CRISPR/Cas9-mediated knockout of Mct8 reveals a functional involvement of Mct8 in testis and sperm development in a rat

Abstract

Thyroid hormone (TH) has long been believed to play a minor role in male reproduction. However, evidences from experimental model of thyrotoxicosis or hypothyroidism suggests its role in spermatogenesis. Cellular action of TH requires membrane transport via specific transporters such as monocarboxylate transporter 8 (MCT8). SLC16A2 (encodes for MCT8) inactivating mutation in humans can lead to Allan-Herndon Dudley-syndrome, a X-linked psychomotor and growth retardation. These patients present cryptorchidism which suggests a role of MCT8 during spermatogenesis. In this study, we found that Mct8 is highly expressed during early postnatal development and decreases its expression in the adulthood of testis of wild-type male rats. Histological analysis revealed that spermatogonia largely lacks MCT8 expression while spermatocytes and maturing spermatids highly express MCT8. To further understand the role of Mct8 during spermatogenesis, we generated Slc16a2 (encodes MCT8) knockout rats using CRISPR/Cas9. Serum THs (T3 and T4) level were significantly altered in Slc16a2 knockout rats when compared to wild-type littermates during early to late postnatal development. Unlike Slc16a2 knockout mice, Slc16a2 knockout rats showed growth delay during early to late postnatal development. In adult Slc16a2 knockout rats, we observed reduced sperm motility and viability. Collectively, our data unveil a functional involvement of MCT8 in spermatogenesis, underscoring the importance of TH signaling and action during spermatogenesis.

ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction

“…The study of ZP4 was not possible until now in animal models because, while the KO technology was well-developed in the house mouse ( Mus musculus ), which has a pseudogenized Zp4 , the technique was very difficult to perform in rat, hamster and rabbit. However, development of CRISPR-cas9 technology made it possible to develop the KO technique in species that possess ZP4 (Fan et al, 2014 ; Bae et al, 2020 ). In fact, we have recently reported the phenotype of the female rabbit without the ZP4 gene (Lamas-Toranzo et al, 2019 ).…”

Recent Advances in the Production of Genome-Edited Rats

“…They named this technology “Technique for Animal Knockout system by Electroporation (TAKE)”. Since then, many GE rats have been produced using EP-based GE technology [ 54 , 90 , 91 , 95 , 111 , 117 , 121 , 125 , 131 , 140 , 141 , 144 ]. Notably, in vitro EP can also be applied to frozen mouse and rat zygotes for the efficient introduction of CRISPR components, without affecting the embryo viability or development [ 173 , 174 ].…”

“…If the RNP enters the nuclei of cells, GE should commence quickly, which will, in turn, lead to increased GE efficiency. Using this RNP-based GE system, a number of GE rats have been produced to date [ 57 , 58 , 68 , 90 , 91 , 95 , 111 , 113 , 115 , 117 , 121 , 126 , 131 , 142 , 150 ].…”

Involvement of TauT/SLC6A6 in Taurine Transport at the Blood–Testis Barrier

“…These pieces of evidence indicate the relevance of taurine to the healthy maintenance and pathogeneses of sperm, suggesting the importance of regulating the concentration of such antioxidants in the seminiferous tubules, where Sertoli cells form the blood-testis barrier (BTB) to separate germ cells from the circulating blood [19,20]. At the BTB, Sertoli cells are assumed to form tight junctions to suppress non-specific transport via the paracellular route [21] and have been reported to express various transporter molecules, such as glucose transporters (GLUTs/SLC2As), L-type amino acid transporter 1 (LAT1/SLC7A5), monocarboxylate transporter 8 (MCT8/SLC16A2), sodium-dependent vitamin C transporters (SVCTs/SLC23As), concentrative nucleoside transporters (SLC28As), equilibrative nucleoside transporters (SLC29As), P-glycoprotein (P-gp/MDR1/ABCB1), and multidrug resistance-associated protein 1 (MRP1/ABCC1), involved in the transport of nutrients, metabolites, and xenobiotics at the BTB [22][23][24][25][26][27], suggesting the involvement of certain transporters in regulating taurine concentration in the seminiferous tubules.…”