**5. Effects of melatonin on the male reproductive system**

Various studies have shown that melatonin has some effects on the male reproductive system as well as the female reproductive system. It has been determined that this effect is by binding to the receptors of Sertoli and Leydig cells in the testicles and via the hypothalamus/pituitary axis.

#### **5.1 Direct effects of melatonin on the male reproductive system**

When the direct effects of melatonin on testicular tissue are examined, it has been stated that melatonin affects testicular development by binding to the relevant receptors in the testis [45] and in a study, exogenous melatonin administration in rats caused a decrease in testicular size [46, 47]. Various studies have shown that MT1 and MT2 are expressed in the testes of young and adult rats [45]. In a study, it was observed that the external application of Melatonin had negative effects on the seminiferous tubules in the testicles of old male mice [48]. It has also been reported that the administration of melatonin causes shrinkage in testicular tissue and low spermatid count in rats [49]. In another study, significant morphological changes occurred in the tubular and interstitial compartments of the testicles in hamsters exposed to short-term sunlight for a long time [50]. In addition, it was determined that melatonin significantly reduced the volume of mitochondria and nongranular endoplasmic reticulum, which are organelles containing enzymes that have an important role in androgen biosynthesis in mice Leydig cells [51]. Looking at the mechanism of testosterone production, it was determined that the LH hormone released from the adenohypophysis stimulates the cAMP signal in Sertoli cells [52]. It was also stated that melatonin did not suppress the activity of P450scc and thus decreased steroid synthesis. It was determined that StAR protein expression regulated by melatonin LH or cAMP was significantly reduced [53]. Another pathway that is effective in the testosterone release mechanism is that GnRH does so by increasing cytosolic Ca2+ concentrations and activating protein kinase C [52]. In some studies using the fluorescent Ca2+ indicator, it has been reported that melatonin suppresses the release of GnRH-dependent Ca2+ from intracellular stores, thereby reducing cellular Ca2+ levels and suppressing testosterone release [54]. In addition to the aforementioned mechanisms, melatonin has been reported to regulate testosterone production by interacting with the CRH system in the testicles [55, 56]. It has been determined that the CRH hormone is produced in the testicles as well as the hypothalamus. CRH released from the testicles acts as an important negative autocrine regulator of LH-induced testosterone production [57]. In a study, it was reported that Melatonin administration significantly increased the levels of mRNA related to CRH in Leydig cells [58]. Another hormone involved in testicular function is Estrogen. In immature males, the main production site of estrogen is Sertoli cells. Estrogen receptor-alpha (ERα), one of the estrogen receptors, was found in Leydig cells, while ERβ, another estrogen receptor subtype, was found in Sertoli and germ cells [59, 60]. Studies show that ERβ plays a role in the regulation of Leydig cell proliferation and testosterone production in adult mouse testicles. The cytochrome P450 aromatase (P450arom) enzyme is a key enzyme found in the endoplasmic reticulum of various tissues and is responsible for the production of estrogen from androgens. P450arom enzyme has been identified in Leydig cells of several species [61]. Melatonin has been reported to reduce estrogen biosynthesis by inhibiting the activity and expression of aromatase [62–64]. It has been determined that the administration of melatonin in Leydig and Sertoli cells obtained from rams increases testosterone production by increasing the expression of stem cell factor and insulin-like growth factor-1 [65]. In addition, it was determined that melatonin administration regulated lactate metabolism in Sertoli cells. Lactate released from Sertoli cells provides nutritional support to cells and prevents apoptosis [66]. As a result of the studies, it has been determined that the level of melatonin in the semen is related to infertility [67–70]. Especially in rams, melatonin level is directly related to sperm quality. Although rams can produce semen throughout the year, the quality of sperm outside the breeding season has been found to be low [71]. In the studies, it was determined that the application of melatonin outside of the breeding season increased sperm volume in the semen [72–74]. It was also determined that the dividing ability of oocytes fertilized by spermatozoa treated with melatonin increased. This increase is mediated by the increase of hyaluronidase enzyme activity by the administration of melatonin [75, 76].

#### **5.2 Indirect effects of melatonin on the male reproductive system**

When the indirect effects of melatonin on the male reproductive system are examined, it has been determined that there is an effect, especially on the hypothalamus-pituitary axis. In a study, it was determined that the application of melatonin to the hypothalamus significantly reduced testicular weight [77–79]. It was concluded that it showed this effect by suppressing GnRH secretion. In another study, it was determined that the administration of melatonin to mice caused a decrease in testicular and seminal vesicle mass, thus causing a decrease in sperm count [80]. GnIH, a hormone that suppresses the release of GnRH, was determined in a study conducted on quails in 2000 [81–83]. It has been determined that the effect of melatonin on GnRH secretion is mediated by this hormone. It has been determined that melatonin stimulates the release of GnIH by binding to the MT1 receptor, thus suppressing the release of GnRH [84, 85]. It has been determined that melatonin hormone androgen receptor and ABP levels are decreased in animals with seasonal reproduction. In some animals, it has been determined that tonin has positive effects on the male reproductive system. For example, it has been determined that it has indirect effects as well as direct effects on rams. Melatonin administration has been found to increase the testosterone concentration of somatic cells in ram testis tissue [86, 87]. Kispeptins, which are stimulators of GnRH neurons, have been found to be highly effective in transmitting the melatonin message [88–90]. In a study, it was determined kisspeptin expression decreased and atrophy occurred in the gonaon in winter days when the daylight decreased in Syrian hamsters. It has been determined that the level of testosterone increases with the injection of kisspeptin [91, 92]. In a study with zebrafish, it was determined that melatonin administration could induce the expression of Kiss1 and Kiss2 and GnRH3 genes in brain tissue and an increase in LH-β in the pituitary gland [93]. This induced gonad development. However, in a study in rats, it was reported that the administration of melatonin suppressed the release of FSH and LH, thus suppressing spermatogenesis in Sertoli cells and delaying sexual maturation [94]. Again, it was determined that the administration of melatonin to rats in the fetal period suppressed LHRH release and significantly reduced the LH level [95, 96]. Melatonin administration is thought to inhibit LH release by reducing Ca2+ flow and cAMP concentrations in the pituitary gland [97].
