Dynamic cytoplasmic projections connect mammalian spermatogonia in vivo.

Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25 µm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.

Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice.

We previously described a novel germ cell-specific X-linked reproductive homeobox gene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating a Rhox13 gene knockout (KO) mouse. Rhox13 KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7-8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

Ion-pair reversed phase liquid chromatography with ultraviolet detection for analysis of ultraviolet transparent cations.

This paper describes the use of an anionic ion-pair reagent (IPR) to impove the ultraviolet (UV) detection and hydrophobic retention of polar and UV transparent cations. Anionic IPR added to the mobile phase forms an ion-pair with cations. Formation of the ion-pair causes a redshift in the absorption wavength, making it possible for direct UV detection of UV-inactive cations. The ion-pairs with increased hydrophobicity were separated by reversed phase liquid chromatography (RPLC). Different perfluorinated caboxylic acids (trifluoroacetic acid, heptafluorobutyric acid, nonafluoropentanoic acid) were evaluted as IPR in the separation and detection of the common cations sodium, ammonium and Tris(hydroxymethyl)aminomethane (Tris). The effects of the IPR type and concentration on separation and detection have been investigated to understand the separation and detection mechanisms. The optimal separation and detection condtions were attained with mobile phase containing 0.1% nonafluoropentanoic acid and with the UV detection at 210nm. UV detection and charged aerosol detection (CAD) were compared in the quantitation of the cations. The limit of quantitation (LOQ) of sodium and Tris with UV detection is comparable to that by CAD. The LOQ of ammonium with UV detection (1ppm or 3ng) is about 20-fold lower than that (20ppm or 60ng) by CAD. The RPLC-UV method was used to monitor ammonium clearance during ultrafiltration and diafiltration in the manfucaturing of biopharmceutical drug substance.

The effect of fat on the measurement of bone mineral density by digital X-ray radiogrammetry (DXR-BMD).

OBJECTIVE: We have previously shown that surrounding fat causes an increase of up to 21% in bone mineral density (BMD) measured by Lunar 'Intelligent DXA' (iDXA), one of the latest generation dual energy X-ray absorptiometry (DXA) scanners [1]. The purpose of our study was to see if it was possible to avoid this artifact when measuring the BMD of metacarpals II, III, and IV by digital X-ray radiogrammetry (DXR). METHODS: We took X-rays of the bones of a cadaveric left hand which were immobilized in a wooden cradle to preserve an approximate in vivo configuration. The X-rays were digitized into Digital Imaging and Communications in Medicine (DICOM) files which were analyzed using dxr-online (dxr-online, Sectra, Sweden) which uses the same DXR-BMD algorithm previously used by Pronosco X-posure v2 and Sectra Osteoporosis package. The X-rays were repeated four times. We then surrounded the bones with a layer of lard, and again X-rayed four times. This process was repeated with the bones were covered by two layers, and then three layers of lard. RESULTS: The measured DXR-BMD increased by a maximum of 0.44% when the metacarpals were covered by either two or three layers of lard compared with when the metacarpals were not covered by lard. CONCLUSION: The measurement of metacarpal BMD measured by DXR is minimally affected by surrounding lard. The measurement of metacarpal BMD by DXR seems to be a way of avoiding the artifactual change in BMD caused by fat, when it is measured by DXA.