5.2.1 Basal spermatogonia of p73KO mice retain mitotic ability
To enable constant production of meiotic sperm cells, a certain pool of basal spermatogonia has to go through asymmetric mitotic divisions. This process ensures creation of two types of spermatogonia. Type A spermatogonia will stay in the proliferation status, intermediate spermatogonia will differentiate into type B spermatogonia, which will progress into meiosis to produce haploid sperm cells (Borg et al., 2010, Hermo et al., 2010). We therefore wanted to check whether the decrease of post-meiotic sperm cells in p73KO testis could be due to spermatogonia, losing their ability to proliferate. To gain information about the amount of mitotic cells within p73KO testis we applied IHC to testis sections, using the proliferation marker Ki67 (Gerdes et al., 1984). Comparing KO with WT mice, we could not find any differences in the proliferation ability of basal cells (Figure 5.6 A). Quantitation of Ki67 positive tubules per testis section did show comparable levels of proliferating tubules for p73KO, heterozygous (Het) and WT mice. This observation was independent of age (Figure 5.6 C). The loss of sperm cells within the seminiferous epithelium of p73KO mice cannot be explained by a reduction or loss of the basal mitotic rate.
5.2.2 The meiotic rate is not changed in p73KO testis
Haploid sperm cells derive from diploid spermatogonia by going through meiosis. To permanently produce mature sperm cells for reproduction, constantly a number of basal spermatogonia have to pass through two meiotic divisions (Borg et al., 2010, Hermo et al., 2010). Loss of sperm cells in p73KO mice could be a result of a decreased meiotic rate within the seminiferous epithelium. To test this hypothesis, testis sections of p73KO and WT mice were stained for phosphorylated Histone 3 (H3-phosphoSer10) performing IF. H3 is only phosphorylated at Serine 10 during chromosome condensation, which occurs during mitosis as well as meiosis (Hendzel et al., 1997, Wei et al., 1999). H3Ser10 positive cells within the seminiferous epithelium, excluding basal mitotic cells (also refer to 5.2.1), can therefore be counted as meiotic spermatocytes. Comparing p73KO and WT mice we could observe no difference in H3Ser10 staining pattern of positively stained tubules (Figure 5.6 B).
Quantitation of H3Ser10 positive cells per testis section revealed comparable numbers for p73KO, Het and WT mice, independent of age (Figure 5.6 D). Therefore the meiotic rate seems to be normal in p73KO testis.
WT
p73KO
Ki67
6 weeks
n.s. n.s. n.s.
A
B
WT
p73KO
H3Ser10 merge DAPI
10 weeks
C D
n.s.
n.s.
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5.2.3 No impairment of the hormonal axis of p73 and TAp73KO mice
Germ cell development is tightly regulated by a complex hierarchic hormone system.
Gonadotropin releasing hormone (GnRH) is produced in the hypothalamic neurons and its secretion will activate the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland. Both gonadotropins will travel through the blood stream and stimulate testicular cells (also refer to 2.1.4). FSH is influencing spermatogenesis by activating Sertoli cells, while LH is binding to its receptor on intertubular Leydig cells, which will subsequently produce testosterone. Testosterone itself is also important for activation of spermatogenesis (Borg et al., 2010). As already described, p73KO mice are known to show severe neural defects in the brain (refer to 2.2.3). To exclude a secondary effect for the observed testicular phenotype by deregulation of the hormonal axis starting in the brain, we wanted to measure mRNA expression and serum levels of these hormones.
Quantitation of the expression levels of GnRH mRNA, isolated from brain, by performing qPCR revealed no difference for WT and p73KO mice (Figure 5.7 A). The same was true for FSH and LH mRNA, isolated from the pituitary gland of TAp73KO, Het and WT mice (Figure 5.7 B). To affirm physiological levels of the secreted hormones in KO mice, serum levels of FSH and LH were determined by antigen-specific ELISA assays. Results for FSH as well as LH showed comparable levels of both hormones in the blood stream of TAp73KO and WT mice (Figure 5.7 C and D). Additionally, serum testosterone levels of p73KO, TAp73KO and WT mice were determined by ELISA. Independent of the genotype the measurements displayed high variance for the data points of each group, but a similar distribution comparing WT and KO mice. No significant difference in testosterone levels could be observed (Figure Fig. 5.6 Loss of sperm cells in KO mice is not a result of decreased proliferation or impaired meiosis.
A) Immunohistochemistry staining of testis sections from adult p73KO and WT mice. Antibody against Ki67 (mitotic cells) was applied to sections. Basal proliferation is also observed in p73KO mice.
Magnification: 400x
B) Immunofluorescence staining of testis sections from adult p73KO and WT mice. Antibody directed against phosphorylated H3 (mitotic and meiotic cells) was applied to sections. Meiosis is not impaired in p73KO mice. Magnification: 400x
C) Quantitation of proliferating cells in p73KO versus WT and Het mice of different age. No significant difference in number of Ki67 positive tubules could be detected. n=2-5 mice were analyzed per
5.7 E and F). In sum, the described hormonal axis important for germ cell development seems to function normally in p73 and TAp73KO mice.
A B
C D
E F
Fig. 5.7 The hormonal axis is not affected in p73 and TAp73KO mice.
A+B) Quantitation of mRNA isolated from brain (GnRH, n=3) or pituitary gland (FSH and LH, n=5-10) of adult KO and WT mice, performing qPCR. Expression levels of all three hormones are physiologically normal in p73 and TAp73KO mice. GOI = gene of interest
C+D) Measurement of LH and FSH levels applying ELISA assay. n=4-5 adult mice were analyzed per genotype. In KO mice serum levels of both hormones are comparable to WT.
E+F) Measurement of testosterone levels via ELISA assay. No change in testosterone serum levels can be observed for adult p73WT/Het and TAp73 KO mice. The indicated numbers of mice were analyzed per genotype. To all quantitations the student´s t-test was applied. p > 0.05