Figure 3. Expression of PNLDC1.

Panel A shows the testicular cell types that express PNLDC1, as determined by interrogation of single-cell RNA sequencing on adult testis.³¹ In the horizontal box plots, the vertical solid lines are the medians and the left and right sides of the boxes are the interquartile ranges of the normalized read counts for each cell type. The I bars indicate the minimum and maximum counts, and when the third quartile is zero, the mean is indicated with a dotted line. Each cell type identified by the analysis³¹ is marked with a different color, and the number in brackets indicates sequential developmental stages of a given cell type. Panel B shows an in situ hybridization analysis performed with probes targeting PNLDC1 in a testicular-biopsy sample from a control with complete spermatogenesis (top) and a testicular-biopsy sample from Patient 4 (bottom). PNLDC1 shows high expression in pachytene spermatocytes (each red dot represents a single transcript) in the control but is nearly absent in Patient 4. The bars on the left images represent 50 μm, and on the right images 10 μm. Panel C shows a reverse-transcriptase–quantitative-polymerase-chain-reaction (RT-qPCR) analysis performed with primers targeting PNLDC1 on RNA isolated from fixed testis samples with complete spermatogenesis from controls (Table S3 in the Supplementary Appendix) and testicular RNA isolated from Patients 1, 3, and 4 (RNA was not available from Patient 2). The analysis was performed in duplicate. An RT-qPCR analysis of the housekeeping gene RPS20 showed nonsignificant differences in expression between the patients and controls, a finding that indicates that the RNA quality was not compromised. Panel D shows an RT-qPCR analysis performed with primers targeting PNLDC1 on three samples from controls with complete spermatogenesis, two samples of tissue devoid of germ cells (Sertoli cell–only pattern [SCO]), an ovary tissue sample, and different somatic tissue samples (liver, skin, and ductus deferens); the findings indicate that PNLDC1 expression is specific to germ cells. In Panels C and D, the data are presented as relative to the controls, as indicated by the dashed line. Each blue dot represents a measurement (performed in duplicate per sample), and the vertical bars indicate the 95% confidence intervals, which were calculated by means of bootstrapping. NS denotes not significant.