Prps1l1, a Testis-specific Gene, Is Dispensable for Mouse Spermatogenesis

Phosphoribosyl pyrophosphate synthetase (PRPS) plays an important role in nucleotide biosynthesis by transferring pyrophosphate groups from adenosine triphosphate (ATP) to ribose-5-phophate to form 5-phosphoribosyl-1-pyrophosphate (PRPP)(Cunningham et al. 2014; de Brouwer et al. 2010). The PRPS family contains three members encoded by Prps1, Prps2 and Prps1l1 (de Brouwer et al. 2010; Taira et al. 1989). Prps1 and Prps2 are X-linked genes expressed in a wide variety of organs/tissues, whereas Prps1l1 on chromosome 12 is exclusively expressed in the testis (Taira et al. 1989). Despite its testis-specific expression, the function of Prps1l1 remains unknown.

To explore the physiological role of Prps1l1, we examined its expression profile and generated a global Prps1l1 knockout (KO) mouse line. Prps1l1 and Prps1, but not Prps2, were abundantly expressed in the testis, and unlike Prps1 which was also expressed in other organs, Prps1l1 expression appeared to be restricted to the testis (Fig. 1A). The onset of Prps1l1 mRNA expression was at postnatal day 21 (P21) and its levels remained high thereafter (Fig. 1B), suggesting that Prps1l1 is mainly expressed in haploid male germ cells, i.e., round and elongating/elongated spermatids, in adult murine testes. In contrast, levels of Prps1 mRNAs were lower prior to P7 and became much higher thereafter (Fig. 1B), suggesting that it is expressed in all types of spermatogenic cells in adult murine testes. The cellular origin of Prps1 and Prps1l1 mRNA expression was further confirmed by qPCR analyses using pachytene spermatocytes, round and elongating spermatids purified from the adult murine testes (Fig. 1C). Next, we generated Prps1l1 global knockout (KO) mice using the CRISPR/Cas9-based genome editing technology. A PCR-based genotyping method was developed such that presence of external and absence of internal PCR products would indicate homozygous Prps1l1 KO (Fig. 1D and Supplemental Table S1). In addition to genotyping assays, the complete lack of Prps1l1 mRNAs in the testes of Prps1l1 KO males was further confirmed using qPCR analyses (Fig. 1E).

Figure 1.

Prps1l1 is dispensable for spermatogenesis despite its high expression in the testis. A. qPCR analyses of mRNA expression levels of three PRPS genes (Prps1, Prps2 and Prps1l1) in ten organs of adult mice. Expression levels were normalized to Gapdh. Data are presented as means±SEM, n=3. B. qPCR analyses of mRNA expression levels of three PRPS genes (Prps1, Prps2 and Prps1l1) in developing murine testes at postnatal day 0 (P0), P7, P14, P21, P28, P35 and adulthood. Expression levels were normalized to Gapdh. Data are presented as means±SEM, n=3. C. qPCR analyses of Prps1l1 mRNA levels in pachytene spermatocytes, round and elongating spermatids purified from WT adult murine testes. Expression levels were normalized to Gapdh. Data are presented as means±SEM, n=3. D. Schematic illustration of the generation of Prps1l1 KO mouse and a representative genotyping result of Prps1l1 heterozygous (+/−), homozygous (−/−) and wild type (+/+). M, marker. The red lightning bolt represents gRNAs used, and its right and left orientations indicate the gRNAs targeting the reverse and forward strands of the genomic DNA, respectively. Blue arrows show the position of external primers, while light green arrows indicate that of internal primers. The expected size of PCR products is indicated in the same color. E. qPCR analyses of mRNA expression levels of the three PRPS genes (Prps1, Prps2 and Prps1l1) and two related genes (Ppat and Ahcy) in Prps1l1 KO testes. Expression levels were normalized to Gapdh. Data are presented as means±SEM, n=3, ***p<0.001. F. Normal morphology of WT and Prps1l1 KO testes. One unit on the ruler is 1mm. G. A representative image of Haematoxylin and Eosin (HE)-stained WT and Prps1l1 KO testes section. Scale bar=50 µm. H. A representative phase-contrast micrograph showing normal morphology of WT and Prps1l1 KO sperm. Scale bar=100 µm. I. Sperm motility and concentration assays on adult Prps1l1 KO males.

A 3-month-long fertility test showed that Prps1l1 KO males had normal fertility (data not shown). Testicular weight and histology of adult Prps1l1 KO males were comparable to those of WT males (Fig. 1F and Fig. 1G). Prps1l1 KO males also displayed normal sperm counts and motility, as well as sperm morphology (Fig.1H and Fig. 1I). Levels of Prps1 and Prps2 mRNAs were slightly, although not significantly, increased in Prps1l1 KO compared to WT testes (Fig. 1E). No significant upregulation of either Ppat or Ahcy, two genes known to encode proteins that can bypass PRPP (de Brouwer et al. 2010), was observed either (Fig. 1E). Taken together, our data suggest that, despite its testis-exclusive expression, Prps1l1 is dispensable for spermatogenesis in mice.