Hypoxanthine phosphoribosyltransferase (HPRT)‐deficiency is associated with impaired fertility in the female rat

The purine hypoxanthine plays important role in regulating oocyte maturation and early embryonic development. The enzyme hypoxanthine phosphoribosyltransferase (HPRT) recycles hypoxanthine to generate substrates for nucleotide synthesis and key metabolites, and here we show that HPRT deficiency in the rat disrupts early embryonic development and causes infertility in females.

Purine metabolites play critical roles in regulating early embryonic development in mammals. The levels of cyclic AMP, cyclic GMP and hypoxanthine regulate meiotic arrest of mouse oocytes in vivo, whilst elevated levels of hypoxanthine, adenine, or inosine can disrupt the first cleavage stages during embryonic development in vitro (Dienhart & Downs, 1996;Wigglesworth et al., 2013). The enzyme hypoxanthine phosphoribosyltransferase (HPRT) is an essential component of the purine salvage pathway, involved in recycling hypoxanthine and guanine to provide substrates for the synthesis of nucleic acids and key metabolites including second messengers. The HPRT gene is located on the X chromosome and when mutated in humans causes the debilitating neurological disorder Lesch-Nyhan disease in males (Lesch & Nyhan, 1964). Here, we report that absence of HPRT disrupts early embryonic development leading to impaired fertility in female Hprt knock-out (KO) rats.
We previously described the generation of Hprt KO rats using targeted rat DA embryonic stem cells (Meek et al., 2016). The Hprt  et al., 2020). In contrast, Hprt KO female rats mated with wild-type males produced many fragmented embryos and <50% expanded blastocysts at day E4.5, which corresponded with reduced litter sizes at term (Figures 1b and 1e). Interestingly, male pups were represented in these litters, albeit at slightly reduced numbers, indicating that "rescue" did not rely on the contribution of an intact Hprt allele from X-chromosome-bearing sperm (Figure 1a). In matings with wild-type males from a transgenic line carrying a Rex1-EGFP knock-in reporter gene that is first expressed at the 4-8-cell stage (Meek et al., 2020), Rex1-EGFP fluorescence was evident in almost all fragmented embryos (Figure 1d,e). This confirmed that fertilization of most HPRT-deficient oocytes had taken place, and zygotic gene activation had begun in the majority of the degenerating embryos.

The failure to recover intact blastocysts from crosses between
Hprt KO rats demonstrated that HPRT activity is essential for proper progression through the initial cleavages of early embryonic Normal embryo development of some KO oocytes after fertilization with wild-type sperm suggests that Hprt-derived products carried by either sperm or the seminal fluid can rescue the HPRT-deficient embryos. The retention of cytoplasmic bridges between developing spermatids allows equilibration of mRNA, protein, and metabolites between maturing sperm, making post-meiotic spermatids phenotypically equivalent (Braun, Behringer, Peschon, Brinster, & Palmiter, 1989). In this way, Hprt-derived RNA, protein, or HPRT-dependent purine metabolites provided by any sperm could rescue HPRT-deficient embryos. Alternatively, metabolites or factors in the seminal fluid or in sperm-associated extracellular microsomes could be provided in trans by Hprt-expressing somatic support cells of wild-type males.
Rescue by wild-type sperm was incomplete pointing to a sensitized state in the HPRT-deficient embryos, either due to variation in the sensitivity of HPRT-deficient embryos or the penetrance of rescue factors. The nature of the early HPRT-deficient sensitized state and the identity of the rescue factor(s) requires further investigation.
However, based on previous experiments in mouse embryos (Dienhart & Downs, 1996;Wigglesworth et al., 2013), it is tempting to speculate that imbalances in the levels of purines or derivative metabolites contribute to a sensitized state that compromises early embryonic development in the rat. HPRT-deficient rat embryos may, therefore, provide a new in vivo experimental system in which to investigate how purine metabolism and the uterine environment influence early embryo development in mammals.