The cause for hypospermatogenic testes can be changes in germ cell development, Sertoli cell function or germ cell colonization. Defects in germ cell colonization can be excluded
since pre-pubertal KO mice do not show abnormal testis morphology and the testis morphology, shape and localization as such is not compromised (Figure 5.1 A).
p53 family members are known to be involved in maintaining stem ness and proliferation capacity. ΔNp63 for example is necessary to keep the stem cell pool of keratinocytes in squamous epithelia and ΔNp73 is an important survival factor in brain development (Pozniak et al., 2002, Senoo et al., 2007, Tissir et al., 2009). Additionally, ΔNp73 and ΔNp63 are both shown to be overexpressed in several tumor types, promoting cell survival and proliferation (Hibi et al., 2000, Moll et al., 2004, Stiewe et al., 2002a). Furthermore, p73, especially the TAp73 isoform, was recently described to ensure adult neurogenesis by promoting long-term maintenance of neural stem cells in the brain (Fujitani et al., 2010, Talos et al., 2010).
However, in testis development TAp73 seems to be dispensable for maintaining proliferation capacity of basal cells and ensuring a stable pool of spermatogonia, because (TA)p73KO mice show comparable numbers of Ki67 and GCNA1 positive cells as WT mice (Figure 5.5 and 5.6 A and C). Since we also could show that ΔNp73 is dispensable for normal testis development (Figure 5.2 B), p73 isoforms do not appear to influence the testicular stem cell pool. If TAp73 does not influence the number of basal proliferating cells, it might be necessary for sperm differentiation. In contrast to the function of ΔNp63 on epithelial stem ness, the role of TAp63 in skin development is connected to keratinocyte differentiation (Candi et al., 2006). TAp73 is involved in neural differentiation; TAp73KO mice display reduction in number of hippocampal neurons, TAp73 drives differentiation of cortical neurons in vitro and its expression increases during synaptogenesis in the postnatal brain (Agostini et al., 2010, Tomasini et al., 2008, Agostini et al., 2011). Endogenous TAp73 levels increase during retinoic acid-induced differentiation of neuroblastoma cells as well as myeloid leukemic cells (De Laurenzi et al., 2000, Tschan et al., 2000). Likewise, TAp73 might also possess differentiating functions during testis development, ensuring production of mature sperm cells. In contrast to the female germ line, where depletion of TAp73 leads to spindle described to show neural loss in the cortex, hippocampus and thalamic eminescence (Tissir et al., 2009, Tomasini et al., 2008, Yang et al., 2000). The hypothalamus and therefore GnRH expression might also be affected, disturbing the entire GnRH-FSH/LH-testosterone hormone axis (also refer to 2.1.4), and thereby causing the defect in sperm development. Analyses of
mRNA expression levels of all involved hormones of the brain (GnRH, FSH, LH) as well as serum hormone measurement do not reveal differences in (TA)p73KO mice compared to WT littermates, suggesting that TAp73 is acting directly on physiologic testis homeostasis (Figure 5.7).
Maturating sperm cell numbers are decreased in the seminiferous epithelium of TAp73KO testis and in parallel less mature sperm as well as increased apoptotic and immature sperm can be found in the lumen of the epididymis (Figure 5.8). This observation points towards a retention failure of developing sperm cells in the testis. We also describe that the seminiferous epithelium of TAp73KO mice does loose its coordinated structure, since sperm cells are usually arranged in specific layers close to each other according to their developmental stage, which is called the seminiferous cycle (also refer to 2.1.2) (Figure 5.2 A, 5.4 B and 5.11 A). The loss of maturating sperm cells is accompanied by a change in Sertoli cell morphology, Sertoli cells displaying shortened cytoplasmic arms and increased vacuolization (Figure 5.10 and 5.11 B). Taking these observations into account we can hypothesize that TAp73KO Sertoli cells fail to provide physical support for developing sperm cells, which therefore cannot stay in proper contact with the seminiferous epithelium. This might lead to premature detachment of developing sperm from the testicular epithelium.
Some of the immature sperm cells are flushed to the epididymis, but likely the main part will be eliminated by apoptosis. Since Sertoli cells also harbour phagocytic function, this could be the reason why the lost sperm cells are not detectable anymore, but appear to “vanish” from the testis and epididymis (Chemes, 1986). The increased phagocytic activity of TAp73KO Sertoli cells could also explain their change in morphology, which is characterized by increased cytoplasmic vacuolization (Figure 5.11 B). The idea that sperm cells might fail to stay attached to Sertoli cells and the seminiferous epithelium is furthermore reinforced by the fact that the structure of Sertoli sperm junctions at the apical ES is impaired in TAp73KO mice (Figure 5.16). Elongating spermatids are attached to Sertoli cells by the apical ES (also refer to 2.1.3.3). If they have completed their testicular maturation they will be released from the seminiferous epithelium. Restructuring of junctions by Sertoli cells is necessary for spermiation and defects in this process would support premature release of sperm cells.
Premature sloughing of sperm cells is for example described in mice deficient of the enzyme α-mannosidase IIx. The carbohydrate N-glycan adhesion molecules, synthesized by this enzyme, are lost and therefore Sertoli-sperm adhesion is impaired (Akama et al., 2002). It might be possible that TAp73 regulates the homeostasis of adhesion molecules, ensuring attachment of sperm to Sertoli cells. However, it cannot be said if abnormal Sertoli cell
function is the primary cause of the phenotype or a secondary effect due to abnormal sperm development.
Taken together, TAp73 loss leads to hypospermatogenesis with decreased numbers of spermatocytes and spermatids, accompanied by abnormal structure of Sertoli cells. The decrease of developing sperm numbers is explained by a retention failure and premature sloughing of sperm cells.