| The testis is a principle organ in the male reproductive system, and is responsible for the formation of spermatozoa and the synthesis of steroid hormones (particularly androgens). The former process is called spermatogenesis, and contains three successive phases: mitotic (spermatogonia), meiotic (spermatocytes) and post-meiotic (spermatids),from the periphery to the lumen of seminiferous tubules. The latter is steroidogenesis,which is mainly characterized by producing androgens in the Leydig cells. Testosterone is the most important androgen, which is essential to normal male reproductive development and fertility. Recent studies showed that testosterone production is not only governed by hypothalamus-hypothesis-testis axis, but also regulated locally by multiple signaling pathways in Leydig cells. Other studies have detected the taste receptor type 1 subunit 3(T1R3) and its associated heterotrimeric G protein ?-gustducin (G?) in extra-oral tissues,especially in testes. Genetic absence of both TAS1R3 and GNAT3 (which encodes T1R3 and G?) resulted in male specific sterility with disorganized or exfoliated germinal epithelium,a lower sperm concentration, sperm immotility, and pathological changes in sperm. These prompted that T1R3 and Ga could be regarded as potential sites of regulating male reproduction, but related reports are very limited. Considering the potential roles of testicular T1R3 and Ga, the increasing consumption of sweeteners in our daily life may be associated with increased male reproductive disorders risk, it is urgently needed for us to understand the potential roles of testicular T1R3 and G? in male reproduction. Therefore,this paper explored expression patterns of testicular T1R3 and G? in mice during development to acquire the possibilities about these two proteins involved in testicular biologic functions, and investigated the relationship between testicular biologic functions and its T1R3 and Ga after daily exposure to sweeteners. Furthemore, both the potential mechanism of testicular T1R3 and Ga involved in its steroidogenesis in Leydig cells, and associations between testicular T1R3-G? and steroidogenesis in puberty by administration of neonatal mice with saccharin during lactation were deciphered in this study. All the above studies have preliminarily revealed that the novel functions of sweet tasting molecules T1R3 and G? in the testis, which facilitates our understanding of the two proteins-involved male fertility, and contributes to formulating a reasonable additive dose of sweeteners in our daily life. The main results are as follows:1 Expression patterns of T1R3and Ga in the mouse testis during developmentThe expression patterns of T1R3 and Ga in the mouse testis at critical stages of postnatal life (neonatal-7 d; weaning-21 d; puberty-35 d; adult-56 d; aged-1.5 yr), and throughout the spermatogenic cycle were investigated in the present study. The results indicated that T1R3 and G? exhibited a stage-dependent expression pattern during development, and a cell-specific pattern during the spermatogenic cycle. Western blotting results showed that the two protein expressions were increased significantly from pre-pubertal to pubertal periods, and decreased significantly in aged mice. It has been observed in IHC experiment that the above changes were mainly attributed to the differential expression of T1R3 or Ga in elongated spermatids and Leydig cells at different stages of the spermatogenic cycle. In addition, the expression of T1R3 and Ga were first observed in residual bodies of spermatozoa and endothelial cells of blood vessels at post-pubertal mice, while Ga was located in apoptotic spermatogonia of postnatal mice.2 Effects of daily exposure to sweeteners on testicular biologic functions and its sweet tasting T1R3 and G? in MiceIt was found that dose-response relationships existed for with regard to administration of saccharin or sucrose to mice for 35 d, and this association involved testes-expressed sweet-tasting molecules (T1R3 and Ga) in this experiment. Mouse body weights and testis weights in middle- and low-dose saccharin-treated groups were increased with up-regulations of molecules involved in testicular sweet taste and steroidogenic (middle saccharin: StAR, CYP11A1 and CYP17A1; low saccharin: StAR). Moreover, a high-dose saccharin-related decline in reproductive hormone levels, and injuries to testis and sperm were observed to be associated with suppressions of testicular T1R3 and Ga, as well as steroidogenic related factors (StAR, CYP11A1, 3?-HSD, CYP17A1 and 17(3-HSD), and activation of cleaved caspase-3. However, abnormalities of the testis and sperm in high-and middle-dose sucrose-exposed mice were related to the increased-cleaved caspase-3, but independent of T1R3 and/or G?. Collectively, our results clearly suggest that saccharin-induced physiologic effects on testis are associated with testicular T1R3 and G?.3 Acute effect of sweet tasting T1R3 and Ga-activated by sweeteners on Leydig cell steroidogenesis in the mouseUsing the intratesticular microinjection, the relationship between testicular sweet tasting molecules T1R3-G?-involved steroidogenesis and its cellular cAMP were explored in present study. The IHC results showed that testicular sweet tasting molecules (T1R3 and Ga) and steroidogenesis related factor (StAR, 3?-HSD,CYP17A1 and 17?-HSD) were co-localized on Leydig cells in adult mouse. Furher, there was no difference between each treatments in fasting blood glucose levels after 0 h, 3 h, 6 h and 12 h adiminstration, under the circumstance of intratesticular injection without impact on the testicular index and morphology. RIA resluts showed that compared to saline and control groups, a higher serum testosterone and estrodiol levels induced by saccharin were only found in 6 h saccharin-treated group. Additionally, an increase on serum E2 level was observed in 6 h hCG and hCG-saccharin groups, but their increased serum T was detected at exposure 3 h,6 h and12 h. Although the serum T level in 6 h saccharin-treated group was lower than hCG and hCG-saccharin groups, it was still higher than saline-injected and non-injected groups.In addition, the serum E2 level in saccharin and/or hCG groups at 6 h was significantly increased, compared with saline-injected and non-injected groups. In agreement, the western blot and ELISA relults showed that both testicular sweet tasting molecules (T1R3 and Ga) and steroidogenesis related factor (StAR, CYP11A1, 3?-HSD, GYP17A1 and 17?-HSD) in 6 h saccharin-treated group were increased with a higher cAMP level,compare to saline and control gorups. Furthermore, compare to saline and control groups,the 6 h saccharin-hCG-treated mice testes showed suppressions of saccharin-activated sweet proteins (T1R3 and G?) and saccharin-/hCG-induced steroidogenic enzymes(3p-HSD, CYP17A1 and 17?-HSD) with a lower cAMP concentration, and thereby down-regulation of hCG-induced testosterone sythesis in the testis. These results indicated that a cAMP signaling and steroidogenic enzymes (3(3-HSD, CYP17A1 and 17?-HSD) play important roles in Leydig cell steroidogenesis mediated by sweet T1R3 and G? proteins.Consequently, testicular T1R3 and G? involved Leydig cell steroidogenesis is related to their cAMP signaling.4 Administration of neonatal mice with saccharin sodium during lactation induced testicular steroidogenesis in puberty via a sweet tasting pathwayAfter administration of neonatal mice with saccharin during lactation, the relationship between testicular sweet taste sensing and its steroidogenesis in puberty was investigated in the present study. The results showed that administration of neonatal mice with high-/low-dose saccharin during lactation increased their weaning weights, puberty testicular index,and the higher testicular index in high-dose saccharin-treated mice is related to their serum T andE2and testicular T increased, as well as testicular LH decreased, wherea in low-dose saccharin-treated group, the higher testicular index was mainly associated with the increase of testicular T, serum T and E2. From IHC results showed that the testicular sweet sensing molecules (T1R3 and Ga) were co-expressed with its steroidogenic related protein (like StAR, 3?-HSD, CYP17A1 and 17?-HSD) in Leydig cells of puberty mice. The Western blot and real time-PCR results indicated that testicular sweet tasting molecules (T1R3 and Ga) and its steroidogenesis related factors (StAR, CYP11A1, 3(3-HSD, CYP17A1 and 17?-HSD) were significantly up-regulated in puberty mice after the high-/low-dose saccharin-injected during lactation, but their testicular T1R2 was considerably stable.ELISA results showed that the high-/low-dose saccharin-injected during lactation induced an increase of testicular cAMP level in puberty, but there was no effect on testicular TG and CHO. These results indicated that saccharin stimulated the testicular sweet tasting molecules (T1R3 and Ga), increased testicular cAMP level and up-regulated the expression of down-stream related steroidogenesis enzymes (StAR, CYP11A1, 3?-HSD, CYP17A1 and 17?-HSD), but there was no effect of sweet T1R2 receptor on' the testis. Briefly,administration of neonatal mice with saccharin during lactation induces testicular steroidogenic involved sweet tasting T1R3/Ga-cAMP pathway, and is independent on its T1R2 receptor, thereby finally alters their reproductive hormone levels. In addition, the saccharin-treated during lactation also elevates puberty mouse fasting glucose level and exacerbates its glucose intolerance without changing insulin sensitivity. |
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