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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.

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).

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.

The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial dedifferentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate–dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility. Summary Sentence Loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, suggesting PTEN regulates epididymal water, ion, and organic solute transporter activities.

The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

Two nuclear genes (NtODC-1 and NtODC-2) encoding ornithine decarboxylase (ODC, EC 4.1.1.17) were characterized from tobacco (Nicotiana tabacum L., and their differential induction by methyl-jasmonate (MeJA) was analyzed. ODC transcript levels increased in BY-2 cells following treatment with MeJA, with the highest levels of transcript observed approximately 5 h post-treatment. Analysis of transgenic BY-2 cell lines expressing a β-glucuronidase (GUS) reporter gene under the control of either the NtODC-1 or NtODC-2 gene promoter revealed that expression of the NtODC-1::GUS transgene was highly induced (8-fold) by treatment with MeJA, whereas the NtODC-2::GUS transgene showed little MeJA induced GUS expression. Removal of auxin (AUX, 2,4-D) from the cell growth media led to an increase in NtODC-1 and NtODC-2 transcript levels, whereas high levels of 2,4-D repressed overall ODC gene expression and suppressed the MeJA-induced expression of NtODC-1. Deletion analysis identified an upstream region (-924 to -1424 bp relative to the start site of transcription) of the NtODC-1 promoter that confers MeJA-responsiveness. No similar region was identified in the NtODC-2 promoter. Sequence analysis revealed that the NtODC-1 promoter fragment contains both G-box and TGACG-motif elements similar to those found in other MeJA-responsive genes. Our findings are discussed in terms of potential factors mediating different phytohormone-activated signal transduction pathways in the formation of nicotine and related compounds in tobacco.[PUBLICATION ABSTRACT]

Two nuclear genes (NtODC-1 and NtODC-2) encoding ornithine decarboxylase (ODC, EC 4.1.1.17) were characterized from tobacco (Nicotiana tabacum L., and their differential induction by methyl-jasmonate (MeJA) was analyzed. ODC transcript levels increased in BY-2 cells following treatment with MeJA, with the highest levels of transcript observed approximately 5 h post-treatment. Analysis of transgenic BY-2 cell lines expressing a β-glucuronidase (GUS) reporter gene under the control of either the NtODC-1 or NtODC-2 gene promoter revealed that expression of the NtODC-1::GUS transgene was highly induced (8-fold) by treatment with MeJA, whereas the NtODC-2::GUS transgene showed little MeJA induced GUS expression. Removal of auxin (AUX, 2,4-D) from the cell growth media led to an increase in NtODC-1 and NtODC-2 transcript levels, whereas high levels of 2,4-D repressed overall ODC gene expression and suppressed the MeJA-induced expression of NtODC-1. Deletion analysis identified an upstream region (-924 to -1424 bp relative to the start site of transcription) of the NtODC-1 promoter that confers MeJA-responsiveness. No similar region was identified in the NtODC-2 promoter. Sequence analysis revealed that the NtODC-1 promoter fragment contains both G-box and TGACG-motif elements similar to those found in other MeJA-responsive genes. Our findings are discussed in terms of potential factors mediating different phytohormone-activated signal transduction pathways in the formation of nicotine and related compounds in tobacco.[PUBLICATION ABSTRACT]