The p53 homolog p73 takes hold of the male germ line – a novel function of TAp73 in protecting sperm cell adhesion, migration and maturation within the seminiferous epithelium of the testis

Abstract

The p53 family of transcription factors possesses diverge functions in tumorigenesis and development. The evolutionarily conserved role of the family member p63 to protect the genetic stability of germ cells and influence germ cell development is thought to be the ancestral protein function. Using p53, p63 and p73 knockout (KO) mice, the impact of these transcription factors on the female and male germ line was previously investigated. While mice deficient for p53 and p63 are fertile, loss of either all p73 isoforms or of the transcriptionally active TAp73 isoform alone was shown to cause infertility. Concerning its cause, female TAp73KO mice were reported to be infertile due to impaired ovulation, oocyte spindle defects and abnormal blastocyst development. However, the effect of TAp73 loss in the male germ line is unknown. Here we identify a hitherto unknown function of the transcription factor TAp73 in the development and maintenance of the adult male germ line. Working with total p73KO and isoform-specific KO mice, we find that TAp73, but not ΔNp73 deficiency, leads to a strong loss of developing sperm cells in the testicular tubules of mice 6 weeks and older. Whereas the basal spermatogonia and pachytene spermatocytes of the germ cell epithelium are unaffected (normal cell number and cell proliferation), the numbers of late spermatocytes and spermatids are strongly reduced in global p73- and TAp73-deficient testis. Concomitantly, a higher amount of apoptotic and immature sperm cells accumulate in the lumen of the epididymis of both KO mice, indicating aberrant premature sloughing of sperm cells from the seminiferous epithelium. This finding is reinforced by the observation that the sperm cell nurse cells, the Sertoli cells, display shortened cytoplasmic arms and abnormal vacuolated morphology by electron microscopy. Moreover, the seminiferous epithelium is loosened with loss of tight packaging of sperm cells and strongly disorganized Sertoli-sperm cell junctions. This impaired epithelial structure with aberrant loss of sperm cells appears to be the result of a defective blood testis barrier (BTB), as revealed by in vivo BTB permeability assay and aberrant Sertoli-Sertoli tight junctions. Functional loss of the BTB disturbs the epithelial polarity, the microenvironment of developing sperm cells and their upward migration via attachment-reattachment cycles in the germ cell nursing pockets of Sertoli cells. The molecular explanation for imbalanced junctional restructuring was obtained by quantitative whole genome expression profiling of TAp73 target genes comparing wildtype (WT) versus TAp73KO testes tissue. Loss of TAp73 induces upregulation of adhesion- and migration-related genes including integrins and protease inhibitors like Timp1 and Serpins, known to be involved in disassembly and reassembly of cell-cell junctions in the testis. TAp73 is primarily expressed in the sperm cell fraction and seems to act on Sertoli cells in a paracrine fashion, as revealed by isolated primary Sertoli cell culture, where upregulation of TAp73 target genes declined over time in long-term cultures. In conclusion, we identify for the first time an indispensable role of TAp73 in adult spermatogenesis. Specifically, TAp73 orchestrates a transcriptional program of adhesion and migration-related genes, ensuring the cohesion of the seminiferous epithelium and preventing premature sloughing of sperm. Together, this enables proper germ cell maturation. Conversely, TAp73 loss leads to severe attachment defects of developing sperm within the seminiferous epithelium, explaining the infertility of p73KO and TAp73KO mice.