Puberty is defined as the “first reproductive period”, which marks the maturity of the genitals, the development of secondary sexual characteristics, the acceleration of linear growth, emotional changes, and the occurrence of first spermatorrhea or menarche (1). The onset time of puberty has shown an increasing trend of being earlier worldwide (2). In the past 150 years, the average age of menarche of European children has declined from approximately 16–17 years in the middle of the 19th century to 12 years in the middle of the 20th century (3, 4), which is estimated to be advanced by 3 years every 100 years (5). A cohort study of Swedish boys showed that the starting age of puberty was 1.5 months earlier for every decade increase in birth year (6). Moreover, the average age at which Danish boys initiated puberty was 3 months earlier from 2006 to 2008 than before (7), and that of Chinese urban boys was 2 years earlier from 2003 to 2005 than 1979 (8). The early onset of puberty not only affects mental health in children and adolescents but also increases the risk of certain diseases in adulthood, such as metabolic syndrome, cardiovascular disease, osteoporosis, and testicular cancer (9, 10), which has developed into a serious public health problem.

With the development of ‘Fetal Origin of Adult Disease’ (FOAD) and ‘Development Origin of Health and Disease’ (DOHaD) (11, 12), an increasing number of studies have linked the endocrine environment during pregnancy to the long-term effects on future generations (13–16). With lifestyle changes and the effects of exogenous endocrine disruptors (17), the proportion of women with high androgen levels during pregnancy was significantly increased (18–20). Exposure to prenatal high androgen environments may lead to changes in hormone levels and pubertal development in offspring (21).

Leptin is a protein product of the obesity (Ob) gene and plays an important role in pubertal development and reproduction (22). Recent studies have found that leptin can regulate the synthesis and release of GnRH in the hypothalamic arcuate (ARC) nucleus (23). GnRH secretion is insufficient in mice lacking the leptin gene (24). Studies have directly shown that leptin can trigger the beginning of puberty in boys (25). However, studies have found that there is no leptin receptor expression or very little expression in ARC nucleus GnRH neurons (26). Therefore, leptin cannot directly act on GnRH neurons, but it is involved in regulating puberty onset (27, 28). However, leptin may indirectly affect the activity of GnRH neurons by affecting NPY neurons (29). NPY was identified as the main regulator of GnRH pulse secretion (30), which is the ‘gatekeeper’ of adolescence (31). As a regulator, NPY neurons are located in the ARC (32), which can directly exert an inhibitory effect on GnRH neurons (33). This effect is achieved by the direct binding of NPY to NPY1R on GnRH neurons (34). Moreover, recent studies have demonstrated that leptin receptors exist in NPY neurons (24), which indicates that leptin can directly regulate NPY neurons (35) and thus play an important role in regulating reproduction (36).

However, whether high androgen exposure during pregnancy can participate in puberty onset by affecting the levels of leptin and NPY in offspring has rarely been reported. Therefore, this study administered androgen intervention to maternal rats during pregnancy to shed light on the mechanism of leptin and NPY in puberty onset of offspring male rats after androgen intervention during pregnancy, which provides a powerful clue for exploring the relationship between prenatal androgen and the onset of puberty in male offspring.

The early onset of puberty has become a trend. The role of the intrauterine environment during pregnancy in the onset of puberty in offspring has received increasing attention. In the current study, we found that testosterone intervention in pregnant rats leads to an early time of onset of puberty in male offspring rats, a positive association between the time of onset of puberty and AR mRNA, an increase in hypothalamic NPY2R and leptinR protein and a decrease in AR and NPY protein expression. This showed that male offspring rats after androgen intervention during pregnancy might be more sensitive to androgens, leptin, and NPY, thus leading to early puberty.

It has become a consensus that puberty onset is correlated with body shape indicators (37); that is, puberty begins when body weight reaches a certain threshold. In this study, we found positive associations between the time of onset of puberty and weight, body length, and abdominal fat in OOG, which was consistent with previous studies. However, there were positive correlations between the time of onset of puberty and serum DHT, DHEA levels, and FAI in TG, which showed that offspring male rats after androgen intervention during pregnancy might be more sensitive to androgens. At the same time, the protein expression of AR decreased in the TG, which also suggested that the time of onset of puberty of male offspring rats was more sensitive to androgen after testosterone intervention during pregnancy. It follows that the control of puberty onset of male offspring may no longer be the feedback of body shape changes but more through the feedback regulation of sex hormones in offspring male rats after androgen intervention during pregnancy (38).

It is well known that leptin sends a negative feedback signal to the brain center when the body has sufficient energy storage, thereby maintaining the body’s energy balance by reducing food intake and increasing energy expenditure (39). Moreover, related studies have shown that androgen can inhibit the expression of leptin (40). Likewise, in a study on adipocytes of 3T3-L1 mice, it was found that dihydrotestosterone (DHT) significantly decreased leptin transcription and protein expression (41). In normal men, leptin levels may be limited by androgen, resulting in lower leptin levels than in women (42), and if serum leptin levels are above a certain threshold, leptin will in turn inhibit testicular function (43). Taken together, these signs suggest that androgen has an inhibitory effect on leptin. It was found that testosterone intervention reduced the protein expression of AR and upregulated the protein expression of leptinR at the onset of puberty in male offspring rats, which may be caused by the weakening of the inhibitory effect of low levels of androgens on leptin after testosterone intervention during pregnancy. In addition to its role in regulating energy balance, leptin also plays an important role in puberty onset by stimulating gonadotropin expression by upregulating the mRNA levels of GnRHR, activin, and FSHB (44). In this study, the expression of hypothalamic leptinR in the TG was higher than that in the OOG, indicating that the binding of hypothalamic leptin to leptinR may be enhanced after testosterone intervention, which affects the onset time of puberty.

Interestingly, related studies (45) have shown that there is no leptinR protein expression on GnRH neurons; therefore, by which mediator is leptin’s regulation of puberty onset time achieved? NPY, a 36-amino acid orexin-producing protein, is a key feeding center in the hypothalamic arcuate nucleus (46) and binds to its receptors Y1 and Y2 to regulate reproduction and food intake, respectively (47), in which binding to NPY1R can directly inhibit the release of GnRH (28). Moreover, kisspeptin and POMC neurons are also regulated by leptin (48, 49). However, a relevant study showed that increased Kiss1 mRNA levels were detected only after the onset of puberty (50), and Kiss1 does not express substantial amounts of leptin receptors (51), indicating that kisspeptin neurons may play a role after puberty. Another study also noted that the leptin-POMC pathway has sex specificity in terms of reproductive control, that is, it is difficult to play a role in male mice (52). However, for NPY neurons, related studies (29, 53) have provided evidence that serum leptin and hypothalamic leptin receptors are involved in the regulation of GnRH release by NPY and that NPY levels are elevated in leptin-deficient (ob/ob) mice (54). As shown in the results of this study, at the onset of puberty, the protein expression of leptinR and NPY2R in the hypothalamus increased, whereas the expression of NPY protein decreased in the TG, which may be due to the increased sensitivity of offspring male rats to leptin, thus suggesting that the binding of leptin to the leptin receptor was enhanced in the hypothalamus. The signal was transmitted to NPY neurons to inhibit NPY mRNA encoding the NPY protein, resulting in a decrease in NPY protein expression. Furthermore, in the environment of increased expression of NPY2R protein, the binding ability of NPY2R to NPY is enhanced, which mainly plays a role in energy expenditure and locomotion (55); thus, the inhibition of GnRH may be weakened followed by GnRH increasing rapidly during puberty; therefore, puberty starts earlier.

In this study, we detected only NPY2R, not NPY1R, so we cannot draw specific conclusions directly through the binding of NPY and NPY1R. Moreover, the intervention of testosterone was only in the third trimester of pregnancy, and it did not reflect the effect of testosterone on the puberty onset of offspring during pregnancy. At the same time, we sampled at the onset of puberty and reflected the differences between groups in the form of standardization, which may reduce the effectiveness of reflecting differences between groups. Finally, the association results were based on too few samples, which had limitations in the interpretation of associations.

Testosterone intervention in pregnant rats led to an earlier time of onset of puberty in male offspring rats. There were positive correlations between the time of onset of puberty and serum DHT, DHEA levels, and FAI, increased NPY2R mRNA, leptinR, and NPY2R protein expression, and decreased AR and NPY protein expression in male offspring rats after testosterone intervention during pregnancy. These results indicate that high androgen during pregnancy leads to early puberty onset in male offspring, which may be related to the decreased protein expression of AR, the increased expression of leptinR in the hypothalamus, and the enhanced inhibition of NPY neurons, as well as the enhanced binding of NPY2R and NPY. This could render male offspring after testosterone intervention during pregnancy more sensitive to androgens, leptin, and NPY at the onset of puberty. Therefore, the inhibition of GnRH may be weakened, and puberty onset occurs earlier (Figure 3).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Ethics Committee of Bengbu Medical College.

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.