Quantitative analysis of claudin-11 localization in contact with the basal membrane in men with impaired spermatogenesis compared to normal spermatogenesis.

<p>(A) The total number of patches per tubule as well as (B) the number of patches reaching the basal membrane differs highly significant between NSP and SCO. (C) The ratio of the total number of patches versus patches with contact to the basal membrane. The ratio is significantly higher for MA and SCO. Analysis is based on a cohort of 100 patients (NSP n = 25, Hyp n = 24, MA n = 24, SCO n = 19, and SGA n = 8). Columns represent mean values with SEM indicated. Kruskal-Wallis-test was used to calculate p-values, p ≤ 0.05 was considered significant (*), p ≤ 0.001 highly significant (**), respectively, p ≤ 0.0001 (***).</p

Scheme of claudin-11 expression in the seminiferous epithelium used for patch analysis.

<p>A claudin-11 patch is defined as the claudin-11 localization pattern between two points crossing the fictive line above the spermatogonia (black arrows). In the drawing the patches are indicated by brackets, letters (a-e) and triangles. If claudin-11 is in contact with the basal membrane (a & e), the patch was evaluated as positive (filled triangles), all other types negative (b-d, white triangles).</p

Electron microscopical analysis reveals intact blood-testis barrier (BTB) and blood-epididymis barrier (BEB).

<p>(<b>A</b>) Ultrastructural analysis shows that intercellular tracer penetration does not extend beyond the junctional complex of the BTB (arrow in inset) within the seminiferous epithelium of UPEC infected rats (x3,000 magnification, inset x20,000 magnification). SC = Sertoli cells, orientation of the luminal and basal compartment are highlighted (<b>B</b>) Ultrastructural analysis of a UPEC infected epididymis demonstrates intercellular tracer penetration (x3,000 magnification). Inset is a magnification of the area represented in the black frame showing the tight junctions. (x20,000 magnification).</p

Increase of TUNEL positive cells in UPEC infected testis.

<p>(<b>A</b>) DNA strand breakage in testicular cells from control (upper panel) and UPEC infected (lower panel) rats were analyzed using TUNEL assay. Nuclei were counterstained with DAPI (blue). TUNEL (+) cells (green) with ring-like nuclear stain are indicated with arrows. (<b>B</b>) Numbers of TUNEL (+) cells are presented as mean ± SD/seminiferous tubule. Student’s t-test was used for statistical analysis and the level of significance is indicated as **<i>p</i><0.01. (x20 objective).</p

Primer information.

<p>Primer information.</p

Presence of UPEC inside testes of infected rats 7 days after inoculation in the vas deferens.

<p>(<b>A</b>) Genomic DNA was extracted from testicular tissue and 200 ng DNA from each sample were amplified with PCR using UPEC pili primers. DNA isolated from one explanted testis immediately after direct UPEC injection served as a positive control. PCR products were separated on 1.5% agarose gel and stained with ethidium bromide. The same amount of DNA from each sample without PCR amplification was subjected to agarose gel electrophoresis and served as a loading control. Lane 1∶100 bp DNA marker; lane 2–4: samples from saline injected animals; Lane 5∼7: samples from UPEC infected rat testes; Lane 8: UPEC positive control; Lane 9: negative control. (<b>B</b>) Testicular homogenates from saline injected (left panel) and UPEC infected rats (right panel) were streaked on agar plates without antibiotics and kept at 37°C overnight. Colonies were counted under translucent light. (<b>C</b>) Cryosections of testis from control (left panel) and UPEC infected rats (right panel) were probed with anti-<i>E. coli</i> antibody and decorated with secondary anti-rabbit IgG antibody conjugated to Cy-3 (orange). DAPI (blue) was used for nuclear counterstain (x20 objective). (<b>D</b>) Semithin cross-section of a seminiferous tubule (asterisk) with adjacent interstitial space. Microbial presence in interstitial space is visible (arrow in the black frame, x20 objective). Inset: Electron microscopical examination on the same area (primary magnification x3,000).</p

NF-κB pathway is not activated in UPEC infected testis.

<p>(<b>A</b>) Total testis proteins were separated on 10% SDS-PAGE. Immunoblots were labeled with mouse anti-IkBα antibody. ß-actin served as a loading control. (<b>B</b>) The intensity of target bands was measured with the ImageJ software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) and results are presented as the relative intensity = intensity of p65/intensity of ß-actin. (<b>C</b>) Testis cryosections were probed with anti-p65 antibody labeled with Cy3-linked secondary antibody (orange) and the nuclei were counterstained with DAPI (blue, images taken with x40 objective). Representative results from at least two independent experiments are shown.</p

Morphological changes and histological evaluation of the testis and epididymis.

<p>(<b>A</b>) Testicular weight of control (n = 8) and infected rats (n = 10) are presented as mean ± standard deviation (SD). Student’s t-test was employed for statistical analysis and the level of significance is indicated as **<i>p</i><0.001. (<b>B</b>) Sperm concentration was assessed in seven animals of each group and the results are presented as mean ± SD. Statistical analysis was performed with Student’s t-test and statistical significance is denoted as *<i>p</i><0.05. (<b>C</b>) Tissue sections of paraffin embedded testes were stained with hematoxylin and eosin. Histopathological assessment was performed on control (n = 5) and UPEC infected (n = 9) testes using light microscopy. The images were captured using Axioplan 2 Imaging system at magnification x20 and representative figures are shown. Various forms of impairment of spermatogenesis are visible exemplified by a Sertoli cell only tubule (star) and a hypospermatogenic tubule (triangle). (<b>D</b>) Histopathological images of caput (D top panels) and cauda epididymis (D bottom panels, x20 objective). Representative results from control (n = 5) and infected (n = 9) rats are depicted.</p

RNA expression pattern of the <i>bcl-2</i> family genes in the testis.

<p>The expression of the anti-apoptotic gene <i>bcl-2</i> (<b>A</b>), pro-apoptotic genes <i>bax</i> (<b>B</b>), <i>bid</i> (<b>C</b>), <i>bim</i> (<b>D</b>) and <i>bak</i> (<b>E</b>) in the testis were determined with quantitative real time PCR. Target gene expression levels were normalized with the endogenous control ß-2-microglobulin (ß2M). Data are present as 2^ΔCt, ΔCt = Ct_{target gene}-Ct_ß2M. The Mann-Whitney U test was employed for statistical analysis (* <i>p</i><0.05). Each single symbol (circle and triangle) represents one individual testis sample.</p

Katanin-like 2 (KATNAL2) functions in multiple aspects of haploid male germ cell development in the mouse

<div><p>The katanin microtubule-severing proteins are essential regulators of microtubule dynamics in a diverse range of species. Here we have defined critical roles for the poorly characterised katanin protein KATNAL2 in multiple aspects of spermatogenesis: the initiation of sperm tail growth from the basal body, sperm head shaping via the manchette, acrosome attachment, and ultimately sperm release. We present data suggesting that depending on context, KATNAL2 can partner with the regulatory protein KATNB1 or act autonomously. Moreover, our data indicate KATNAL2 may regulate δ- and ε-tubulin rather than classical α-β-tubulin microtubule polymers, suggesting the katanin family has a greater diversity of function than previously realised. Together with our previous research, showing the essential requirement of katanin proteins KATNAL1 and KATNB1 during spermatogenesis, our data supports the concept that in higher order species the presence of multiple katanins has allowed for subspecialisation of function within complex cellular settings such as the seminiferous epithelium.</p></div