Figure 6. ANN assessment of quadrilateral CatSper nanodomains and Δ fluorescent intensity in sperm population along the cleared female tract immunolabeled for CatSper1 conforms to findings by other approaches used in this study.

(A) 3D perspective schematic views of quadrilateral CatSper nanodomains. A cross-section (left). A side view (right). (B) A schematic diagram describing the image processing procedure. (C) An illustration of generating a heatmap from the pre-processed micrographs. (D) 20 processed micrographs of the CatSper1 signal from the sperm cluster from middle isthmus of the cleared oviduct. (E) Processed micrographs (top) and their corresponding heatmaps (bottom) from 20 spermatozoa from the oviduct close to UTJ (left), middle isthmus (middle), and ampulla (right). (F) An example of fluorescent intensity analysis of processed images showing the four peaks corresponding to four CatSper1 quadrilateral domains and calculated averaged Δ value (transversal red line). (G) Analysis of the fluorescent intensity differences (Δ values; red area in panel F) among three sperm populations from ampulla, middle isthmus, and isthmus close to UTJ. Graph represents Mean ± SEM. with individual measurements are plotted as dots (N = 60). (H) A topological heatmap showing the integrity of the quadrilateral CatSper domain organization represented by Δ values of the fluorescent intensity along the morphometrical space of the cleared oviduct (left, N_sperm = 152) with the corresponding inferential statistical analysis of the differences of the signal intensities (Δ values, right) among four sperm populations (Amp – Ampulla, DI – Distal Isthmus close to ampulla, MI – middle isthmus, UTJ – utero-tubal junction). Bars denote Mean ± SEM. The baseline indicates homogenous angular distribution of the CatSper fluorescent signal with no quadrilateral distributions. Statistical significance was calculated using KW-ANOVA, (***p<0.001), N_animals = 4. See also Figure 6—source datas 1 and 2.

Figure 6—figure supplement 1. Analysis of the transversal quadrilateral CatSper1 domains organization in the tail of sperm inside the cleared female reproductive tract.

(A) Demonstration of the individual round analytical areas within the transversal projection of the CatSper1 tail signal intensity heatmap. Individual circles indicate the ROI areas of the signal intensity integration and calculation (N_showed = 8; N_total = 80) with the starting point in the ROI with the highest intensity (top right corner). (B) Comparison of the transversal quadrilateral CatSper1 domains organization quality among sperm populations from three regions of the cleared oviduct. Solid lines represent Mean and dotted lines denote ± SEM. N = 60. See also Figure 6—source data 2.