Low-mobility sperm phenotype in the domestic turkey: Impact on sperm morphometry and early embryonic death.

The sperm mobility assay measures the ability of sperm to swim through a dense layer of Accudenz® , and the sperm mobility phenotype has been shown to predict fertility and other sperm performance traits in roosters and turkeys. In this study, we examined turkey sperm morphometry and rates of early embryonic death associated with high- and low-mobility semen. We also assessed whether the hypo-osmotic stress test, which evaluates the structural integrity of the sperm plasma membrane, may be used as a faster and simpler assay for sperm mobility and viability. We confirmed previous work that found that high-mobility sperm are faster and swim more linearly than low-mobility sperm, and that mobility traits were repeatable within males. In contrast to previous studies, we did not find higher rates of fertility, but low-mobility sperm was associated with higher rates of early embryonic death, though this trend was not significant. High-mobility sperm had longer sperm heads, explained by longer nuclei, despite shorter acrosomes. Although these sperm were faster, midpiece length and flagellum length did not differ between high- and low-mobility sperm. Finally, mobility was not found to be associated with sperm performance in the hypo-osmotic stress test.

Correction: Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation.

[This corrects the article DOI: 10.1371/journal.pone.0123282.].

Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa.

Fluorescence-activated flow cytometry analyses were developed for determination of reactive oxygen species (ROS) formation and membrane lipid peroxidation in live spermatozoa loaded with, respectively, hydroethidine (HE) or the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C(11)BODIPY(581/591) (BODIPY). ROS was detected by red fluorescence emission from oxidization of HE and membrane lipid peroxidation was detected by green fluorescence emission from oxidation of BODIPY in individual live sperm. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeSo4/ascorbate (FeAc), because menadione and H(2)O(2) had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H(2)O(2); FeAc had no effect. The incidence of basal or spontaneous ROS formation and membrane lipid peroxidation were low in boar sperm (<1% of live sperm) in fresh semen or after low temperature storage; however the sperm were quite susceptible to treatment-induced ROS formation and membrane lipid peroxidation.

Expression and regulation of glucocorticoid-induced leucine zipper in the developing anterior pituitary gland.

The expression profile of glucocorticoid-induced leucine zipper (GILZ) in the anterior pituitary during the second half of embryonic development in the chick is consistent with in vivo regulation by circulating corticosteroids. However, nothing else has been reported about the presence of GILZ in the neuroendocrine system. We sought to characterize expression and regulation of GILZ in the chicken embryonic pituitary gland and determine the effect of GILZ overexpression on anterior pituitary hormone levels. Pituitary GILZ mRNA levels increased during embryogenesis to a maximum on the day of hatch, and decreased through the first week after hatch. GILZ expression was rapidly upregulated by corticosterone in embryonic pituitary cells. To determine whether GILZ regulates hormone gene expression in the developing anterior pituitary, we overexpressed GILZ in embryonic pituitary cells and measured mRNA for the major pituitary hormones. Exogenous GILZ increased prolactin mRNA above basal levels, but not as high as that in corticosterone-treated cells, indicating that GILZ may play a small role in lactotroph differentiation. The largest effect we observed was a twofold increase in FSH beta subunit in cells transfected with GILZ but not treated with corticosterone, suggesting that GILZ may positively regulate gonadotroph development in a manner not involving glucocorticoids. In conclusion, this is the first report to characterize avian GILZ and examine its regulation in the developing neuroendocrine system. We have shown that GILZ is upregulated by glucocorticoids in the embryonic pituitary gland and may regulate expression of several pituitary hormones.

Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) by using fluorescence-activated flow cytometry.

A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (Deltapsi(m)), >80-100 mV using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear stain propidium iodine (PI). This flow cytometric method is described in detail here. When in contact with membranes possessing a high Deltapsi(m), JC-1 forms aggregates (J(agg)) that are fluorescent at 590 nm in response to 488 nm excitation. We found that the reactive oxygen species generator, menadione reduced sperm motility and reduced Deltapsi(m) in a dose responsive fashion that was closely correlated with the loss of motility.

Effect of db-camp on embryonic development of bovine oocytes fertilized with sex sorted semen. / Efecto del db-cAMP Sobre el Desarrollo Embrionario de Ovocitos Bovinos Fecundados con Semen Sexado.

The objective of this study was to improve sexed bovine embryo production with sorted sperm in chemically defined conditions by supplementing the IVF medium with db-cAMP. Cumulus-oocyte complexes (COCs) were matured for 18 h in supplemented TCM-199 and fertilized with X- or Y-bearing sperm in the presence of heparin (10 µg/ml), db-cAMP (1 µM) or no treatment (control). Presumptive zygotes were cultured 54 h in g-SOF. From 72 to 144 h post- insemination (hpi) embryos were cultured in c-SOF+NEA and from 144 to 192 hpi embryos were placed in maturation medium without hormones. No significant differences were found among treatments for Y-sperm when compared to controls. A significant (P<0.01) improvement in the proportion of cleaved oocytes was found for X-sperm treated with db-cAMP (70.83%) compared to the Y-sperm inseminated oocytes treated wit db-cAMP (46.37%). Treatment with db-cAMP enabled a better (P<0.05) blastocyst formation rate (19.29%) compared to control (8.47%) and heparin (10.44%). Treatment of db-cAMP significantly increased the rate of blastocysts in X-sperm inseminated oocytes (30.77%) compared to Y-sperm inseminated oocytes treated the same (9.68%) and compared to X- and Y-sperm treated with heparin (5.88% and 15.15%, respectively) and not treated (9.68% and 7.14%, respectively, P<0.05). These results suggest that db-cAMP may prove to be an effective treatment of sorted sperm for in vitro production of female bovine embryos under chemically defined conditions.

El objetivo de este estudio fue mejorar la producción de embriones bovinos con semen sexado bajo condiciones químicamente definidas mediante la suplementación del medio de fecundación con db-cAMP. Los complejos ovocitos cumulus (COCs) fueron madurados por 18 horas en TCM-199 suplementado y fueron fecundados con espermatozoides X o Y en presencia de heparina (10 g/ml), db-cAMP (1 µM) o sin tratamiento alguno (control). Los presuntivos cigotos fueron cultivados por 54 horas en g-SOF. Desde las 72 a las 144 horas post-inseminación (hpi) los embriones se cultivaron en c-SOF+NEA y desde 144 a 192 hpi fueron colocados en medio de maduración pero sin hormonas. No se observaron diferencias entre tratamientos para los oocitos fecundados con espermatozoides Y cuando se compararon a los controles. Se observó una mejora significativa (P<0,01) en la proporción de ovocitos que se dividieron cuando fueron fecundados con espermatozoides X tratados con db-cAMP (70,83%) en comparación con los fecundados con espermatozoides Y tratados con db-cAMP (46,37%). El tratamiento con db-cAMP fue capaz de inducir una mayor (P<0,05) tasa de formación de blastocistos (19.29%) en comparación a los tratamientos control (8,47%) y heparina (10,44%). El tratamiento con db-cAMP incrementó (P<0,05) la tasa de embriones de cuatro células alcanzando el estadio de blastocisto cuando los oocitos fueron fecundados con espermatozoides X (30,77%) en comparación a cuando la fecundación se realizó con espermatozoides Y (9,68%) y en comparación a cuando se llevó a cabo con espermatozoides X o Y en presencia de heparina (5,88% y 15,15%, respectivamente) o en el tratamiento control (9,68% y 7,14%, respectivamente). Estos resultados sugieren, que el db-cAMP puede ser un tratamiento efectivo para el semen sexado, a fin de incrementar la producción in vitro de embriones bovinos hembra bajo condiciones químicamente definidas.

CAG repeat lengths in X- and Y-bearing sperm indicate that gender bias during transmission of Huntington's disease gene is determined in the embryo.

The size of the CAG tract at the Huntington's disease (HD) locus upon transmission depends on the gender of the parent. However, the basis for the parent-of-origin effect is unknown. To test whether expansion and contraction in HD are "imprinted" in the germ cells, we isolated the X- and Y-bearing sperm of HD transgenic mice. Here we show that CAG repeat distributions in the X- and Y-bearing spermatozoa of founding fathers do not differ. These data show that gender-dependent changes in CAG repeat length arise in the embryo.