Although the negative effects of obesity on reproductive function were first documented many years ago by Hippocrates , the mechanism underlying this relation has not yet been thoroughly investigated. The relation between leptin and reproduction has received much attention in recent years. Some studies have reported that leptin primarily targets the hypothalamus [18, 26] and that reproductive function is influenced by the hypothalamic–pituitary–gonadal axis [8, 27, 28]. Herrid et al. reviewed that leptin has direct effects on the regulation of testicular development and reported that testosterone production is diverse in the immature and adult mouse testes [29, 30]. However, little is known regarding the potential mechanisms of the direct action of leptin on the testicular structure and function at different ages in mice. The observation that leptin receptors are present in male gonadal tissue [12, 13] provided a basis for our study. Accordingly, we conducted this study to explore whether leptin regulates the testes directly during maturation from infancy to adulthood and, if so, to study the mechanism that is responsible for this effect.
Methods that are used to establish models of obesity in animals involve neuroendocrine, dietary, or genetic manipulation . MSG treatment is one neuroendocrine method to induce obesity. Several studies had established and successfully utilised the MSG-induced obesity model [10, 32, 33], which results in a syndrome that is characterised by obesity and hypogonadism . On the other hand, MSG is a polar solute whose dose in the blood vessels of tissue, including the hypothalamus and testes, is limited to <1%. Meanwhile, the blood/brain barrier (BBB) restricts and adjusts the flux of substrates between the circulation and the central nervous system. As MSG crosses lipid cellular membranes or is transported by selective BBB carriers, MSG is depleted . In addition, our results exclude the direct role of MSG in testosterone production in vitro. Compared with dietary methods, ip MSG treatment is much easier to control and is more likely to induce obesity. To maintain the endogenic leptin state, we could exclude the methods of genetic change and direct leptin injection.
The mice were treated with MSG each day from d0 to d14, d28, or d56, which represent the prepubertal, pubertal, and postpubertal stages of maturation, respectively. Consistent with a previous report , we observed alterations that accompanied MSG-induced endogenous hyperleptinemia through the course of development; MSG treatment elevated leptin concentration on d14 and d28, and markedly elevated leptin concentration on d56. We could observe that epididymal adipose accumulated more in MSG-treated mice; however, there was no significant difference in the body weight, which might be limited by the short period of observation . Although some studies revealed that the ip administration of MSG in animals showed a reduction in dietaty intake and body weight [9, 11]. The common aspect of these studies was that the total fat mass significantly increased, which paralleled with concentrated leptin circulation in the blood [10, 37]. Additionally expression of LEPR is detected in most tissues, including hypothalamus and testes. Circulatory leptin can cross the blood/brain or blood/testes barrier to combine with LEPR. Thus intra-testicular concentrations of leptin can mirror circulatory concentrations in part .
Testosterone, which is the major androgen, is produced by Leydig cells in the testes. To measure the effect of MSG on testicular function, we measured the plasma testosterone concentration at corresponding time points. We found that testosterone secretion in MSG-treated mice displayed an increasing trend from prepuberty to puberty (d14 to d28), but then decreased from puberty to adulthood (d28 to d56). Our results from the in vitro incubation of testicular tissue showed that testosterone secretion was stimulated by a lower concentration (10 nM) of leptin, but was inhibited by a higher concentration (100 nM) of leptin. This result is consistent with those results from our in vivo study of MSG-treated mice. These findings suggest that the production of testosterone is affected by the timing of MSG treatment. MSG-treatment to d28 slightly increased leptin and testosterone production, but that with continued MSG treatment to d56, the high level of leptin inhibited testosterone production. This result indicates that MSG-induced hyperleptinemia affected Leydig cells, which are the major cells that produce testosterone, through a direct or indirect approach.
The decreases in testicular weight and volume showed that hyperleptinemia impaired testicular development; however, this effect was reversible in part by the withdrawal of MSG. The testicular weight appears to be the most sensitive indicator of drug toxicity , and the testicular volume is regarded as an index of spermatogenesis [40–42]. Although MSG induced testosterone secretion during prepuberty and puberty, there were also negative effects on testis development, which were obvious through adulthood. The histopathological analysis of the testes showed that hyperleptinemia had negative effects on the testicular structure from infancy; persistent MSG treatment caused changes in the testicular structure, such as looser seminiferous tubule distribution and smaller tubule diameter. Additionally, MSG treatment prevented the proliferation and differentiation of spermatocytes and sperm. Variations in the seminiferous tubules have a negative effect on reproduction . However, the testicular structure and function recovered partly after withdrawal of the MSG treatment, including increased testosterone level, testicular weight, volume, number of offspring, diameter of seminiferous tubules, number of spermatocytes, sperm and Leydig cells. To our knowledge, this report is the first to demonstrate such reversible changes. Leydig cell steroidogenesis is one functional parameter in reproduction [43, 44]. The enzymatic complex 3β-HSD, which is in the endoplasmic reticulum and mitochondria, plays an essential role in the biosynthesis of testosterone and is detected in Leydig cells [45, 46]. We found a reduced number of 3β-HSD-positive Leydig cells in the testes of mice that were treated with MSG continuously from infancy to adulthood (MSG-d56 group) and an increased number in the MSG-d28 group; however, these variations were not found in the MSG-d14 group. The number of offspring that were produced by normal female mice that mated with MSG-treated male was lower in MSG-treated than in NS-treated mice. These results show that MSG-treated hyperleptinemia has adverse effects on testicular development, function and fecundity.
In this study, it has been shown that the testicular expression of SOCS3 is regulated by leptin. SOCS3 was previously identified as a potential mediator of central leptin resistance. SOCS3 negatively regulates leptin signalling and plays important roles in mediating leptin sensitivity, glucose homeostasis, and energy expenditure . Consistent with some studies, leptin induces the expression of SOCS3 and then SOCS3 negatively regulates the leptin level . When leptin resistance emerged, the regulatory role was attenuated. Then, leptin concentration suddenly increased and induced the expression of SOCS3. To rule out the contribution of other regulatory signals, we used a static in vitro system to incubate testicular tissue, and we confirmed that leptin induced the expression of SOCS3 directly. In addition, some studies have reported that SOCS3 regulates the JAK–STAT pathway in the hypothalamus. Our results clearly showed that SOCS3 and pSTAT3 were also expressed in the testes. By down- and upregulating SOCS3, we confirmed that SOCS3 regulates testosterone secretion in the testes through the STAT pathway. This result has demonstrated that SOCS3 regulation and STAT3 phosphorylation play important roles in testosterone secretion and, consequently, affect the development of the testes.