RNA-Seq analysis on ets1 mutant embryos of Xenopus tropicalis identifies microseminoprotein beta gene 3 as an essential regulator of neural crest migration.

The proto-oncogene ets1 is highly expressed in the pre-migratory and migratory neural crest (NC), and has been implicated in the delamination and migration of the NC cells. To identify the downstream target genes of Ets1 in this process, we did RNA sequencing (RNA-Seq) on wild-type and ets1 mutant X. tropicalis embryos. A list of genes with significantly differential expression was obtained by analyzing the RNA-Seq data. We validated the RNA-Seq data by quantitative PCR, and examined the expression pattern of the genes identified from this assay with whole mount in situ hybridization. A majority of the identified genes showed expression in migrating NC. Among them, the expression of microseminoprotein beta gene 3 (msmb3) was positively regulated by Ets1 in both X. laevis and X. tropicalis. Knockdown of msmb3 with antisense morpholino oligonucleotides or disruption of msmb3 by CRISPR/Cas9 both impaired the migratory streams of NC. Our study identified msmb3 as an Ets1 target gene and uncovered its function in maintaining neural crest migration pattern.

Generation and analysis of serine protease inhibitor kazal type 3-cre driver mice.

Serine protease inhibitor Kazal type 1 (SPINK1; mouse homologue Spink3) was initially discovered as a trypsin-specific inhibitor in the pancreas. However, previous studies have suggested that SPINK1/Spink3 is expressed in a wide range of normal tissues and tumors, although precise characterization of its gene expression has not been described in adulthood. To further analyze Spink3 expression, we generated two mouse lines in which either lacZ or Cre recombinase genes were inserted into the Spink3 locus by Cre-loxP technology. In Spink3(lacZ) mice, ß-galactosidase activity was found in acinar cells of the pancreas and kidney, as well as epithelial cells of the bronchus in the lung, but not in the gastrointestinal tract or liver. Spink3(cre) knock-in mice were crossed with Rosa26 reporter (R26R) mice to monitor Spink3 promoter activity. In Spink3(cre);R26R mice, ß-galactosidase activity was found in acinar cells of the pancreas, kidney, lung, and a small proportion of cells in the gastrointestinal tract and liver. These data suggest that Spink3 is widely expressed in endoderm-derived tissues, and that Spink3(cre) knock-in mice are a useful tool for establishment of a conditional knockout mice to analyze Spink3 function not only in normal tissues, but also in tumors that express SPINK1/Spink3.

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity.

Serine protease inhibitor Kazal-type (SPINK3)/P12/PSTI-II is a small secretory protein from mouse seminal vesicle which contains a KAZAL domain and shows calcium (Ca(2+))-transport inhibitory (caltrin) activity. This molecule was obtained as a recombinant protein and its effect on capacitated sperm cells was examined. SPINK3 inhibited trypsin activity in vitro while the fusion protein GST-SPINK3 had no effect on this enzyme activity. The inactive GST-SPINK3 significantly reduced the percentage of spermatozoa positively stained for nitric oxide (NO) with the specific probe DAF-FM DA and NO concentration measured by Griess method in capacitated mouse sperm; the same effect was observed when sperm were capacitated under low Ca(2+) concentration, using either intracellular (BAPTA-AM) or extracellular Ca(2+) (EDTA) chelators. The percentage of sperm showing spontaneous and progesterone-induced acrosomal reaction was significantly lower in the presence of GST-SPINK3 compared to untreated capacitated spermatozoa. Interestingly, this decrease was overcome by the exogenous addition of the NO donors, sodium nitroprusside (SNP), and S-nitrosoglutathione (GSNO). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, however, only GSNO was able to reverse this effect. Sperm progressive motility was not significantly diminished by GST-SPINK3 or BAPTA-AM but enhanced by the addition of SNP. This is the first report that demonstrates that SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration and independently of SPINK3 trypsin inhibitory activity.

Roles of serine protease inhibitor Kazal type 1 (SPINK1) in pancreatic diseases.

Serine protease inhibitor Kazal type 1 (SPINK1) was originally identified as a trypsin inhibitor by Kazal et al. in 1948. SPINK1 is strongly elevated in pancreatitis and the elevation correlates with the severity of disease. In 2000, mutations in the SPINK1 gene were shown to be associated with chronic pancreatitis. Since then, there have been many reports on association between mutations in the SPINK1 genes and patients with pancreatitis. In 1982, SPINK1 was shown to be identical to tumor associated trypsin inhibitor (TATI). In addition, sequence similarities were detected between human epidermal growth factor (EGF) and human SPINK1 in 1983. Actually, SPINK1 was shown to stimulate growth of several cell lines including cancer cells in 1985. Recent clinical studies showed that high levels of SPINK1 protein in serum or urine were associated with adverse outcome in various cancer types. However, there was little evidence that showed in vivo function of SPINK1. Surprisingly, mice deficient in Spink3 (a mouse homologue gene of human SPINK1) showed excessive autophagy, but not pancreatitis in the exocrine pancreas, leading to autophagic cell death. We also demonstrated that SPINK1 acts as a growth factor through EGFR signaling. These data indicate that the role of the SPINK1 is not just as a trypsin inhibitor, but also as a growth factor as well as a negative regulator of autophagy. In this review, we summarize the roles of SPINK1/Spink3 in pancreatic diseases based on the data obtained from analyses using mouse models.

Signaling aggression.

From psychological and sociological standpoints, aggression is regarded as intentional behavior aimed at inflicting pain and manifested by hostility and attacking behaviors. In contrast, biologists define aggression as behavior associated with attack or escalation toward attack, omitting any stipulation about intentions and goals. Certain animal signals are strongly associated with escalation toward attack and have the same function as physical attack in intimidating opponents and winning contests, and ethologists therefore consider them an integral part of aggressive behavior. Aggressive signals have been molded by evolution to make them ever more effective in mediating interactions between the contestants. Early theoretical analyses of aggressive signaling suggested that signals could never be honest about fighting ability or aggressive intentions because weak individuals would exaggerate such signals whenever they were effective in influencing the behavior of opponents. More recent game theory models, however, demonstrate that given the right costs and constraints, aggressive signals are both reliable about strength and intentions and effective in influencing contest outcomes. Here, we review the role of signaling in lieu of physical violence, considering threat displays from an ethological perspective as an adaptive outcome of evolutionary selection pressures. Fighting prowess is conveyed by performance signals whose production is constrained by physical ability and thus limited to just some individuals, whereas aggressive intent is encoded in strategic signals that all signalers are able to produce. We illustrate recent advances in the study of aggressive signaling with case studies of charismatic taxa that employ a range of sensory modalities, viz. visual and chemical signaling in cephalopod behavior, and indicators of aggressive intent in the territorial calls of songbirds.

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy.

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.

Fine mapping and functional analysis of a common variant in MSMB on chromosome 10q11.2 associated with prostate cancer susceptibility.

Two recent genome-wide association studies have independently identified a prostate cancer susceptibility locus on chromosome 10q11.2. The most significant single-nucleotide polymorphism (SNP) marker reported, rs10993994, is 57 bp centromeric of the first exon of the MSMB gene, which encodes beta-microseminoprotein (prostatic secretory protein 94). In this study, a fine-mapping analysis using HapMap SNPs was conducted across a approximately 65-kb region (chr10: 51168330-51234020) flanking rs10993994 with 13 tag SNPs in 6,118 prostate cancer cases and 6,105 controls of European origin from the Cancer Genetic Markers of Susceptibility (CGEMS) project. rs10993994 remained the most strongly associated marker with prostate cancer risk [P = 8.8 x 10(-18); heterozygous odds ratio (OR) = 1.20, 95% confidence interval (CI): 1.11-1.30; homozygous OR = 1.64, 95% CI: 1.47-1.86 for the adjusted genotype test with 2 df]. In follow-up functional analyses, the T variant of rs10993994 significantly affected expression of in vitro luciferase reporter constructs. In electrophoretic mobility shift assays, the C allele of rs10993994 preferentially binds to the CREB transcription factor. Analysis of tumor cell lines with a CC or CT genotype revealed a high level of MSMB gene expression compared with cell lines with a TT genotype. These findings were specific to the alleles of rs10993994 and were not observed for other SNPs determined by sequence analysis of the proximal promoter. Together, our mapping study and functional analyses implicate regulation of expression of MSMB as a plausible mechanism accounting for the association identified at this locus. Further investigation is warranted to determine whether rs10993994 alone or in combination with additional variants contributes to prostate cancer susceptibility.

[Effect of ventral prostate secretory proteins on oviductal fluid glycoproteins in golden hamsters].

OBJECTIVE: To investigate the effect of the secretory proteins of the ventral prostate on the glycoproteins in the oviductal fluid of golden hamsters. METHODS: Male golden hamsters were divided into four groups: sham operation (SH), total removal of accessory sex glands (TX), and retainment of the ventral prostate only (VP). Oviductal fluid was collected from female hamsters at 0.5, 2, 4 and 6 h after mating with the males of different operated groups with or without ventral prostate. Glycoproteins were probed with a panel of lectins and their changes in the oviductal fluid were analyzed by Western blot. RESULTS: The 47 000, 52 000, 81 000 and 128 000 WGA-binding proteins were observed in the oviductal fluid of the 6 h TX group, the 32 000, 35 500, 47 000 and 52 000 WGA-binding glycoproteins noted in the 6 h VP group, the 47 000, 68 000, 95 000 and 128 000 pisum sativum agglutinin (PSA)-binding glycoproteins shown in the 6 h TX and VP groups, two extra 32 000 and 37 500 bands detected in the 6 h VP group, the 47 000 and 52 000 dolichos biflorus agglutinin (DBA)-binding glycoproteins present in the 6 h VP but absent in the 6 h TX group. CONCLUSION: Ventral prostate secretory proteins affect acetylglucosamine, N-acetylgalactosamine/galactose and mannose in the oviductal fluid collected 6 hours after mating. And these glycoproteins may play an important role in the development of embryos.

beta-Microseminoprotein-related molecules may participate in formation of the mesoderm in the chick embryo.

It has previously been shown that human beta-microseminoprotein enhances development of mesodermal structures in the chick embryo. The present study was carried out to elucidate the mechanism of action of human beta-microseminoprotein in the chick embryo. beta-Microseminoprotein brought about significant modulation of expression of Brachyury in gastrulating embryos. In approximately 50% of the treated embryos, Brachyury expression was enhanced around the Hensen's node. These cells not only expressed higher levels of Brachyury, but also appeared to switch off Brachyury expression prematurely, postinvagination. The spatial modulation of Brachyury is not clearly reflected in the northern blots, indicating that beta-microseminoprotein treatment results in redistribution of available transcripts or that the upregulation is compensated for by early switching off of Brachyury postinvagination. Because higher levels of Brachyury during gastrulation are believed to result in early exit of cells from the primitive streak, beta-microseminoprotein treatment appeared to have stimulated morphogenetic movements by upregulating Brachyury around the Hensen's node. This deduction was confirmed by scanning electron microscopic analysis that showed that altered morphogenetic movements accompany modulation of Brachyury. The specific responses elicited by beta-microseminoprotein indicate presence of a structurally related molecule in the chick. By western blotting, similar molecules were indeed detected in the chicken seminal plasma and in chick embryos. These data strongly suggest that beta-microseminoprotein-related molecule(s) participates in mesoderm formation in the chick embryo.

Ab initio structure of human seminal plasma prostatic inhibin gives significant insight into its biological functions.

Human seminal plasma prostatic inhibin (HSPI) is a protein isolated from the human prostate gland. Despite its profound biomedical and biotechnological importance, the 3D structure of this protein of 94 amino acids remains undeciphered. The difficulties in extracting it in pure form and crystallizing it have restrained the determination of its structure experimentally. The homology-based computational methods are also not applicable, as HSPI lacks sufficient sequence homology with known structures in the protein data banks. We have predicted the structure of HSPI by a knowledge-based method using nonparametric multivariate statistical techniques. Stereochemical and other standard validation tests confirm this to be a well-refined structure. The biophysical properties exhibited by this structure are in good agreement with the NMR experimental observations. Docking and other computational studies on this structure provide significant explanation and insight into its binding activities and related biological and immunogenic functions and offer new directions for its potential applications.

Advancing perspectives on prostate cancer: multihormonal influences in pathogenesis.

Nonandrogenic hormones are implicated in the growth and function of the prostate, which is itself an endocrine gland that synthesizes and secretes hormones and growth factors, including follicle-stimulating hormone (FSH) and prostatic inhibin peptide (PIP). Findings of increased FSH concentrations and receptor expression in diseased prostate tissue suggest a role for FSH in prostate cancer growth. Not only does PIP suppress circulating levels of FSH, but it responds to and modulates prostatic FSH, suggesting a close interlinkage of these compounds in controlling both healthy and diseased prostate cells. Other focuses of endocrinologic research include androgen receptors, vitamin D, growth factors (including insulin-like growth factors I and II), and retinoids. Issues such as optimal therapy timing, intermittent administration, and the adoption of a multihormonal approach to the management of prostate cancer remain to be resolved.