Identification of germ cell tumor modifier genes from the 129.MOLF-Chr19 consomic mouse strain

Naturally occurring genetic variants confer susceptibility to disease in the human population, including in testicular germ cell tumor development. Disease susceptibility loci for testicular germ cell tumors have been identified by genetic mapping in humans and mice. However, the identity of many of the susceptibility genes remains unclear. My study utilized a chromosome substitution strain, the 129.MOLF-Chr 19 (or M19 strain), to identify candidate testicular germ cell tumor susceptibility genes. Males of this strain have a high incidence of germ cell tumors in the testes. By forward genetic approaches, five susceptibility loci were fine-mapped and the genetic interactions were dissected. In addition, I identified three protein-coding genes and one micro-RNA as testicular tumor susceptibility genes by genomic screening. Using reverse genetic approaches, I verified one of the candidates, Splicing factor 1, as a modifier of testicular tumor. Deficiency of SF1 significantly reduces the incidence of testicular tumors in mice. This study highlights the advantage of the 129.MOLF-Chr 19 consomic strain in disease gene identification and validation. It also sets the stage to elucidate the molecular mechanisms of tumorigenesis in the testis. ^

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Evidence for the involvement of proximal chromosome 3P loci in the progression of Von Hippel-Lindau related tumors

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of retinal and central nervous system hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic islet cell tumors (PICT). The VHL gene maps to chromosome 3p25 and has been shown to be mutated in 57% of sporadic cases of RCC, implicating VHL in the genesis of RCC. We report a multigeneration VHL kindred in which four affected female siblings developed PICT at early ages. Analysis of the three coding exons of the VHL gene in this family revealed a single, missense mutation in codon 238. Inheritance of the 238 mutation has been reported to correlate with a 62% risk of pheochromocytoma development. In this kindred, all affected individuals carried the mutation as well as one additional sibling who showed no evidence of disease. Clinical screening of this individual indicated small ($<$1 cm) pancreatic and kidney tumors. Results suggest that inheritance of the codon 238 mutation does not correlate with early onset pheochromocytoma. Rather, the only individual in the pedigree with pheochromocytoma was the proband's mother who developed bilateral pheochromocytoma at the age of 62. Thus, the VHL codon 238 mutation may predispose to late onset pheochromocytoma in this family; however, it does not explain the preponderance of PICT in the third generation since this mutation has not been reported to increase the risk of developing pancreatic lesions. This suggests that inheritance of the codon 238 mutation and subsequent somatic inactivation of the wild type allele of the VHL gene may not be sufficient to explain the initiation and subsequent progression to malignancy in VHL-associated neoplasms. Since the two tumor types that most frequently progress to malignancy are RCC and PICT, we asked whether loss of heterozygosity (LOH) could be detected proximal to the VHL gene on chromosome 3 in distinct regions of 3p previously implicated by LOH and cytogenetic studies to contain tumor suppressor loci for RCC. LOH was performed on high molecular weight DNA isolated from peripheral blood and frozen tumor tissue of family members using microsatellite markers spanning 3p. Results indicated LOH for all informative 3p loci in tumor tissue from affected individuals with PICT. LOH was detected along the entire length of the chromosome arm and included the proximal region of 3p13-14.2 implicated in the hereditary form of renal cell carcinoma.^ If 3p LOH were a critical event in pancreatic islet cell tumorigenesis, then it should be expected that LOH in sporadic islet cell tumors would also be observed. We expanded LOH studies to include sporadic cases of PICT. Consistent LOH was observed on 3p with a highest frequency LOH in the region 3p21.2. This is the first evidence for an association between chromosome 3 loci and pancreatic islet cell tumorigenesis. (Abstract shortened by UMI.) ^

AN INVESTIGATION INTO THE MOLECULAR MECHANISMS OF ANDROGEN ACTION IN THE SERTOLI CELL

Steroid hormones regulate target cell function via quantitative and qualitative changes in RNA and protein synthesis. In the testis, androgens are known to play an important role in the regulation of spermatogenesis. The Sertoli cell (SC), whose function is thought to be supportive to the developing germ cell, has been implicated as an androgen target cell. Although cytoplasmic androgen receptors and chromatin acceptor sites for androgen-receptor complexes have been found in SC, effects on RNA synthesis have not previously been demonstrated. In this study, SC RNA synthetic activity was characterized and the effect of testosterone on SC nuclear transcriptional activity in vitro assessed. SC exhibited two fold increases in RNA and ribonucleotide pool concentrations during sexual maturation. These changes appeared to correlate with a previously observed increase in protein concentration per cell over an age span of 15-60 days. Following incubation with ('3)H-uridine, SC from older animals incorporated more label into RNA than SC from younger animals. Since the relative concentration of cytidine nucleotides was higher in SC from older rats, the age-related increase in tritium incorporation may reflect an associated increase in incorporation of ('3)H-CMP into RNA. Alternatively, the enhanced labeling may be the result of either a change in the base composition of the RNA resulting in a higher proportion of CMP and UMP in the RNA, or compartmentalization of the nucleotide pools. The physiologic consequences of these maturational alterations of nucleotide pools remains to be elucidated. RNA polymerase activities were characterized in intact nuclei obtained from cultured rat SC. (alpha)-Amanitin resistant RNA polymerase I+III activity was identical when measured in low or high ionic strength (0.05 M or 0.25 M ammonium sulfate (AS)) in the presence of MnCl(,2) or MgCl(,2), with a divalent cation optimum of 1.6 mM. RNA polymerase II was most active in 0.25 M AS and 1.6 mM MnCl(,2). The apparent Km of RNA polymerase II for UTP was 0.016 mM in 0.05 M AS and 0.037 mM in 0.25 M AS. The apparent Km values for total polymerase activity was 0.008 mM and 0.036 mM at low and high ionic strenghts, respectively. These data indicate that Sertoli cell RNA polymerase activities have catalytic properties characteristic of eukaryotic polymerase activities in general. In the presence of 21 (mu)M testosterone, RNA polymerase II activity increased two fold at 15 minutes, then declined but was still elevated over control values six hours after androgen addition. Polymerase I+III activity was not greatly affected by testosterone. The stimulation of polymerase II measured at 15 minutes was dose-dependent, with a maximum at 0.53 nM and no further stimulation up to 10('-5) M (ED(,50) = 0.25 nM testosterone), and was androgen specific. The results of preliminary RNA isolation and characterization experiments suggested that the synthesis of several species of RNA was enhanced by testosterone administration. These findings have great potential importance since they represent the first demonstration of a direct effect of androgens on the transcriptional process in the Sertoli cell. Furthermore, the results of these studies constitute further evidence that the Sertoli cell is a target for androgen action in the testis. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Spinal cord injury causes sustained disruption of the blood-testis barrier in the rat.

There is a high incidence of infertility in males following traumatic spinal cord injury (SCI). Quality of semen is frequently poor in these patients, but the pathophysiological mechanism(s) causing this are not known. Blood-testis barrier (BTB) integrity following SCI has not previously been examined. The objective of this study was to characterize the effects of spinal contusion injury on the BTB in the rat. 63 adult, male Sprague Dawley rats received SCI (n = 28), laminectomy only (n = 7) or served as uninjured, age-matched controls (n = 28). Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), BTB permeability to the vascular contrast agent gadopentate dimeglumine (Gd) was assessed at either 72 hours-, or 10 months post-SCI. DCE-MRI data revealed that BTB permeability to Gd was greater than controls at both 72 h and 10 mo post-SCI. Histological evaluation of testis tissue showed increased BTB permeability to immunoglobulin G at both 72 hours- and 10 months post-SCI, compared to age-matched sham-operated and uninjured controls. Tight junctional integrity within the seminiferous epithelium was assessed; at 72 hours post-SCI, decreased expression of the tight junction protein occludin was observed. Presence of inflammation in the testes was also examined. High expression of the proinflammatory cytokine interleukin-1 beta was detected in testis tissue. CD68(+) immune cell infiltrate and mast cells were also detected within the seminiferous epithelium of both acute and chronic SCI groups but not in controls. In addition, extensive germ cell apoptosis was observed at 72 h post-SCI. Based on these results, we conclude that SCI is followed by compromised BTB integrity by as early as 72 hours post-injury in rats and is accompanied by a substantial immune response within the testis. Furthermore, our results indicate that the BTB remains compromised and testis immune cell infiltration persists for months after the initial injury.

Estimation of male-to-female ratios of mutation rate in primates and rodents

Haldane (1935) developed a method for estimating the male-to-female ratio of mutation rate ($\alpha$) by using sex-linked recessive genetic disease, but in six different studies using hemophilia A data the estimates of $\alpha$ varied from 1.2 to 29.3. Direct genomic sequencing is a better approach, but it is laborious and not readily applicable to non-human organisms. To study the sex ratios of mutation rate in various mammals, I used an indirect method proposed by Miyata et al. (1987). This method takes advantage of the fact that different chromosomes segregate differently between males and females, and uses the ratios of mutation rate in sequences on different chromosomes to estimate the male-to-female ratio of mutation rate. I sequenced the last intron of ZFX and ZFY genes in 6 species of primates and 2 species of rodents; I also sequenced the partial genomic sequence of the Ube1x and Ube1y genes of mice and rats. The purposes of my study in addition to estimation of $\alpha$'s in different mammalian species, are to test the hypothesis that most mutations are replication dependent and to examine the generation-time effect on $\alpha$. The $\alpha$ value estimated from the ZFX and ZFY introns of the six primate specise is ${\sim}$6. This estimate is the same as an earlier estimate using only 4 species of primates, but the 95% confidence interval has been reduced from (2, 84) to (2, 33). The estimate of $\alpha$ in the rodents obtained from Zfx and Zfy introns is ${\sim}$1.9, and that deriving from Ube1x and Ube1y introns is ${\sim}$2. Both estimates have a 95% confidence interval from 1 to 3. These two estimates are very close to each other, but are only one-third of that of the primates, suggesting a generation-time effect on $\alpha$. An $\alpha$ of 6 in primates and 2 in rodents are close to the estimates of the male-to-female ratio of the number of germ-cell divisions per generation in humans and mice, which are 6 and 2, respectively, assuming the generation time in humans is 20 years and that in mice is 5 months. These findings suggest that errors during germ-cell DNA replication are the primary source of mutation and that $\alpha$ decreases with decreasing length of generation time. ^

Esx1 dosage impacts reproductive fitness: Effects on viability and fertility

Numerous genes expressed in placenta or testis localize to the X-chromosome. Both tissues undergo specialized X-chromosome inactivation (imprinted paternal inactivation in placenta and MSCI in testicular germ cells). When the X-chromosome is duplicated or improperly inactivated, defects in placentation, growth and spermatogenesis are noted, suggesting tight control of X-chromosome gene dosage is important for reproduction. ^ Esx1 is a mouse homeobox gene on the X-chromosome with expression limited to extraembryonic tissues and testicular germ cells. Here, we examine the effects of increased and decreased Esx1 dosage on placental and testicular development, the role of genetic background on Esx1 function and characterize the human orthologue of Esx1. ^ Previously, by targeted deletion, Esx1 was shown to be an X-chromosome imprinted regulator of placental development and fetal growth. We show C57Bl6-congenic Esx1 mutants display a more severe phenotype with decreased viability and that the 129 genetic background contains dominant modifier genes that enhance Esx1 mutant survival. ^ Varying Esx1 dosage impacts testicular germ cell development. Esx1 hemizygous null mice are fertile, but we show their testes are two-thirds normal size. To examine the effect of increased Esx1 dosage, Esx1 BAC transgenic mice were generated. Increased Esx1 dosage results in dramatic deficits in testicular germ cell development, leading to sterility and testes one-fourth normal size. We show germ cell loss occurs through apoptosis, begins between postnatal day 6 and 10, and that no spermatocytes complete meiosis. Interestingly, increased Esx1 dosage in testes mimics germ cell loss seen in Klinefelter's (XXY) mice and humans and may represent a molecular mechanism for the infertility characteristic of this syndrome. ^ Esx1 dosage impacts reproductive fitness when maternally transmitted. Three transgenic founder females were unable to transmit the transgene to live offspring, but did produce transgenic pups at earlier stages. Additionally, one line of Esx1 BAC transgenic mice demonstrated decreased embryo size and fitness when the transgene is inherited compared to wild type littermates. ^ It is possible that Esx1 plays a role in human disorders of pregnancy, growth and spermatogenesis. Therefore, we cloned and characterized ESX1L (human Esx1), and show it is expressed in human testis and placenta. ^

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

The epidemiology of uterine cervical cancer among Anglo and Hispanic women in Texas

This study describes the incidence and mortality of uterine cervical cancer among Texas Anglo and Hispanic women, compares these data with respective data from the U.S. SEER Program, and determines factors which explain observed differences between the Texas ethnic groups and between Texas and SEER women. A total of 1,052 invasive and 1,852 in situ cervical cancer cases diagnosed during 1976-1985 among Texas residents were identified from the Texas Cancer Registry for study.^ The effect of ethnicity on the incidence of cervical cancer was found to be strongly modified by age. Texas Hispanic women 35 years and older were found to be at significantly greater risk (two- to four-fold) of invasive cervical cancer than Texas Anglos, and the risk was greatest among women 55-69 years. Compared with SEER females, both Texas ethnic groups exhibited excess risks of invasive cancer, but the magnitude varied with age. In contrast, Texas females were diagnosed less frequently with in situ cervical cancer than SEER females, and Hispanics had the largest differentials.^ As an indicator of differences in screening utilization between Texas and SEER ethnic groups, comparisons of in situ with invasive rates revealed both Texas ethnic groups in all age groups to have lower ratios than respective SEER females. Texas Hispanics had the lowest ratios. A larger percentage of squamous cell tumors were diagnosed among SEER females compared with Texas females, also supporting the finding of less screening. Texas invasive cases did not differ by ethnic group in the distribution of cell types. Hispanics 35-54 years had higher rates than Texas Anglos and SEER Hispanics for all four cell types.^ Declines in the incidence of invasive tumors over time were seen among Texas Anglos 35-54 years and Hispanics 55+ years. The mortality of cervical cancer also declined among Texas Anglo and Hispanic females 55+ years, but the rates still remained highest among these groups.^ In summary, these data indicate increased risks of invasive cervical cancer and less screening among subgroups of Texas females. Prevention efforts should be directed toward these Texas women at high risk of invasive cervical tumors. ^

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.

Expression and function of $\beta$-1,4-galactosyltransferase during wild-type and T/{\it t\/} -mutant spermatogenesis

In the mouse, gamete recognition is mediated in part by the binding of sperm surface $\beta$1,4 galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida ZP3. The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion. In this study, the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis was characterized and the role of GalTase was analyzed in transmission ratio distortion. It was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases and meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice.^ The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from +/t males. Results show that +- and t-spermatids have similar levels of both GalTase RNA and protein, indicating that transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.^ The presence of GalTase on the surface of an early spermatogenic cells before it is required on the mature sperm to perform its function during gamete binding suggests a separate function for GalTase in Sertoli-germ cell adhesion. Studies indicate that cell surface GalTase partly mediates the initial adhesion of pachytene spermatocytes, but not haploid spermatids, to Sertoli cells. ^

Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^