Taurine and its transporter TAUT positively affect male reproduction and early embryo development

Abstract

Study question: Are taurine and its transporter TAUT associated with spermiogenesis and early embryo development?

Summary answer: Morphologically abnormal spermatozoa increased after local functional interference by intratesticular injection, and taurine depletion significantly reduced the normal embryo numbers in vivo and blastocyst formation rate in vitro.

What is known already: Taurine is one of the most abundant amino acids in the male reproductive system and it has been demonstrated that taurine can efficiently improve spermatogenic function in rat models of testicular injury. However, limited information is known about the role of taurine and its transporter TAUT in spermatogenesis and early embryo development.

Study design, size, duration: Clinical characteristics from 110 couples who have experienced recurrent pregnancy loss (RPL) were collected from December 2014 to March 2018. According to whether a fetal heartbeat was seen in the previous pregnancy under ultrasonic monitoring, patients with RPL were divided into two groups: an RPL without heartbeat (pregnancy with no fetal heartbeat, ROH) group, and an RPL with heartbeat (one or more pregnancies with fetal heartbeat, RWH) group. Semen samples (21 ROH and 20 RWH) were finally used for metabolomic analysis. Furthermore, semen samples were obtained from 30 patients with teratozoospermia (normal sperm morphology <4%) seeking evaluation for infertility and 25 age-matched control subjects with normal semen quality for western blotting. Animal experiments were performed in CD-1/ICR mice.

Participants/materials, setting, methods: Metabolomics was performed to determine the metabolic changes between the ROH and RWH groups. Sperm proteins from patients with teratozoospermia and healthy controls were extracted for detecting TAUT expression using western blot analysis. Immunofluorescence was used to characterize the localization of TAUT in the testis and ejaculated spermatozoa. Functional analysis in mice was performed by intratesticular injection of siRNAs or antagonist (β-alanine) and 5% β-alanine was provided in drinking water to 3-week-old male mice for 5 weeks with the aim of depleting taurine. Murine epididymal spermatozoa were stained with hematoxylin and eosin for morphological assessment. IVF and mating tests were performed in mice for assessing fertility.

Main results and the role of chance: Metabolomic analysis demonstrated that the taurine content was lower in spermatozoa but higher in seminal plasma from the ROH than the RWH group. TAUT expression was lower in spermatozoa from patients with teratozoospermia than controls. Immunofluorescence showed that TAUT was localized to the manchette in mouse elongated spermatids functional analysis showed that morphologically abnormal spermatozoa increased after interference, and this defect increased after supplementation with 5% β-alanine but was improved by 5% taurine supplementation. Supplementation with 5% β-alanine significantly reduced the normal embryo number in the mouse uterus as well as blastocyst formation rate in vitro.

Large scale data: N/A.

Limitations, reasons for caution: The sample size was low and larger cohorts are needed to confirm the positive effect of taurine on human sperm quality. A comprehensive safety examination should be performed to evaluate whether taurine is a possible treatment for teratozoospermia. Furthermore, the specific molecular mechanism of TAUT involvement in spermiogenesis remains to be clarified.

Wider implications of the findings: The study provides new insights into the role of taurine and its transporter TAUT in male reproduction and embryo development. The results also indicate that TAUT is a promising molecular candidate for the assessment of sperm quality, which may contribute to the diagnosis and treatment for teratozoospermia.

Study funding/competing interest(s): This work was supported by grants from the National Natural Science Foundation of China (no. 81774075, 31900605, 81971451), Jiangsu Science and Technology Program Grant (BK20190654) and Maternal and child health scientific research of Jiangsu Province (F202121). The authors declare no competing financial interests.

Keywords: embryo development; recurrent pregnancy loss; spermiogenesis; taurine; taurine transporter; teratozoospermia.

Figure 1.

Analysis of semen parameters in male partners of women experiencing recurrent pregnancy loss without a fetal heartbeat. Receiver operating characteristic (ROC) curve analyses for (A) abnormal sperm morphology, (B) total sperm motility, (C) progressive sperm motility, (D) sperm concentration, (E) sperm count and (F) sperm volume. AUC values are presented for each parameter.

Figure 2.

Summary of altered metabolic pathway analysis in spermatozoa and seminal plasma of patients with recurrent pregnancy loss. (A) Receiver operating characteristic (ROC) curve for taurine content of spermatozoa in ROH patients (recurrent pregnancy loss without heartbeat, i.e. pregnancy with no fetal heartbeat). (B) ROC curve for taurine content of seminal plasma in ROH patients. (C) Metabolic pathways in spermatozoa. (D) Metabolic pathways in seminal plasma.

Figure 3.

Levels of TAUT as analyzed by western blotting. (A) Detection of TAUT (taurine transporter) in mouse and human spermatozoa. (B) Representative results of western blotting analysis of TAUT protein in spermatozoa from teratozoospermic men and healthy donors. GAPDH was used as a loading control. (C) Levels of TAUT protein in controls and men with teratozoospermia. (D) Expression of TAUT in different tissues and cells from human and mouse. No negative control was used because of the wide expression of TAUT protein. All data are expressed as mean ± SD; ***P < 0.001 versus normal group by Student’s t-test.

Figure 4.

Levels of TAUT in mouse testes at different developmental stages and immunofluorescent staining of TAUT in mouse spermatogenic cells and mature spermatozoa. (A) Western blot analysis of TAUT (taurine transporter) protein in mouse testes at different developmental stages (w: weeks). No negative control was used because of the wide expression of TAUT protein. (B) Quantification of the level of TAUT protein using GAPDH as a loading control. #The time of siRNA or antagonist intratesticular injection. (C) Double immunofluorescent staining of TAUT (red) and PNA (green) in mature spermatozoa. TAUT was localized in the axoneme of the sperm tail and acrosome, colocalized with PNA in the acrosome. Scale bar, 5 μm. (D, E) Distribution of TAUT (red) in spermatogenic cells at different developmental stages. Scale bar, 10 μm. All data are expressed as mean ± SD of three independent experiments. All nuclei were stained with DAPI (blue).

Figure 5.

Co-localization of TAUT and α-tubulin in different stages of mouse elongated spermatids. Signals for TAUT (taurine transporter: red) coincided with α-tubulin (green) in step 8-16 elongated spermatids. The nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Figure 6.

Effect on mouse sperm morphology of interference with taurine transport in vivo by intratesticular injection. (A) Diagram of the intratesticular injection. Approximately 70% of the seminiferous tubules in 3-week-old mouse testes were injected with siRNAs or antagonist (β-alanine) mixed with indicator (blue). (B, C) Inhibition efficiency of three Taut (taurine transporter) siRNAs on GC-2 cell line. (D, E) Inhibition efficiency of two selected Taut siRNAs on testicular TAUT protein. (F) Effect of Taut siRNA injection and taurine depletion on sperm morphology. (G) Effect of antagonist injection and taurine depletion on sperm morphology. All experiments were performed in triplicate in individual mice. (H) Shapes of spermatozoa from the cauda epididymidis of mice treated with Taut siRNA 1#. Scale bars, 10 μm. One-way ANOVA followed by Bonferroni’s post hoc comparisons tests and Brown-Forsythe and Welch ANOVA with Tamhane’s T2 post hoc multiple comparison tests were performed for statistical analyses. Non-parametric data were analyzed with the Kruskal–Wallis test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7.

Reproductive abnormalities in mice treated with 5% β-alanine. (A) Quantification of fertilization and blastocysts in IVF assays of the siRNA scheme. (B) Mating tests of male mice in four groups from the siRNA scheme with normal adult female mice. (C) Representative images of embryonic Day 11.5 embryos of the four groups. (D) Representative images of appearance of viable and resorbed embryos. ▲, viable embryos in the uterus; ◆, resorbed embryos in the uterus. (E) Quantification of fertilization and blastocysts in IVF assays of the antagonist scheme. (F) Mating tests of male mice in four groups from the antagonist scheme with normal adult female mice. All experiments were performed in triplicate in individual mice. One-way ANOVA followed by Bonferroni’s post hoc comparisons tests and Brown-Forsythe and Welch ANOVA with Tamhane’s T2 post hoc multiple comparison tests were performed for statistical analyses. Non-parametric data were analyzed with the Kruskal–Wallis test. *P < 0.05; **P < 0.01.