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There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 −/− sperm, the short one (Mw 36 kDa) is mis-localized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2 −/− sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/2/3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 proteins or SUN4 expression in Prm2 −/− testes. Furthermore, the Prm2 −/− sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone–protamine 1/2 ratio, modified levels of cytoskeletal proteins and we detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions. In conclusion, our findings present potential interaction between Septin 12 and Protamine 2 or Lamin B2/3 and describe a new connection between their expression and localization, contributing likely to low sperm motility and morphological abnormalities. [ABSTRACT FROM AUTHOR]

Simple Summary: In our study, we investigated the association between pepsin levels in bronchoalveolar lavage and factors such as hemorrhagic/non-hemorrhagic character of BAL, type of lung disease, tumor location, patient demographics, and smoking history. Our objective was to investigate the possible influence of extraesophageal reflux on the development of lung adenocarcinoma. Contrary to our hypothesis, we did not find significant differences in pepsin levels among patients with lung adenocarcinoma, pulmonary metastases, and lung sarcoidosis. Pepsin levels were even lower in lung adenocarcinoma patients after excluding hemorrhagic samples. By rigorously excluding smokers and carefully classifying patients, we minimized biases common in similar studies. However, our study was limited by small sample sizes, especially in the sarcoidosis group, and the absence of standardized thresholds for BAL pepsin as a reflux biomarker. The significance of extraesophageal reflux as a risk factor in lung adenocarcinoma has been understudied. In this study, we investigated whether extraesophageal reflux leads to higher pepsin concentrations in bronchoalveolar lavage (BAL) in patients with lung adenocarcinoma compared to controls. Subjects were recruited from non-smoker patients (lifelong non-smokers and ex-smokers with more than 5 years of non-smoking history) who had undergone bronchoscopy due to pulmonary abnormalities on a CT scan and met the inclusion criteria. Based on histological verification of the lung process, the patients were divided into three groups: (1) lung adenocarcinoma, (2) pulmonary metastases, and (3) lung sarcoidosis. Lung adenocarcinoma cases were further categorized as central or peripheral. BAL samples collected during bronchoscopy were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA) to measure pepsin levels. No statistically significant difference in pepsin concentration was observed between the lung adenocarcinoma group and control groups (p = 0.135). After excluding hemorrhagic BAL samples, the pepsin concentration was significantly the lowest in patients with lung adenocarcinoma (p = 0.023) compared to the control groups. The results of the study do not support the hypothesis of a higher occurrence of extraesophageal reflux (evaluated as the amount of pepsin in BAL) in non-smoker patients with lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 -/- sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2 -/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in Prm2 -/- testes. Furthermore, the Prm2 -/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone- protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.

Objectives: Ondansetron is an antagonist of 5-HT3 receptors mostly used as an antiemetic yet known to modulate metabolism and appetite. We tested the metabolic effects of ondansetron in newly derived congenic rat strain, carrying limited chromosome 8 regions of (PD) Brown Norway (BN) and polydactylous (PD) strain origins (including variant serotonin receptor Htr3b gene) within the genomic background of highly inbred model of metabolic syndrome, the spontaneously hypertensive rat (SHR)., Methods: Adult, standard diet-fed male rats of SHR and the congenic SHR.(PD/BN)8 strains received ondansetron (2mg/kg body weight/day) or vehicle (n=6/strain/treatment) via oral gavage for 14 days while we followed their metabolic and morphometric profiles including glucose tolerance and triacylgycerol and cholesterol concentrations in 20 lipoprotein fractions., Results: We fine-mapped the chromosome 8 differential segment in the new SHR.(PD/BN)8 congenic strain: it comprises BN-derived region together with an adjacent 422kb stretch of PD origin. The SHR.(PD/BN)8 rats were heavier than SHR, the fasting glucose was significantly higher in ondansetron-treated congenic than in SHR (post-hoc Tukey's HSD p=0.02). Compared to SHR, ondansetron induced significantly more robust increases of cholesterol and triacylglycerol concentrations in total, chylomicron, VLDL and HDL particles in the SHR.(PD/BN)8 congenic strain., Conclusion: We established new congenic model with distinct pharmacogenetic profile related to metabolic effects of ondansetron, facilitating thus the search for responsible genetic variants within the limited genomic region demarcated by the differential segment.

Dexamethasone (DEX) is known to induce diabetes and dyslipidemia. We have compared fasting triacylglycerol and cholesterol concentrations across 20 lipoprotein fractions and glucose tolerance in control (standard diet) and DEX-treated 7-month-old males of two rat strains, Brown Norway (BN) and congenic BN.SHR-(Il6-Cd36)/Cub (BN.SHR4). These two inbred strains differ in a defined segment of chromosome 4, originally transferred from the spontaneously hypertensive rat (SHR) including the mutant Cd36 gene, a known target of DEX. Compared to BN, the standard-diet-fed BN.SHR4 showed higher cholesterol and triacylglycerol concentrations across many lipoprotein fractions, particularly in small VLDL and LDL particles. Total cholesterol was decreased by DEX by more than 21% in BN.SHR4 contrasting with the tendency to increase in BN (strain*DEX interaction p = 0.0017). Similar pattern was observed for triacylglycerol concentrations in LDL. The LDL particle size was significantly reduced by DEX in both strains. Also, while control BN and BN.SHR4 displayed comparable glycaemic profiles during oral glucose tolerance test, we observed a markedly blunted DEX induction of glucose intolerance in BN.SHR4 compared to BN. In summary, we report a pharmacogenetic interaction between limited genomic segment with mutated Cd36 gene and dexamethasone-induced glucose intolerance and triacylglycerol and cholesterol redistribution into lipoprotein fractions.

Endogenous retroviruses (ERVs) contribute to a range of germline, as well as somatic mutations in mammals. However, autonomous retrotransposition of potentially active elements has not been demonstrated in the rat genome. We cloned an insertion that disrupted the normal splicing of the Cntrob gene that was subsequently identified as a nonautonomous, novel endogenous retrovirus of the RnERV-K8e family. The RnERV-K8e family is closely related to the recently reported MmERV-K10c elements, but differs from the autonomous mouse MusD or IAP families. In addition, we identified a novel, unexpectedly close relative of RnERV-K8e in the mouse, suggesting ERV-K cross-species transmission between mice and rats. We cloned a potentially autonomous RnERV-K8e element identified by in silico analysis and, using an in vitro retrotransposition assay, demonstrated that it is capable of retrotransposition. This particular element (named Rat-rho, pronounced "retro") encodes a retroviral envelope gene (env); however, env is not required for de novo retrotransposition events. Significant levels of RnERV-K8e-associated genetic polymorphisms were detected among inbred rat strains, suggesting ongoing retrotransposition in the rat genome. This study identifies an ERV-K-type family in rats that shows obvious signs of recent activity. Ongoing retrotranspositional activity may significantly add to genomic variability among inbred rat strains.

In human, congenital malformations of the limbs are ranked among the most prevalent of all congenital birth defects. Substantial portion of these defects has genetic origin. Increasing knowledge about the particular mutations responsible for limb malformations in human results in the increasing availability of DNA diagnostic procedures for confirmation of clinical diagnosis and family counselling. Increasing understanding of the underlying developmental processes revealed by studying limb defects both in human and animal models may offer better therapeutic options in the future. This review concentrates on the role of Hox genes in limb development. Man, as well as other mammals, has 39 HOX genes, divided into 4 complexes (clusters). HOX genes play a major role in body plan layout and development of many organ systems. Experimental data show that during the limb development, HOX genes influence patterning along the proximodistal and anteroposterior (thumb-little finger) axis of the limb bud. In human, limb malformation was described in patients with mutations in HOXA11, HOXA13, HOXD10, and HOXD13 genes. Most frequent among these malformations are hand-foot-genital syndrome caused by HOXA13 gene mutation, and synpolydactyly caused by HOXD13 mutation. Severity of the phenotype manifestation of these diseases is variable, and depends on the particular mutation type, where point mutations, polyalanine expansions and deletions can take part.

Aims: Therapeutic administration of retinoids is often accompanied with undesirable side effects, including an increase in lipid levels in up to 45% of treated patients. We tested the hypothesis of whether spontaneously hypertensive rat (SHR) and congenic SHR.PD-(D8Rat42-D8Arb23)/Cub (SHR-Lx) strains, differing only in a 14-gene region of chromosome 8 and previously shown to display differential sensitivity to the teratogenic effects of retinoic acid, could serve as a pharmacogenetic model set of the metabolic side effects of retinoid therapy., Materials & Methods: Male, 15-week old rats (n = 12/strain) of SHR and SHR-Lx strains were fed a high-sucrose diet for 2 weeks and subsequently treated either with all-trans retinoic acid (15 mg/kg) or only with a vehicle for 16 days (n = 6/strain/treatment), while still on the high-sucrose diet. We assessed the morphometric and metabolic profiles of all groups, including glucose tolerance tests, levels of insulin, adiponectin, free fatty acids, concentrations of triglycerides and cholesterol in 20 lipoprotein fractions under conditions of both high-sucrose diet and high-sucrose diet plus all-trans retinoic acid administration., Results & Conclusion: SHR-Lx displayed substantially greater sensitivity to a number of all-trans retinoic acid-induced metabolic dysregulations compared with SHR, resulting in impairment of glucose tolerance, increased visceral adiposity, and substantially greater increase of circulating triglyceride concentrations, accompanied by a shift towards their less favorable distribution into the lipoprotein fractions. These observations closely mimic the common side effects of retinoid therapy in humans, rendering SHR-Lx an experimental pharmacogenetic model of atRA-induced dyslipidemia.

The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein. During the final phase of spermiogenesis, centrobin localizes to the manchette, centrosome, and the marginal ring of the spermatid acroplaxome, where it interacts with keratin 5-containing intermediate filaments. Mutant spermatids show a defective acroplaxome marginal ring and separation of the centrosome from its normal attachment site of the nucleus. This separation correlates with a disruption of head-tail coupling apparatus, leading to spermatid decapitation during the final step of spermiogenesis and the absence of sperm in the epididymis. Cntrob may represent a novel candidate gene for presently unexplained hereditary forms of teratozoospermia and the "easily decapitated sperm syndrome" in humans.

Lx mutation in SHR.Lx rat manifests in homozygotes as hindlimb preaxial polydactyly. It was previously mapped to a chromosome 8 segment containing the Plzf gene. Plzf (promyelocytic leukemia zinc finger protein) influences limb development as a direct repressor of posterior HoxD genes. However, the Plzf coding sequence is intact in the Lx mutants. Using linkage mapping in F2 hybrids, we downsized the segment containing Lx to 155 kb and sequenced conserved noncoding elements (CNEs) inside. A 2,964-bp deletion in Plzf intron 2, never detected in control animals, is the only candidate for Lx. The deletion removes the most deeply conserved CNE in the 155-kb segment, suggesting a regulatory influence on Plzf expression. Correspondingly, using in situ hybridization and quantitative real-time polymerase chain reaction, we found a decrease of Plzf expression in Lx/Lx limb buds with concomitant anterior expansion of expression domains of its targets, Hoxd10-13 genes, in the absence of ectopic Sonic hedgehog expression. Upstream regulation of Plzf in limb buds is currently unknown. We present here the first candidate Plzf cis-regulatory sequence., ((c) 2009 Wiley-Liss, Inc.)

Purpose: We isolated an autosomal semi-dominant cataract from our inbred SHR/OlaIpcv rat colony. Heterozygotes express pulverulent cataract with smaller eyes; homozygotes express marked microphthalmia with hypoplastic lens. We call this mutation Dca (for dominant cataract). In this study, we focus on the identification of the responsible gene., Methods: We performed linkage mapping using 93 F2(SHR-Dca x PD) hybrids and a panel of microsatellite markers. In a separate group of animals with a SHR genetic background, we examined the lenses histologically using Epon semi-thin sections and toluidine blue staining. We also assessed the weight of the eyes as an immediate measure for microphthalmia., Results: We mapped the Dca gene to chromosome 2, spanning 8.6 Mbp between markers D2Rat134 and D2Rat186. By sequencing the most plausible candidate gene, Gja8 (coding for connexin 50), we found a T to A transversion at codon 7, leading to a substitution of glutamine for leucin (L7Q). L7Q lies within the NH(2)-terminal cytosolic domain, presumably involved in voltage gating. Histology revealed disturbances in cell to cell contacts in the lens., Conclusions: L7Q is a novel mutation in connexin 50 (Gja8), causing semi-dominant pulverulent cataracts. Dca rats can serve as a model for cataract development. A study on the properties of the mutant protein may offer an insight into the connexin channel function.

We have developed a new, double-congenic rat strain BN-Lx.SHR2, which carries two distinct segments of chromosome 2 introgressed from the spontaneously hypertensive rat strain (SHR) into the genetic background of congenic strain BN-Lx, which was previously shown to express variety of metabolic syndrome features. In 16-wk-old male rats of BN-Lx and BN-Lx.SHR2 strains, we compared their glucose tolerance and triacylglycerol and cholesterol concentrations in 20 lipoprotein subfractions and the lipoprotein particle sizes under conditions of feeding standard and high-sucrose diets. Introgression of two distinct SHR-derived chromosome 2 segments resulted in decreased adiposity together with aggravation of glucose intolerance in the double-congenic strain. The BN-Lx.SHR2 rats were more sensitive to sucrose-induced rise in triacylglycerolemia. Although the total cholesterol concentrations of the two strains were comparable after the standard diet and even lower in BN-Lx.SHR2 after sucrose feeding, detailed analysis revealed that under both dietary conditions, the double-congenic strain had significantly higher cholesterol concentrations in low-density lipoprotein fractions and lower high-density lipoprotein fractions. We established a new inbred model showing dyslipidemia and mild glucose intolerance without obesity, attributable to specific genomic regions. For the first time, the chromosome 2 segments of SHR origin are shown to influence other than blood pressure-related features of metabolic syndrome or to be involved in relevant nutrigenomic interactions.

Germinal epithelium of seminiferous tubules in adult, infertile hypodactylous males displays significant reduction in the number of germ-line cells. Detection of apoptosis in the germ-line cells during postnatal differentiation was performed to elucidate the mechanism of the decreased number of germ cells in the testes of adult rats. Evaluation of DNA fragmentation and expression of activated caspase-3 in germ cells did not confirm marked germ cell death during the onset of spermatogenesis as a main cause of significant reduction of germ cells in Hd/Hd testes of adult males. The primary cause of spermatogenesis defect seems to be rather associated with a disorder in the cell cycle regulation and interrelation of germ-line cells with Sertoli cells.