RGS9 Knockout Causes a Short Delay in Light Responses of ON-Bipolar Cells

RGS9 and R9AP are components of the photoreceptor-specific GTPase activating complex responsible for rapid inactivation of the G protein, transducin, in the course of photoresponse recovery from excitation. The amount of this complex in photoreceptors is strictly dependent on the expression level of R9AP; consequently, the knockouts of either RGS9 or R9AP cause comparable delays in photoresponse recovery. While RGS9 is believed to be present only in rods and cones, R9AP is also expressed in dendritic tips of ON-bipolar cells, which receive synaptic inputs from photoreceptors. Recent studies demonstrated that knockouts of R9AP and its binding partner in ON-bipolar cells, RGS11, cause a small delay in ON-bipolar cell light responses manifested as a delayed onset of electroretinography b-waves. This led the authors to suggest that R9AP and RGS11 participate in regulating the kinetics of light responses in these cells. Here we report the surprising finding that a nearly identical b-wave delay is observed in RGS9 knockout mice. Given the exclusive localization of RGS9 in photoreceptors, this result argues for a presynaptic origin of the b-wave delay in this case and perhaps in the case of the R9AP knockout as well, since R9AP is expressed in both photoreceptors and ON-bipolar cells. We also conducted a detailed analysis of the b-wave rising phase kinetics in both knockout animal types and found that, despite a delayed b-wave onset, the slope of the light response is unaffected or increased, dependent on the light stimulus intensity. This result is inconsistent with a slowdown of response propagation in ON-bipolar cells caused by the R9AP knockout, further arguing against the postsynaptic nature of the delayed b-wave phenotype in RGS9 and R9AP knockout mice

Does varicocele repair improve conventional semen parameters? A meta-analytic study of before-after data

Purpose The purpose of this meta-analysis is to study the impact of varicocele repair in the largest cohort of infertile males with clinical varicocele by including all available studies, with no language restrictions, comparing intra-person conventional semen parameters before and after the repair of varicoceles. Materials and Methods The meta-analysis was performed according to PRISMA-P and MOOSE guidelines. A systematic search was performed in Scopus, PubMed, Cochrane, and Embase databases. Eligible studies were selected according to the PICOS model (Population: infertile male patients with clinical varicocele; Intervention: varicocele repair; Comparison: intra-person before-after varicocele repair; Outcome: conventional semen parameters; Study type: randomized controlled trials [RCTs], observational and case-control studies). Results Out of 1,632 screened abstracts, 351 articles (23 RCTs, 292 observational, and 36 case-control studies) were included in the quantitative analysis. The before-and-after analysis showed significant improvements in all semen parameters after varicocele repair (except sperm vitality); semen volume: standardized mean difference (SMD) 0.203, 95% CI: 0.129–0.278; p<0.001; I2=83.62%, Egger’s p=0.3329; sperm concentration: SMD 1.590, 95% CI: 1.474–1.706; p<0.001; I2=97.86%, Egger’s p<0.0001; total sperm count: SMD 1.824, 95% CI: 1.526–2.121; p<0.001; I2=97.88%, Egger’s p=0.0063; total motile sperm count: SMD 1.643, 95% CI: 1.318–1.968; p<0.001; I2=98.65%, Egger’s p=0.0003; progressive sperm motility: SMD 1.845, 95% CI: 1.537%–2.153%; p<0.001; I2=98.97%, Egger’s p<0.0001; total sperm motility: SMD 1.613, 95% CI 1.467%–1.759%; p<0.001; l2=97.98%, Egger’s p<0.001; sperm morphology: SMD 1.066, 95% CI 0.992%–1.211%; p<0.001; I2=97.87%, Egger’s p=0.1864. Conclusions The current meta-analysis is the largest to date using paired analysis on varicocele patients. In the current meta-analysis, almost all conventional semen parameters improved significantly following varicocele repair in infertile patients with clinical varicocele. Keywords Controlled before-after studies; Infertility, male; Meta-analysis; Varicocel

A summary of fitting parameters for the analysis of the maximal slope of ERG b-wave rising phase as function of flash intensity.

<p>The data were fitted using <i>Equation 3</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#s4" target="_blank">Materials and Methods</a>); <i>I_half</i> is the half-saturating flash intensity, <i>S_max</i> is the asymptotic value of the maximal slope at saturating flash intensities, and <i>n</i> is the Hill coefficient (mean ± SEM).</p

A summary of fitting parameters obtained from the analysis of ERG b-wave and a-wave stimulus-response curves in Figure 3B.

<p>The data were averaged from 13 eyes of <i>R9AP</i>^−/−; 13 eyes of <i>R9AP</i>^+/+; 7 eyes of <i>RGS9</i>^−/−; and 7 eyes of <i>RGS9</i>^+/+ mice and were fitted using <i>Equation 1</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#s4" target="_blank">Materials and Methods</a>); <i>I_0.5,1</i> and <i>I_0.5,2</i> are half-saturating flash intensities of rod- and cone-driven responses, <i>R_max,1</i> and <i>R_max,2</i> are the corresponding maximal response amplitudes. Fitting parameters are given as mean ± SEM.</p

Plots of three parameters characterizing the rising phases of b-waves as functions of flash intensity.

<p>(<b>A</b>) Time of b-wave onset. (<b>B</b>) Time required to reach the maximal slope of the rising b-wave. (<b>C</b>) The value of maximal b-wave slope. The data in (A) and (B) were fitted by Equation 2 (fitting parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#pone-0027573-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#pone-0027573-t003" target="_blank">3</a>, respectively); the data in (C) were fitted to Equation 3 (fitting parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#pone-0027573-t004" target="_blank">Table 4</a>). The statistical significance of the difference between the pairs of mean values obtained from knockout mice and their WT littermates was determined by a paired independent t-test, yielding p-values marked as follows: (*) p<0.05; (**) p<0.01; (***) p<0.001.</p

The effects of R9AP and RGS9 knockouts on the b-wave rising phase.

<p>(<b>A</b>) Examples of averaged b-wave responses from <i>R9AP</i>^−/− and <i>RGS9</i>^−/− (red) and WT (black) mice shown on a shorter time scale than in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#pone-0027573-g003" target="_blank">Figure 3A</a> to illustrate differences in the rising phase kinetics. Traces were filtered to remove oscillatory potentials and the a-wave was additionally subtracted from the 100 cd·s/m² flash responses. (<b>B</b>) The first derivatives of the traces in (A) used to calculate maximal slopes of the b-wave rising phase and times required to reach the maximal slope.</p

Electron microscopy analysis of rod and cone synaptic terminals in WT, <i>RGS9</i>^−/− and <i>R9AP</i>^−/− mice.

<p>Shown are representative electron micrographs of cross-sections through rod spherules (smaller images on the left) and through the base of cone pedicles (right). At least 60 rod spherules and 10 cone pedicles were analyzed for each mouse type. Abbreviations are: SR – synaptic ribbon, HC – horizontal cell process, BC – bipolar cell dendrite. Asterisks indicate flat synaptic contacts at the cone terminals. Scale bars: 200 nm.</p

ERG responses of <i>R9AP</i>^−/− and <i>RGS9</i>^−/− mice.

<p>(<b>A</b>) ERG recordings from <i>R9AP</i>^−/− or <i>RGS9</i>^−/− mice (red) and their corresponding WT littermates (black) were averaged from all recordings evoked by a given flash intensity. Arrows indicate the time when light flash was applied. (<b>B</b>) Stimulus-response curves of b-wave amplitudes (upper panel) and a-wave values measured at 8 ms after the flash, a time point preceding the b-wave onset (lower panel). Data for each knockout mouse are shown in red, data for WT littermates are shown in black. Data points (mean ± SEM) were fitted using Equation 1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#s4" target="_blank">Materials and Methods</a>); fitting parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#pone-0027573-t001" target="_blank">Table 1</a>. The data were averaged from 13 eyes of <i>R9AP</i>^−/−; 13 eyes of <i>R9AP</i>^+/+; 7 eyes of <i>RGS9</i>^−/−; and 7 eyes of <i>RGS9</i>^+/+ mice.</p

A summary of fitting parameters for the analysis of the time required to reach the maximal slope of b-wave rising phase as function of flash intensity.

<p>The data were fitted using <i>Equation 2</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027573#s4" target="_blank">Materials and Methods</a>); <i>y_max</i> is the maximal onset value, <i>n</i> is the Hill coefficient, and <i>k</i> and <i>y₀</i> are fitting parameters (mean ± SEM).</p