21135 Whitfield Pl Suite 102
Sterling VA 20165
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Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa. - Reproductive biology and endocrinology : RB&E
Relaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.Spermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.Both receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.Immunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.
Beneficial effects of relaxin on motility characteristics of stored boar spermatozoa. - Reproductive biology and endocrinology : RB&E
Relaxin is detected in seminal plasma of many species and its association with sperm motility may be beneficial in some aspects of assisted reproduction. Here, we immunolocalized relaxin receptors and investigated the effects of exogenous relaxin on motility characteristics, viability, and cAMP content of boar spermatozoa after storage.Commercial doses of boar semen were obtained on the collection day (Day 0) and kept in shipping containers at room temperature for up to 4Â days (Day 4). On Day 0, spermatozoa were fixed for immunofluorescence detection of relaxin receptors RXFP1 and RXFP2 (Experiment 1). Semen aliquots were taken from the same dose at Day 0, Day 1, and Day 2 (Experiment 2a), and Day 2 and Day 4 (Experiment 2b) for analyses. Alive spermatozoa were purified and incubated (1Â h-37Â°C) with 0, 50, or 100Â ng relaxin/ml (Experiment 2a) and 0, 100, or 500Â ng relaxin/ml (Experiment 2b). Afterward, aliquots of each treatment group were subjected to motility (Experiments 2), viability (Experiment 3) analyses, and cAMP quantification (Experiment 4). Data (3-4 independent replicates) were statistically analyzed (ANOVA followed by pairwise comparisons) and p values less or equal to 0.05 was set for significant difference.Both RXFP1 and RXFP2 receptors were immunolocalized on the entire spermatozoon. Relaxin concentration of 100Â ng/ml significantly improved the proportions of motile, progressive, and rapid spermatozoa up to Day 2. Only 500Â ng relaxin/ml provided beneficial effects on Day 4. The viability of spermatozoa was not affected by relaxin (100Â ng/ml) during storage, but the extent of mitochondria membrane damages was significantly decreased. Furthermore, relaxin did not affect the cAMP contents of spermatozoa during storage, in our conditions.Relaxin could be a valuable motility booster of stored- or aged-spermatozoa for assisted reproduction techniques. However, the related-intracellular signaling cascades of relaxin in boar spermatozoa remain undetermined.
The use of digital infrared thermal imaging to detect estrus in gilts. - Theriogenology
Yorkshire/Landrace crossbred gilts (N = 32) were evaluated using digital infrared thermal imaging (DITI) to discriminate between estrus and diestrus phases of the porcine estrous cycle. Gilts (N = 32) were part of an ongoing reproductive efficiency study involving the use of raw soybean (RSB; N = 15) versus soybean meal (SBM; N = 17) as a source of dietary protein. Gilts were monitored daily for signs of estrus using a teaser boar. Thermal images of vulva surface temperatures (TEMP) were recorded at standing estrus and diestrus. Measurements for analysis included minimum (MIN), maximum (MAX), mean (AVG), and standard deviation (SD) of temperature gradients. At imaging, ambient (AMB) and rectal temperatures (RT) were recorded, and blood samples taken for serum progesterone (P(4)) concentration analysis (by RIA) to confirm stage of cycle. Mean serum progesterone values at estrus and diestrus were (mean Â± SD) 1.0 Â± 0.1 and 10.9 Â± 0.8 ng/mL, respectively. Vulva MIN, MAX, and AVG thermal images were positively correlated with one another (P < 0.01), and were positively correlated with ambient temperature (P < 0.01). Vulva MAX and AVG thermal temperatures were greater (P < 0.05) at estrus than at diestrus (36.6 Â± 0.2 Â°C and 33.4 Â± 0.3 Â°C vs. 35.6 Â± 0.3 Â°C and 31.8 Â± 0.6 Â°C, respectively), whereas MIN and SD had no differences (P > 0.05) between stages of the cycle. No differences (P > 0.05) in RT were detected between stages and RT was not significantly correlated with vulva thermal images. Diet had no significant effect on RT or vulva temperature.Copyright Â© 2012. Published by Elsevier Inc.
Otic mesenchyme cells regulate spiral ganglion axon fasciculation through a Pou3f4/EphA4 signaling pathway. - Neuron
Peripheral axons from auditory spiral ganglion neurons (SGNs) form an elaborate series of radially and spirally oriented projections that interpret complex aspects of the auditory environment. However, the developmental processes that shape these axon tracts are largely unknown. Radial bundles are comprised of dense SGN fascicles that project through otic mesenchyme to form synapses within the cochlea. Here, we show that radial bundle fasciculation and synapse formation are disrupted when Pou3f4 (DFNX2) is deleted from otic mesenchyme. Further, we demonstrate that Pou3f4 binds to and directly regulates expression of Epha4, Epha4â»/â» mice present similar SGN defects, and exogenous EphA4 promotes SGN fasciculation in the absence of Pou3f4. Finally, Efnb2 deletion in SGNs leads to similar fasciculation defects, suggesting that ephrin-B2/EphA4 interactions are critical during this process. These results indicate a model whereby Pou3f4 in the otic mesenchyme establishes an Eph/ephrin-mediated fasciculation signal that promotes inner radial bundle formation.Copyright Â© 2012 Elsevier Inc. All rights reserved.
A transgenic insertional inner ear mutation on mouse chromosome 1. - The Laryngoscope
To clone and characterize the integration site of an insertional inner ear mutation, produced in one of fourteen transgenic mouse lines. The insertion of the transgene led to a mutation in a gene(s) necessary for normal development of the vestibular labyrinth.Molecular genetic analysis of a transgene integration site.Molecular cloning, Southern and northern blotting, DNA sequencing and genetic database searching were the methods employed.The integration of the transgene resulted in a dominantly inherited waltzing phenotype and in degeneration of the pars superior. During development, inner ear fluid homeostasis was disrupted. The integration consisted of the insertion of a single copy of the transgene. Flanking DNA was cloned, and mapping indicated that the genomic DNA on either side of the transgene was not contiguous in the wild-type mouse. Localization of unique markers from the two flanks indicated that both were in the proximal region of mouse chromosome 1. However, in the wild-type mouse the markers were separated by 6.3 cM, indicating a sizable rearrangement. Analysis of the mutant DNA indicated that the entire region between the markers was neither deleted nor simply inverted.These results are consistent with a complex rearrangement, including at least four breakpoints and spanning at least 6.3 cM, resulting from the integration of the transgene. This genomic rearrangement disrupted the function of one or more genes critical to the maintenance of fluid homeostasis during development and the normal morphogenesis of the pars superior.
Wocko, a neurological mutant generated in a transgenic mouse pedigree. - The Journal of neuroscience : the official journal of the Society for Neuroscience
Naturally occurring mutations involving the nervous system have provided virtually all of our current understanding of the genetic regulation of neural development (Caviness and Rakic, 1978). The difficulty of isolating the corresponding genes, however, has precluded a molecular analysis of these mutants. Insertional mutagenesis, induced by microinjection of DNA into fertilized ova to produce transgenic animals, provides a molecular tag that marks the site of the mutational event. In this article, we describe a transgenic neurological mutation, designated wocko (Wo), which disrupts the development of the inner ear. These mutant mice display a dominant behavioral phenotype that consists of circling, hyperactivity, and head tossing, reminiscent of the shaker/waltzer class of mutants, and they display a recessive homozygous sublethal phenotype. Anatomical analyses showed that both structural and neural components of the vestibular system were disrupted, while analyses of mutant fetuses showed that these morphological abnormalities were due to aberrant development. Although low levels of transgene expression were detected using a sensitive PCR assay, several nonmutant pedigrees that contain the same construct also expressed the transgene in the inner ear, suggesting that low levels of transgene expression alone were not responsible for the wocko phenotype. Because the integrated transgene provides a marker to clone the wocko mutation, the analysis of this mutant will give unique insight into the molecular genetics of inner ear development and into a broad class of neurological mutations that affect the inner ear.
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