Mutational Insertion of a ROSA26�CEGFP Transgene Leads to Defects in Spermiogenesis and Male Infertility in Mice

Pronuclear injection has been a successful strategy for generating genetically engineered mouse models to better understand the functionality of genes. A characteristic of pronuclear injection is that random integration of the transgene into the genome can disturb a functional gene and result in a phenotype unrelated to the transgene itself. In this study, we have characterized a mouse model containing an insertional mutation that, in the homozygous state, severely affects spermatogenesis as characterized by lack of sperm motility and acrosomal aplasia. Whereas homozygous female mice had normal fertility, male mice homozygous for the insertional mutation were unable to produce pups by natural mating with either homozygous or wild-type female mice. No fertilized embryos were produced by matings to homozygous male mice, and no sperm were present in the reproductive tract of mated female mice. Spermatozoa isolated from homozygous male mice exhibited head and midpiece defects, but no major defects in the principal piece of these sperm. Histologic examination and immunohistochemical staining of the testes revealed vacuolar degeneration of Sertoli cells and loss of structural seminiferous tubule integrity and organization, indicating that spermatogenesis is severely affected in this mouse model. Although the males are always infertile, the severity of the histologic and sperm morphologic defects appeared to be age-related.Abbreviations: EGFP, enhanced green fluorescent protein; GOPC, Golgi-associated proteinThe production of genetically engineered mice has enabled unprecedented advancements in understanding the functionality of genes. Genetically engineered mice can be produced in many ways, but pronuclear injection has been the standard method for many years.^2,4,7,23 Despite its success, pronuclear injection is associated with several problems. One important problem is that random integration of the transgene into the genome can disturb a functional gene and lead to associated problems. However, as we present here, random insertion of the transgene sometimes can result in interesting and important discoveries about gene function.Numerous reports describe problems in spermatogenesis associated with transgene insertion,^10,16,18,24 and these mice have provided powerful research tools with which to study the complexities of male infertility and the production and maturation of spermatozoa. Here we characterize the male infertility phenotype of the FVB/NTac-Tg(Gt(ROSA)26Sor-EGFP)130910Eps/Mmmh strain¹³ The original mouse strain was donated to the University of Missouri Mutant Mouse Regional Resource Center (http://www.mmrrc.org) and assigned designation MMRRC:000366. The founder animal for this strain was generated by random insertion of a transgene construct containing the enhanced green fluorescent protein (EGFP) under control of the mouse ROSA 26 promoter by pronuclear injection of FVB/NTac embryos. The strain has since been maintained for more than 20 generations by backcrossing to FVB/NTac mice. Efforts to cryopreserve the strain revealed that male mice homozygous for the transgene insertion were infertile, thereby leading to the studies presented here.To determine the precise site of integration of the transgene, a chromosome-walking technique³ was used and revealed a single integration site on chromosome 3 within the intronic region of a novel gene (ENSMUSG00000027939). Based on the predicted protein sequence for this gene, it represents a novel nucleoporin with shared similarity to members of the nuclear pore membrane glycoprotein 210 family. Nucleoporins are protein components of the nuclear pore complex and play a key role in nucleocytoplasmic transport. Importantly in the context of the infertility phenotype described for this mouse model, we speculate that the gene disrupted by the mutational insertion may be involved in nucleocytoplasmic trafficking, which is important for male germ cell differentiation.In this report, we describe the infertility phenotype of the FVB/NTac-Tg(Gt(ROSA)26Sor-EGFP)130910Eps/Mmmh strain (ROSA–EGFP) and document age-related sperm defects and Sertoli cell abnormalities that underlie the male infertility seen in animals homozygous for the insertional mutation. These mice exhibited severe defects in spermatogenesis, as manifested by sperm head and midpiece abnormalities and a loss of sperm motility. In addition, we discuss the results of the histologic examinations, which revealed vacuolar degeneration of Sertoli cells, rare multinucleated cells, and reduced numbers of all stages of germinal cells in the seminiferous tubules as well as occasional vacuolation of the epididymal epithelium. The sperm head defects coupled with the Sertoli cell degeneration suggest possible disruption of normal Sertoli cell–spermatid interactions in homozygotes for this transgene insert. Furthermore matings with homozygous male mice failed to produce any pups, whereas heterozygous and homozygous female mice bred with wild-type male mice had normal litter sizes. Functional analysis of the homozygous male mice may provide additional insight into the molecular mechanisms of spermiogenesis, particularly those involving acrosomal biogenesis and the development of functional flagella.     

Enantioselective synthesis and antioxidant activity of 3‐(3,4‐dihydroxyphenyl)‐glyceric acid—Basic monomeric moiety of a biologically active polyether from Symphytum asperum and S. caucasicum

The racemic and enantioselective synthesis of a novel glyceric acid derivative, namely, 2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid as well as the antioxidant activities is described. The virtually pure enantiomers, (+)‐(2R,3S)‐2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid and (?)‐(2S,3R)‐2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid were synthesized for the first time via Sharpless asymmetric dihydroxylation of trans‐caffeic acid derivatives using the enantiocomplementary catalysts, (DHQD)₂‐PHAL and (DHQ)₂‐PHAL. The determination of enantiomeric purity of the novel chiral glyceric acid derivatives was performed by high‐performance liquid chromatographic techniques on the stage of their alkylated precursors. The novel glyceric acid derivatives show strong antioxidant activity against hypochlorite and N,N‐diphenyl‐N‐picryl‐hydrazyl free radical. Their antioxidant activity is about 40‐fold higher than that of the corresponding natural polyether and three‐fold higher of trans‐caffeic acid itself. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibitory effect of dicationic diphenylfurans on production of type I collagen by human fibroblasts and activated hepatic stellate cells

Excessive production of extracellular matrix is responsible for clinical manifestations of fibroproliferative disorders and drugs which can inhibit excessive synthesis of type I collagen are needed for the therapy. Several dicationic diphenylfurans were synthesized and were found to bind RNA. Two of these type compounds were able to reduce synthesis of type I collagen by human fibroblasts and human activated hepatic stellate cells (HSCs). Activated HSCs are responsible for collagen production in liver fibrosis. When added at 40 microM compound 588 reduced intracellular level and secretion of procollagen alpha1(I) by 50%, while compound 654 reduced these parameters by more than 80% at 20 microM. 654 also significantly reduced secretion of fibronectin. Toxic effects were observed at 80 microM for 588 and 40 microM for 654. 654 reduced expression of a reporter gene with collagen signal peptide, while expression of the same gene without signal peptide was unaffected. Also, expression of intracellular proteins tubulin and calnexin was unchanged. 654 accumulated inside the cell in the cytoplasm and did not change the steady-state level of collagen mRNAs. Treatment of cells with proteosome inhibitor MG132 did not change the inhibitory effect of 654, suggesting that 654 acts as suppressor of translation of proteins containing a signal peptide. Most secreted proteins of fibroblasts and activated HSCs are components of extracellular matrix. Therefore inhibition of their production, as shown here for procollagen alpha1(I) and fibronectin, may be a useful property of some of diphenylfurans, making these compounds a basis for development of antifibrotic drugs.     

Calcium intake, food sources and seasonal variations in eastern Croatia

The objective of this study was to examine the quantity of calcium intake among adults, the sources of calcium, differences among seasons, as well as the differences between sexes, correlation with body mass index (BMI), and age. The study included 161 healthy volunteers from the eastern part of Croatia. Each subject completed three food frequency questionnaires (FFQ) with 150 items, at an interval of 3-4 months. The mean calcium intake for whole population for all three FFQs was 965 mg/day. At the same time, the quantity of calcium for all subjects was 14.2 mg/kg, women 14.5 mg/kg, and men 13.6 mg/kg, respectively. There was an inverse relation between calcium intake and age for men (r = -0.32 p = 0.028), but not for women. Correlation between calcium intake and BMI was negative, but not significantly. Milk and dairy products were the main source of calcium. Marginally low mean calcium intake goes to show the needful to educate the population.     

Detection of mouse parvovirus in Mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay

We used primary and nested polymerase chain reaction (PCR) assays to determine the presence of mouse parvovirus (MPV) in mouse sperm, oocytes, preimplantation embryos, and ovarian tissues collected from MPV-infected mice. The primary PCR assay detected MPV in 56% of the sperm samples. MPV was not eliminated by passing sperm samples through a Percoll gradient. After Percoll treatment, MPV was still present in 50% of the samples according to primary PCR assay. Oocyte samples that did not undergo extensive washing procedures had detectable MPV in 7% of the samples based on the primary PCR assay, but nested PCR assay detected higher (28%) infection rate. However, MPV was not detected in oocytes that underwent extensive washing procedures, as assessed by either primary or nested PCR assay. Although primary PCR did not detect MPV in embryos, a nested PCR assay determined that 50% of the embryos were positive for the virus. In addition, ovarian tissues were collected from 3 different mouse colonies with enzootic MPV infection. Ovarian tissue collected from 129CT, 101/R1, and Sencar mice had high incidence (38%, 63%, and 65%, respectively) of MPV infection on the basis of nested PCR amplification. These results demonstrate that mouse gametes, embryos, and ovarian tissues may be contaminated with MPV and therefore caution is necessary when infected germplasm is used for assisted reproductive technologies such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization, ovary transplantation, and intracytoplasmic sperm injection.     

Hantaviruses: an overview

Hantaviruses are a newly emerging group of rodent-borne viruses that have significant zoonotic potential. Human infection by hantaviruses can result in profound morbidity and mortality, with death rates as high as 50%, and potentially long-term cardiovascular consequences. Hantaviruses are carried by peridomestic and wild rodents worldwide and have occasionally been linked to infections in laboratory rodents. Because these viruses have been associated with significant human disease, they have become the subject of intense scientific investigation. In this review the reader is introduced to the hantaviruses, including hantavirus diseases and their pathogenesis. A review of the biology, morphology, and molecular biology of the hantaviruses with a brief overview of the ecology and biology of hantavirus-rodent pairs is also included. The risks of occupational exposure to hantaviruses, diagnosis of hantavirus infections, and methods for handling potentially infected rodents and tissues are discussed as well.     

Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

APOBEC3A (A3A) is a myeloid lineage-specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14⁺-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14⁺ cells were stimulated with interferon. Immunofluorescent microscopy of interferon-stimulated CD14⁺ and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell-wide localization. A3A constructs engineered to be cytoplasmic are also nontoxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses.     

Population Size Estimation of Men Who Have Sex with Men in Tbilisi,Georgia; Multiple Methods and Triangulation of Findings

Introduction

An accurate estimation of the population size of men who have sex with men (MSM) is critical to the success of HIV program planning and to monitoring of the response to epidemic as a whole, but is quite often missing. In this study, our aim was to estimate the population size of MSM in Tbilisi, Georgia and compare it with other estimates in the region.

Methods

In the absence of a gold standard for estimating the population size of MSM, this study reports a range of methods, including network scale-up, mobile/web apps multiplier, service and unique object multiplier, network-based capture-recapture, Handcock RDS-based and Wisdom of Crowds methods. To apply all these methods, two surveys were conducted: first, a household survey among 1,015 adults from the general population, and second, a respondent driven sample of 210 MSM. We also conducted a literature review of MSM size estimation in Eastern European and Central Asian countries.

Results

The median population size of MSM generated from all previously mentioned methods was estimated to be 5,100 (95% Confidence Interval (CI): 3,243 ~ 9,088). This corresponds to 1.42% (95%CI: 0.9% ~ 2.53%) of the adult male population in Tbilisi.

Conclusion

Our size estimates of the MSM population (1.42% (95%CI: 0.9% ~ 2.53%) of the adult male population in Tbilisi) fall within ranges reported in other Eastern European and Central Asian countries. These estimates can provide valuable information for country level HIV prevention program planning and evaluation. Furthermore, we believe, that our results will narrow the gap in data availability on the estimates of the population size of MSM in the region.     

Sterols of Mulberry Leaves and Small Leaf Curl Disease

Free and bound sterols of leaves of five mulberry cultivars differing in their susceptibility to small leaf curl disease have been studied. The total content of sterols in all samples is similar and is not correlated with the resistance of the cultivars. The qualitative composition of particular sterols is also identical. They are represented by cholesterol, campesterol, stigmasterol, sitosterol, and two 4α-methylsterols. The leaves of the most sensitive cultivar are characterized by high cholesterol content. The ratio sitosterol : stigmasterol decreased in proportion to the resistance level of a cultivar.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 460–462.Original Russian Text Copyright © 2005 by Zambakhidze, Sulaberidze, Mzhavanadze, Tsiklauri.