtPMT gene family from tobacco (Nicotiana tabacum L.). BY-2 cell cultures expressing various NtPMT promoter-GUS constructs were examined for their response to treatment with various combinations of methyl jasmonate (MeJA), auxin (AUX), and ethylene (ETH). All four NtPMT gene promoters examined were inducible by MeJA, although the extent of the induction varied dramatically, with the NtPMT1a promoter being the most responsive. High AUX levels in the cell growth media repressed NtPMT::GUS transgene expression and inhibited their MeJA-induced transcription. Treatment of BY-2 cells with ETH alone did not result in a significant alteration in NtPMT::GUS expression. However, similar to AUX, ETH treatment led to the suppression of MeJA-induced transcription. Detailed deletion analysis of the NtPMT1a gene promoter showed that as little as 111 bp upstream of the transcriptional start site were sufficient to confer MeJA-responsiveness. Deletion of a conserved G-box element (GCACGTTG) at -103 to -96 bp completely abolished MeJA-responsiveness. Further mutagenesis studies revealed that in addition to a functional G-box, MeJA-responsiveness of the NtPMT1a promoter also required a TA-rich region and a GCC-motif (TGCGCCC) located at -80 to -69 bp and -62 to -56 bp relative to the start site, respectively. A synthetic G-box tetramer (4 X syn G-box) fused to a -83 bp fragment from the NtPMT1a promoter (containing the TA-rich region, GCC-box, and TATA-box) displayed a 30-fold induction by MeJA treatment, whereas when the 4 X syn G-box was fused to a minimal (-46 bp) promoter fragment derived from the CaMV 35S gene, no induction by MeJA treatment was detected. Our results indicate that multiple intersecting signal transduction pathways and different transcriptional regulatory factors are involved in mediating JA-responsiveness of NtPMT expression in tobacco.

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

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.

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.

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.

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.

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...

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 & 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 TSSK 1 & 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.