During pregnancy, the early loss of embryos is a primary factor that affects litter size in mammals. In the current study, we confirmed that melatonin treatment in pregnant mice significantly increased litter size. This observation was consistent with previous publications on mice and rats [
24,
34]. Rats with a melatonin deficiency caused by pinealectomy had heavier uteri, a thicker endometrium, lower glandular organ weights, higher gland cavities, more uterine epithelial cells and increased variation capacity in attaching to the placenta; melatonin treatment reversed these alterations [
24,
25]. In addition, pinealectomy in mice influenced the serum progesterone and 17β-estradiol levels, the expression of the endometrial progesterone receptor and ovarian corpus luteum numbers, and melatonin supplementation effectively reversed all these changes [
25]. Some reports indicated that pinealectomy resulted in keratinization, continued oestrus and ovulation obstacles in rats [
38]. Rodents with pinealectomy or under continuous illumination tended to show precocious puberty, ovarian atrophy, chronic and persistent oestrus and hyperprolactinemia [
39]. These observations suggest that the melatonin circadian rhythm is essential for regulating reproductive hormones (17β-estradiol, progesterone) and for embryo development and implantation. In the current study, we observed, for the first time, that mice under prolonged light exposure had fewer embryo implantation sites. Prolonged light exposure decreases melatonin production in organisms [
40,
41]. This finding indicated that the decreased embryo implantation sites resulted from melatonin deficiency. Indeed, melatonin supplementation in drinking water (10
−4 M) counteracted the negative effects of prolonged light exposure on pregnant mice and increased their embryo implantation sites back to the control levels. P
4 is essential for embryo implantation and pregnancy maintenance in all mammals, and E
2 has different effects depending on the species and physiological conditions, as mentioned previously [
1]. Both of these hormones are regulated by melatonin via the HPG axis. In the current study, the level of P
4 in the blood wasn’t changed by melatonin treatment; however, melatonin significantly increased the level of E
2. These observations were not consistent with the results obtained in rats [
24] and this may be a species difference. The serum E
2 peak is essential for uterine receptivity. A high level of E
2 is beneficial for the implantation of an embryo, but the duration of uterine receptivity could become short under a high level of E
2 [
3,
4]. The current study showed that melatonin at 10
−5 to 10
−4 M increased the number of embryo implantation sites in pregnant mice. During the time window of uterine receptivity, exogenous melatonin enhanced the level of serum E
2. Based on the results, we concluded that the increased litter size was probably achieved by the promotion of mouse embryo implantation sites induced by exogenous melatonin treatment.
Among the pregnancy outcomes, birth weight is one of the most important indexes. We observed that melatonin treatment did not affect the pups’ birth weights, weaning survival rates, or weaning weights. In addition, the average litter weight was improved in rats exposed to low-nutrient conditions with melatonin treatment [
42]. The authors concluded that melatonin influenced the diastolic function of the blood vessels to enhance the efficiency of the placental nutrition supply [
42]. At the same time, the microenvironment of the uterus was improved by melatonin treatment, which promoted the expression of peroxidase and antioxidant enzymes such as Mn-SOD in the placenta [
42]. In another study, mice treated with melatonin via injection exhibited increased gene expression of
ErbB1, PRA, p53 and
MT2 [
34]. At least it can be concluded that the administration of melatonin does not cause negative effects on offspring.
The activities of melatonin might be mediated by its receptors, since melatonin receptors are present in the ovary and uterine mesangial matrix of rats and mice [
25,
41,
42]. MT1 and MT2 melatonin receptors were differentially expressed in pregnant and non-pregnant human uterine tissue, and affected the circadian rhythms of both uterine contraction and childbirth [
20,
43]. To explore the signal pathway of melatonin in regulating embryo implantation, the protein levels of MT1, MT2 p53 and LIF in uterine tissue were evaluated, and MT1 and MT2 were already expressed in the uteri of mice at day 4.5 of pregnancy. In addition, exogenous melatonin administration significantly promoted the expression of MT1 and MT2. Melatonin had more profound effects on MT2 than on MT1, indicating that MT2 may be the dominant receptor to mediate melatonin’s effect on reproduction. Melatonin was shown to upregulate the expression of p53 and p21 by affecting p38 activity and increasing the phosphorylation level of p53 [
32,
33,
44,
45]. A specific inhibitor of p38 MAPK (PD98059) could block the effect of melatonin on p53 [
45]. The cell selective gene silencing of melatonin receptors or the use of chemical inhibitors such as luzindole could suppress the readjustment of p53 [
44]. Thus, MT1 and MT2 activation by melatonin regulates the expression of p38, resulting in the accumulation of p53 and its phosphorylation [
33]. The results showed that melatonin upregulated the expression of MT1 and MT2 with the enhanced expression of p53 in the mouse uterus. p53 has been identified as a crucial factor for implantation and is the upstream regulator of the expression of
LIF [
9]. The results indicate that p53 may be a downstream element of MT1/2 activation and melatonin could regulate p53 and then upregulate LIF expression to improve embryo implantation. We also recognize the limitations of our study. Currently, we cannot distinguish which melatonin receptors, MT1, MT2 or both, are required for the signal transduction pathway. MT1 and MT2 or both knockout transgenic animal models will be needed for further studies.