Intratesticular creatine maintains spermatogenesis by defining tight junctions

Abstract

One in five couples who wish to conceive is infertile, and half of these couples have male infertility. However, the causes of male infertility are still largely unknown. Creatine is stored in the body as an energy buffer, and the testes are its second-largest reservoir after muscles. Further, even though intratesticular creatine levels have long been known to decrease in male patients with infertility, its role in the testis is unknown. We investigated the intratesticular role of creatine, specifically in the context of the creatine synthesizing enzyme Gamt, and the creatine transporter Slc6a8. The Slc6a8 knockout mice showed no abnormalities in spermatogenesis. While Gamt knockout mice formed spermatozoa, they demonstrated reduced sperm count and decreased sperm motility and fertilization rate. Additionally, intratesticular creatine in Gamt knockout mice was significantly decreased, resulting in the disruption of tight junctions, which could be rectified by creatine supplementation, as was evidenced by the improved sperm count and fertilization rate in these mice. In conclusion, we identified creatine as being required for the maintenance of the tight junction in the testis.

Keywords: Gamt; Creatine; Male infertility; Testis-blood-barrier.

Fig. 1

Evaluation of spermatogenesis in Gamt knockout and Slc6a8 knockout mice. (a) Sperm count, (b) Sperm motility, (c) Testis/ body weight, and (d) Body weight in Gamt knockout and hetero mice (n = 5 per group). (e) Sperm count, (f) Sperm motility, (g) Testis/ body weight, and (h) Body weight in Slc6a8 knockout and control mice (n = 5 per group). (i) Creatine content of Gamt knockout, hetero, and Slc6a8 knockout mice (n = 3 per group). (j) Hematoxylin and eosin staining of the testes in Gamt knockout, hetero, and Slc6a8 knockout mice. All data are expressed as mean ± SD. Two-tailed Student’s t-test for experiments with two groups and the Tukey Kramer method for experiments including ≥ 3 groups were used for analysis as appropriate.

Fig. 2

Evaluation of tight junction by Gamt knockdown in 15P-1 cell. (a) Trans-epithelial electrical resistance (TEER) of siControl and siGamt. Each data point is a mean ± SD from n = 3 experiments. Student’s t-test was used for analysis as appropriate when compared to the corresponding control. (b) Representative immunoblots of GAMT and ACTB of the 15P-1 cell (siControl and siGamt).

Fig. 3

Effect of Creatine diet on Gamt knockout mice. (a) Sperm motility, (b) Sperm count, (c) Testis/ body weight, and (d) Body weight of Gamt knockout mice on normal and creatine diets (n = 5 per group). (e) Creatine content of Gamt knockout on normal diet and Cr diet (n = 3 per group). (f) Fertilization rate of Gamt hetero mice and Gamt knockout mice on normal and creatine diet (n = 3 per group). (g) Hematoxylin and eosin staining of the testes of Gamt knockout on normal diet and creatine diet (h). (i) Representative immunofluorescence images of α-tubulin and Zo-1 staining in the testes of mice in the three groups. White arrow shows abnormal distribution. Data are expressed as the mean ± SD. Two-tailed Student’s t-test for experiments with two groups and the Tukey Kramer method for experiments including ≥ 3 groups were used for analysis as appropriate.

Fig. 4

Graphical abstract. Creatine is required for the maintenance of tight junctions in the testes and creatine deficiency can be corrected to restore this function.

Fig. 5

Generation of Gamt knockout and Slc6a8 knockout mice. (a) Gene map of Gamt. Black and white boxes indicate coding and non-coding regions, respectively. Red arrowheads indicate primers for guide RNAs (gRNAs) for genome editing. (b) Representative immunoblots of GAMT and ACTB from the testes of Gamt knockout mice. (c) Gene map of Slc6a8. Black and white boxes indicate coding and non-coding regions, respectively. Yellow arrowheads indicate loxP. (d) Representative immunoblots of SLC6A8 and ACTB of the testes from Slc6a8 KO mice.