DTA expression in mTas1r3+ cells lead to male infertility

TAS1R taste receptors and their associated heterotrimeric G protein gustducin are strongly expressed in testis and sperm, but their functions and distribution in these tissues were unknown. Using transgenic mouse models, we show that taste signal transduction cascades (mTas1r3-Gnat3-Trmp5) are observed in testis form GFP transgenic mice. It is mTas1rs and mTas2rs, not Gnat3, that was expressed in leydig and sertoli cells. The pattern of mTas1r3 expression was different from that of mTas2r105 expression in seminiferous epithelium. Analysis of the seminiferous epithelium cycle show that both mTas1r3 and mTas2r105 is expressed in the spermatid stage, but mTas2r5 expression is found in spermatocyte stage. Conditional deletion of mTas1r3+ cells leads to male infertility, but do not affect the expression of taste signal transduction cascade during the spermatogenesis. The current results indicate a critical role for mTas1r3+ cell in sperm development and maturation.


Introduction
The Tas1rs are dimeric Class III GPCRs, with large N-terminal ligand binding domains. Three different Tas1rs have been reported, including Tas1r1, Tas1r2 and Tas1r3. So far, it is always believed that these receptors have the biological function in vivo only as heterodimers, with Tas1r3 serving as an obligate partner for both the umami receptor (Tas1r1+Tas1r3) and the sweet receptor (Tas1r2+Tas1r3) (Nelson et al., 2002;Nelson et al., 2001). Furthermore, knockouts of those genes have been used to identify the role of Tas1rs in taste transduction (Damak et al., 2003) . On the other hand, the production of several lines of transgenic mice expressing green fluorescent protein (GFP) from their promoters, particularly Gnat3, Trmp5 and Tas1r3, has revealed an extensive expression in the airways, from the upper airways to the lungs.
Furthermore, Tas1rs signaling has been most extensively studied in indigestive system. The sweet receptor is selectively expressed in enteroendocrine L cells, which can release GLP-1 after sugar ingestion, and GLP-1 in turn augments insulin release from the pancreas (Margolskee et al., 2007). The expression of the Tas1rs and their associated G-protein genes has also been reported in mammalian brain, indicating that the Tas1r2/Tas1r3 is a candidate membrane-bound brain glucosensor (Ren et al., 2009;Voigt et al., 2015).
Recently, several works reveal the expression of taste receptors in male reproductive system. Voigt et al  replaced the mTas1r1 and mTas2r131 open reading frames with the expression cassettes containing the fluorescent proteins mCherry or hrGFP, respectively. With these transgenic mice, Tas1r1 expression was observed in testis, epididymis mature spermatids. Meanwhile, Tas1r1 expression was also detected in earlier developmental stages of spermatogenesis and Sertoli cells. On the other hand, mTas2r131 expression was observed in spermatocyte and spermatid. In taste cells, the expression of mTas1r1 and mTas2r131 is segregated, whereas co-located in male germ cell. In mature spermatozoa, the expression of the mTas1r1 and mTas2r131 is restricted in distinct segments of the flagellum and the acrosomal cap (Meyer et al., 2012). In situ hybridization and real-time PCR revealed the expression of 35 Tas2rs in testis (Xu et al., 2012). With the humanized mTas1r3 transgenic mice, the genetic deletion of mTas1r3 gene and Gnat3 gene, Mosinger et al (Mosinger et al., 2013) revealed a crucial role for these genes in sperm development and maturation. Interestingly, another study have showed the associations between three single nucleotide polymorphisms (SNPs) in taste receptors genes and male infertility (Gentiluomo et al., 2017).
In previous study, we generated two transgenic mice, one expressing Cre/GFP fusion protein from mTas2r105 promoters (Li and Zhou, 2012), and another expressing Cre/GFP fusion protein from mTas1r3 promoters (Li, 2013). The previous study with the mTas2r105-Cre/GFP mouse revealed mTas2r105 expression in testis (Li and Zhou, 2012). We further provide a hypothesis that taste receptors may be involved in the regulation of spermatogenesis (Li, 2013). Here, we analyse the mTas1r3 expression in testis with the mTas1r3-KO and mTas1r3-Cre/GFP mice. The current results show that the genetic modification of mT1R3 or ablation of Tas1r3+ cells do not affect the expression of taste signal transduction cascade during the spermatogenesis. DTA expression in Tas1r3+ cells results in male infertility. Tas1r3 Genotype analyses including R26:lacZbpA flox diphtheria toxin A (DTA) line (Brockschnieder et al., 2006), Tas1r3-KO (Damak et al., 2003), Gnat3-GFP mouse (Wong et al., 1999), Trmp5-GFP (Clapp et al., 2006) were carried out by PCR as previously described. All animal procedures were approved by the committee of

Primary Sertoli and Leydig Cell Isolation
Primary Sertoli and Leydig cells were isolated by Percoll density gradient centrifugation, as previously described (Chang et al., 2011). Testes from 6-to (rabbit sc-22459; Santa Cruz Biotechnology, Santa Cruz, USA). The rabbit polyclonal antibody against Trpm5 (1028-1049 amino acids) was as described by Perez (Perez et al., 2002).

Sperm count.
Cauda epididymides were dissected and minced in phosphate-buffered saline solution. Sperm were squeezed out with fine forceps and allowed to disperse in phosphate-buffered saline at room temperature for 10 min, followed by repeated pipetting. Samples were fixed in 4% paraformaldehyde. Sperm were counted using a hematocytometer according to previous literature (Kovacs and Foote, 1992;Somfai et al., 2002). Sperm counting was performed four times for each sample.

Evaluation of the percentage of sperm motility and observation of sperm morphology
The percentage of sperm motility after 24-h incubation was tested using grading of motility by WHO criteria with respect to the fertilization of oocytes in vitro (Sukcharoen and Keith, 1996). To observe the mass motility, one drop of semen was placed on a pre-warmed (37°C) slide under microscope at 10ⅹ . Mass motility was scored into 0 to 5 scales every two hours according to the protocol suggested by Sukcharoen and Keith (Sukcharoen and Keith, 1996). Routine staining procedure was used to observe sperm morphology (Somfai et al., 2002). Equal drops of trypan blue and diluted semen were mixed on slides with the edge of another slide to make smears.
After drying at room temperature (RT), slides were fixed in 4% Paraformaldehyde for 2 min, then stained in 7.5% Giemsa for 12-20 hrs (overnight) at RT. After drying, they were mounted and coverslipped. Slides were evaluated by bright field light microscopy using 100 × oil immersion objectives, as previously described (Kovacs and Foote, 1992;Somfai et al., 2002).

Taste signal transduction cascades (mTas1r3-Gnat3-Trmp5) are observed in testis form GFP transgenic mice.
Previous study has shown the expression of taste receptors in testis. In order to further verify the expression of taste receptor and signal transduction cascades in the testis, we directly observe the testis from Gnat3-GFP (Fig. 1A), Trmp5-GFP (Figure-1B) and mTas1r3-GFP ( Fig. 1C and 1D) transgenic mice with Stereoanatomical fluorescence microscope. More GFP signals was found in the testis from Gnat3-GFP transgenic mice (Fig. 1A), the fewest GFP signals was observed in the testis from mTas1r3-GFP transgenic mice ( Fig. 1C and 1D). DTA expression lead to the loss of GFP signals in testis from mTas1r3-Cre/GFP-DTA transgenic mice ( Fig.   1E and 1F).
In order to further check the expression of taste receptor and signal transduction cascades in the seminiferous tubules, immunohistochemistry with anti-GFP was employed to analyze GFP expression in seminiferous tubule from Gnat3-GFP ( Fig expression was also detected in spermatid and leydig cell (Fig 2G and 2H).
We separated primary leydig, sertoli cells and spermatogenic cell, the expression of mTas1r1, mTas1r2 and mTas1r3 was examined by immunostaining blotting in these cells (Fig. 3A). Unexpected, Gnat3 expression was only observed in spermatogenic cells (Fig. 3A). We also analyzed the expression of mTas2rs in testis somatic cells with RT-PCR. Several mTas2rs transcripts was detected in leydig and sertoli cells including mTas2r105, mTas2r106 and mTas2r143 (Fig. 3B). Gnat3 transcripts failed to be detected in leydig and sertoli cells (Fig. 3B). We further employ immunostaining to analyze the expression of mTas1rs in primary leydig and sertoli cells. Confocal analysis showed the expression of mTas1r1, mTas1r2 and mTas1r3 in these cells (Fig. 3C-3J). In short, current results reveals the expression of mTas1rs and mTas2rs in leydig, sertoli cells.

DTA expression in mTas1r3+ cells decrease sperm mobility
To investigate the function of mTas1r3 during spermatogenesis, the breed data was analysed in mTas1r3-Cre/GFP transgenic mice， mTas1r3-KO mice and mTas1r3-Cre/GFP-DTA. The infertility was observed in male double transgenic mouse (mTas1r3-Cre/GFP-DTA). Additionally, after counting sperm in epididymis, the mTas1r3-Cre/GFP-DTA mice had less sperm concentration than WT males (Fig.   6A). Ratio of testis to body weight was also not significantly different among the group (Fig. 6B). Subsequently, sperm mobility was also checked. Sperm mobility was not significantly different for fresh sperm just removed from epididymis. However, sperm mobility was significantly decreased after cultured for several hours at 37℃ (Fig. 6C), indicated that spermatogenesis was affected both in mTas1r3-Cre/GFP-DTA and mTas1r3-KO. Area of semiferous tube from the mTas1r3-Cre/GFP-DTA mice is significantly smaller than that from WT C57BL/6 and mTas1r3-Cre/GFP males (Fig. 6D). About 20% of spermatozoa from the double transgenic mouse (mTas1r3-Cre/GFP-DTA) were abnormal (flipped heads, Head tail separation, flagella with tight loops) (Fig. 6H). These are at least twice as many abnormalities as typically observed in WT C57BL/6 ( Fig. 6E) and mTas1r3-Cre/GFP ( Fig. 6F) males. The most significantly abnormalities noted were flipped heads (~8%) and Head without tail (~8%). In a word, DTA expression in mTas1r3+ cells influence on spermatogenesis with an unknown mechanism.
To investigate whether genetic deletion of Tas1r3 or loss of Tas1r3+ cells affected the spermatogenic cycle in mice, Trmp5 expressions were analyzed in adult mice during the 12th spermato-genic cycle. The results indicated that Trmp5 have a unique expression pattern throughout the spermatogenic cycle. Strong Trmp5 immunolabelling was present in the round spermatids at stages VII-VIII and the elongated spermatids at stages 9-15 ( Fig. 8A-8F). After loss of mTas1r3+ cells (Fig.   8A1-8F1) or genetic deletion of Tas1r3 (Fig. 8A2-8F2), positive immunostaining was still observed in the round spermatids at stages VII-VIII and the elongated spermatids at stages 9-15.
These intriguing results indicated that genetic deletion of mTas1r3 or loss of Tas1r3+ cells do not affect the expression of taste signalling transduction cascades including Gnat3, PLC-β2 and Trmp5.

Discussion:
We found that Tas1rs are expressed in Leydig and Sertoli cells. We also detected mRNAs for several mTas2rs including mTas2r105 and mTas2r106 in Leydig and Sertoli cells. These data are in agreement with previous reports, which reveal the expression of mTas1rs and mTas2rs in testis (Iwatsuki et al., 2010;Meyer et al., 2012;Voigt et al., 2012;Xu et al., 2012). It is noteworthy that the pattern of mTas2r105 expression differ in striking ways from that of mTas1r3 during spermatogenesis.
mTas2r105 expression is widely seen during spermatogenesis including spermatogonial, spermatocyte and spermatid phase. On the other hand, Trmp5 expression is limited to the spermatid phase, and Gnat3 expression is not found in spermatocyte phase and testis somatic cells (leydig and sertoli cells). It thus appears that many taste signaling molecules originally identified in taste cells are expressed also in testis, but they are not as concentrated in a certain type of testis cell as in taste bud. Other signaling molecules may be involved in the downstream of the sweet and bitter taste-sensing receptor in testis. The previous study have shown that the T1R3 receptor is likely coupled with Gas, and mediated the anti-adipogenic signal through activation of Gas in 3T3-L1 cells (Masubuchi et al., 2013).
Agreement with our finding, Tas1r1/Tas1r3 has been found to be present broadly in mouse tissues and many types of cultured cells (Max et al., 2001;Wauson et al., 2012). Even robust expression of Tas1r1/Tas1r3 is found in mice heart (Foster et al., 2013). Further study reveals that amino acids bind Tas1r1/Tas1r3 leading to activation of PLC-β, calcium entry, and ERK1/2, thus stimulating the mammalian target of rapamycin complex 1 (mTORC1) (Wauson et al., 2012). Mammalian mTOR is believed to be a central controller of cell growth, metabolism and aging, coordinating cell growth, protein translation, and autophagy with the availability of nutrients, growth factors, and energy (Dazert and Hall, 2011;Duan et al., 2016;Li and Cheng, 2016;Zoncu et al., 2011). We speculate that Tas1r1/Tas1r3 detects extracellular amino acids, transmits that information to mTORC1 and is involved in regulating cell growth and metabolism in spermatogenesis.
The present study also shows that the pattern of mTas2r105 expression was obviously differ from that of mTas1r3 expression during the spermatogenesis.
mTas1r3 expression was mostly observed in mid-later spermatid. In contrast, mTas2r105 expression was observed throughout the spermatogenesis including spermatogonial, spermocyte and spermatid. Thus, DTA expression in mTas2r105+ cells lead to smaller testis (Li, 2013;Li and Zhou, 2012). In taste bud, mTas2r105 is not co-expressed with mTas1r3 Chandrashekar et al., 2000). The previous study has also shown that mTas2rs is observed in a subtype of cells distinguished from Tas1r3 positive cells in extra-oral tissue (Clark et al., 2015;Liu et al., 2015;Voigt et al., 2012). Furthermore, the previous studies have revealed that the expression of mTas1r1 was detected during the earlier developmental stages of spermatogenesis and Sertoli cells (Meyer et al., 2012). The stronger expression of mTas2r131, revealed by hrGFP Fluorescence intensity in mTas2r131 BLiG/BLiG mice, was also found in developmental stages of progenitor cells of spermatozoa. Additionally, the expression of mTas1r1 and mTas2r131, separated in taste cells, can partially co-localize in male germ cells (Meyer et al., 2012;. Another finding in this study is that DTA expression in mTas1r3+ cells lead to male infertility. Although mTas1r3 expression is mainly observed in mid-later spermatid, loss of mTas1r3 positive cells in testis may be critical during spermatogenesis. Two possibilities should be considered. Firstly, taste genes (such as mTas1r1, mTas1r2, mTas1r3 and their associated G-protein) are detected in mammalian brain, including the paraventricular and arcuate nuclei of the hypothalamus, the CA fields and dentate gyrus of the hippocampus, the habenula, and cortex (Ren et al., 2009). mTas1r3 positive cells may play a role in levels of hypothalamo-pituitary-gonadal feedback loop (Wauson et al., 2012). In addition, the present study demonstrated that Tas1r3 is expressed in leydig and sertoli cells. DTA expression should result in loss of function in mTas1r3 positive cells, which may also contribute to male infertility. Recent study also revealed that polymorphism of several mTas2rs and mTas1r2 was proved to be functional and showed a profound effect on human male infertility (Gentiluomo et al., 2017).
In conclusion, our data suggest a detailed expression pattern of mTas1r3, mTas2r105 and other signal molecules. DTA expression in mTas1r3+ cells finally lead to male-infertile. mTas1r3 and mTas2r105 is expressed in a subtype of cells during spermatogenesis, which is distinct from Gnat3 or Trmp5 positive cells, indicating that there existed a different signaling pathway from taste bud. This unique sweet and bitter-sensing receptor may possibly be a potential target for treatment of male infertility.