Identification of programmed cell death 1 and its ligand in the testicular tissue of mice

Abstract

Problem This study aims to determine the expression and localization of programmed cell death 1 (PD‐1) and programmed cell death 1 ligand 1 (PD‐L1) in the testes of mice at different developmental stages. Method of study By means of RT‐qPCR, Western blot and immunofluorescence, the expression and localization of PD‐1 and PD‐L1 were detected in the testicular tissues of mice at different postnatal times: P7, P14, P21, P28, P35, and adulthood. Meanwhile, the level of soluble PD‐L1 (sPD‐L1) was evaluated by ELISA in the testicular interstitial fluid (IF) of the adult mice, culture supernatants of TM4 cell lines (Sertoli cells lines), and primary Sertoli cells at P14. Results Pd‐1 mRNA levels were unexpectedly low. From P7 to P21, there was limited PD‐1 protein detected while PD‐1 was evident at P28 and afterward at significantly higher levels than at P14 and P21 (P < 0.05). Despite being found in the interstitial area at P7, P14, and P21, PD‐1 was also detected in the germ cells of the seminiferous tubules after P28. Pd‐l1 mRNA exhibited age‐related changes, peaking at P21, while PD‐L1 protein was constitutively expressed at any stage, specifically localized in the nucleus of Sertoli cells. Moreover, the level of sPD‐L1 in IF was significantly higher than that in the culture supernatants of both TM4 and primary Sertoli cells at P14. Conclusions PD‐1 and PD‐L1 were present in the testicular tissue of adult mice. The expression and localization of PD‐1 fluctuated with age, and PD‐1 was mainly localized to advanced germ cells, suggesting that it may play a role in spermiogenesis. PD‐L1 was constitutively expressed in the nucleus of Sertoli cells, which could secrete sPD‐L1 into the testicular interstitial space and thus may be involved in testicular immune privilege.

Phenotypic characterization of testicular immune cells expressing immune checkpoint molecules in wild‐type and pituitary adenylate cyclase‐activating polypeptide‐deficient mice

“…Gal‐9 expression was found to be the highest regarding testicular CD4+ T cells in both groups, but none of the investigated immune cells showed a significant difference between PACAP‐deficient and wild‐type mice. Under physiological situations, peritubular cells, spermatocytes, and spermatids in seminiferous tubules of the testis constitutively express or secrete PD‐L1 . PD‐L1 is inducibly expressed by testicular Sertoli cells .…”

“…Under physiological situations, peritubular cells, spermatocytes, and spermatids in seminiferous tubules of the testis constitutively express or secrete PD‐L1 . PD‐L1 is inducibly expressed by testicular Sertoli cells . On the other hand, the testis does not constitutively express PD‐L2 .…”

“…On the other hand, the testis does not constitutively express PD‐L2 . PD‐L1 binding to the PD‐1 receptor expressed by activated CD8+ T cells leads to T‐cell apoptosis, inhibition of proliferation, and cytokine secretion, which represent immune suppression and might play a role in the maintenance of testicular immune privilege . The decreased PD‐1 expression by CD8+ T cells in PACAP‐deficient mice suggests an impaired PD‐1/PD‐L1 pathway where immune cells might shift from immune suppression toward immune activation leading to disturbed testicular immunoregulation.…”

“…The mammalian testis also represents an immune‐privileged organ maintained through the coordination of systemic immune tolerance, the local physical structure, and active local immunosuppression. The local immune modulatory milieu has been intensively investigated, but the mechanisms underlying immune privilege are still not well understood . The blood‐testis barrier (BTB) is much more complex than other blood‐tissue barriers, and its function relies on the complex interaction between the anatomical, physiological, and immunological barriers .…”

“…The blood‐testis barrier (BTB) is much more complex than other blood‐tissue barriers, and its function relies on the complex interaction between the anatomical, physiological, and immunological barriers . Besides the anatomical BTB, testicular somatic cells, including Sertoli cells, peritubular cells, and Leydig cells together with the local active immunosuppression, play vital roles in maintaining the immune‐privileged environment . The testis has most types of immune cells found in interstitial spaces, including macrophages, α/β and γ/δ T lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DCs), and mast cells .…”