Intraovarian activins are required for female fertility

Activins have diverse roles in multiple physiological processes including reproduction. Mutations and loss of heterozygosity at the human activin receptor ACVR1B and ACVR2 loci are observed in pituitary, pancreatic, and colorectal cancers. Functional studies support intraovarian roles for activins, although clarifying the in vivo roles has remained elusive due to the perinatal death of activin betaA knockout mice. To study the roles of activins in ovarian growth, differentiation, and cancer, a tissue-specific knockout system was designed to ablate ovarian production of activins. Mice lacking ovarian activin betaA were intercrossed to Inhbb homozygous null mice to produce double activin knockouts. Whereas ovarian betaA knockout females are subfertile, betaB/betaA double mutant females are infertile. Strikingly, the activin betaA and betaB/betaA-deficient ovaries contain increased numbers of functional corpora lutea but do not develop ovarian tumors. Microarray analysis of isolated granulosa cells identifies significant changes in expression for a number of genes with known reproductive roles, including Kitl, Taf4b, and Ghr, as well as loss of expression of the proto-oncogene, Myc. Thus, in contrast to the known tumor suppressor role of activins in some tissues, our data indicate that activin betaA and betaB function redundantly in a growth stimulatory pathway in the mammalian ovary.     

Female infertility and disrupted angiogenesis are actions of specific follistatin isoforms

The circulating and tissue-bound forms of follistatin (FST315 and FST288, respectively) modulate the actions of activins. FST knockout (KO/null) mice, lacking both isoforms, die perinatally with defects in lung, skin, and the musculoskeletal system. Using constructs of the human FST gene engineered to enable expression of each isoform under the control of natural regulatory elements, transgenic mouse lines were created and crossed with FST null mice to attempt to rescue the neonatal lethality. FST288 expression alone did not rescue the neonatal lethality, but mice expressing FST315 on the KO background survived to adulthood with normal lung and skin morphology and partial reversal of the musculoskeletal defects noted in FST KO mice. The FST315 rescue mice displayed a short period of neonatal growth retardation, impaired tail growth, and female infertility. The latter may be due to failure of corpus luteum formation, a decline in the ovarian follicular population, and an augmented uterine inflammatory response to mating. Failure of corpus luteum formation and impaired tail growth indicate abnormal vascularization and suggest that FST288 is required for the promotion of angiogenesis. The augmented uterine inflammatory response may result from the failure of FST315 to modulate the proinflammatory actions of activin A in the uterus or may result from the altered steroid milieu associated with the ovarian abnormalities. Although we cannot definitively conclude that the remaining defects are due to the absence of a particular isoform or due to variable expression of each, these models have demonstrated novel physiological processes that are influenced by FST.     

Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Disruption of γ-Glutamyl Leukotrienase Results in Disruption of Leukotriene D4 Synthesis In Vivo and Attenuation of the Acute Inflammatory Response

To study the function of gamma-glutamyl leukotrienase (GGL), a newly identified member of the gamma-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGL(tm1)) and in both GGL and GGT (GGL(tm1)-GGT(tm1)) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGL(tm1) show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but approximately 70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C(4) (LTC(4)) to LTD(4), indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD(4). Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC(4) and LTD(4) have distinctly different functions in the inflammatory process.     

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization

Dear Editor, Sperm cooperation has been observed in multiple species(Pizzari and Foster,2008),yet its existence and benefit for reproductive success in mammals remains underexplored.Here,combining tissue-clearing with deep three-dimen-sional (3D) imaging,we demonstrate that postcopulatory mouse sperm congregate into unidirectional sperm cooper-ative clusters at the utero-tubal junction (UTJ),a key physical barrier for passage into the oviduct.Reducing sperm number in male mice by unilateral vasoligation (Uni-Vas) or busulfan-treatment impairs sperm cluster formation and oviduct entry.     

Quantification of the magnification and distortion effects of a pediatric flexible video-bronchoscope

Background

Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.

Methods

Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.

Results

Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.

Conclusion

We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements.     

Analysis of higher-primate phylogeny from transversion differences in nuclear and mitochondrial DNA by Lake's methods of evolutionary parsimony and operator metrics

In the companion paper (Holmquist et al. 1988), we concluded that there is no agreement on either the correct branching order or differential rates of evolution among the higher primates, and we examined in depth why this uncertainty in the evolutionary understanding of our closest living relatives persists. Recently, Lake developed two novel methods, based on group properties of transition and transversion operators, that (a) permit, in principle, objective resolution of problems of the above type and (b) attach a statistical significance level to the conclusions drawn. In the present paper, we develop formulas for using these two methods in tandem and apply them to study transversion differences in (1) nuclear DNA for a 7-kb segment of the psi eta-globin locus and a 3-kb intergenic region between the psi beta- and delta- globin loci and (2) mitochondrial DNA for the 896-bp fragment of Brown et al. Although each of these nucleotide sequence regions has its characteristic tempo and mode of evolution, the nuclear and mitochondrial data together, comprising a total of 10,939 base positions, support a Homo/Pan clade at the 97% confidence level. If we calibrate the divergence point for humans and chimpanzees at 5 Myr, consideration of the transversion branch lengths for the combined nuclear data indicates that the gorilla lineage branched off 600,000- 900,000 years prior to that, although the 2 sigma sampling errors do not preclude either a temporal trifurcation for the three species or a considerably more ancient branch point for the gorilla. To resolve the length of this central branch to a relative accuracy of 25% and 30% will require a factor of 16 and nine times more data, respectively-- i.e., in excess of 100,000 homologous nucleotides for each of the four primates. For the nuclear genes, heterogeneity in evolutionary rates between different parts of the genome is mostly restricted to the human lineage for these two segments. The lineage leading to chimpanzees has evolved 0.4 (3-kb fragment) to 3.5 (7-kb segment) times as rapidly as the lineage leading to humans, and that leading to the gorilla has evolved approximately one-fifth to one-half as rapidly as that leading to chimpanzees. Thus, even local molecular clocks can "tick" badly. As significant is the fact that virtually contiguous parts of the genome tick at markedly different rates.(ABSTRACT TRUNCATED AT 400 WORDS)      

Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides

Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.