Background
Obesity has been one of the most important health problems within the world’s population; the prevalence of overweight and obesity is increasing in reproductive-aged women [
1]. It has detrimental impact on female reproductive health, including ovulatory disorders [
2], decreased rates of conception, infertility [
3], early pregnancy loss etc. [
2]. Therefore, it is of great clinical significance to study the molecular mechanisms of impaired fertility in association with obesity.
Leptin, as an important adipokine, is encoded by ob gene and secreted by adipose tissue. Leptin receptor (lepR), with multiple isoforms (lepR a, b, c, d, e, and f), was encoded by db gene [
4‐
6]. After ligand binding, lepR activates intracellular signal transduction pathways mainly including JAK2-STAT3/STAT5 through three tyrosine residues tyr985, tyr1077 and tyr1138 [
7]. Previous studies reported leptin and lepR-deficient mice, such as ob/ob or db/db mice, all displayed metabolic abnormalities and reproductive disorders [
6,
8]. These fully illustrated that, in addition to controlling energy balance and body weight, leptin also served as a necessary signal to regulate female reproduction. Y123F mice are transgenic mice that are expressed mutant leptin receptors with phenylalanine (F) substitution for all three tyrosine (Y) residues [
9]. Our previous study showed that Y123F female mice were infertile similar with db/db mice, including no estrous cycle, anovulation and a significant decrease in serum estradiol (E2) levels [
10]. But Y123F mice’s symptoms of obesity, hyperphagia, hyperinsulinemia and impairment in glucose tolerance are less severe than db/db mice due to the remaining tyrosine-independent mechanisms. Also, Y123F mice have a longer life span than db/db mice [
9]. Therefore, Y123F mice are more suitable to be a new animal model to investigate the role of leptin and lepR in reproduction, especially in obesity-related infertility.
The expression of lepR has been documented not only in the central nervous system such as hypothalamus or pituitary, but also in the peripheral gonads such as ovary granulosa or embryonic cells of several species [
5,
7,
11]. However, the actions of leptin and lepR in the control of reproductive function are thought to be exerted mainly via the hypothalamic-pituitary-gonadal (HPG) axis, relatively less is known regarding their local effects on the peripheral ovary [
12], especially on ovarian hormone synthesis. Therefore, in this study, we decided to observe effects of leptin and lepR on ovary itself, particularly on its steroid hormone synthesis with Y123F mouse model.
During ovarian steroidogenesis, the first step takes place within mitochondria where steroidogenic acute regulatory protein (STAR) is to facilitate the movement of cholesterol from outer mitochondrial membrane to the inner mitochondrial membrane [
13]. Then, the cholesterol side-chain cleavage enzyme (P450scc or CYP11A1) converts cholesterol to pregnenolone to initiate steroidogenesis, which is a rate-limiting and quantitative regulation step. Next, most enzymes can be divided into two groups to mediate qualitative regulation determining the type of steroid to be produced: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. Among them, CYP19 (P450arom) catalyzes the conversion of the C19 androgen, androstenedione and testosterone, to the C18 estrogen, estrone and estradiol respectively. HSD17B7 primarily activates estrone to estradiol, especially in the luteal phase of the rodent ovarian cycle, which is responsible for the final step in estradiol synthesis [
13,
14]. In our previous study, we verified an elevated expression of site-mutated lepR in Y123F mouse ovaries and showed some accompanied gene profiling changes, particularly including steroid hormone biosynthesis enzyme genes such as Hsd17b7 or Star [
10]. So far there has been no study regarding the relation between lepR and steroidogenic enzymes as well as involved mechanism. Accordingly, in this study, we evaluate sex hormone levels in adult Y123F female mice compared with WT mice; then we focus on Y123F mice ovaries to investigate changes of steroidogenic enzymes and JAK2-STAT3/STAT5 signaling pathway. Finally, we explore whether the decreased fertility of Y123F mouse could be restored by exogenous hFSH supplement.
Discussion
Observational studies from humans or animal models with leptin or leptin-receptor deficiency have fully indicated that leptin-lepR system are involved in maintaining normal reproductive function [
6,
16‐
18]. Although the central effects of leptin on hypothalamic and pituitary function have been examined extensively [
19,
20], the contribution of direct leptin actions on the ovary in obesity-related infertility/subfertility remains incompletely understood. In this study, using the Y123F mouse model, we mainly explored the effects of lepR tyrosine site mutations on serum estrogen levels and local ovary steroidogenic enzymes changes. The novel finding of direct actions of leptin on the ovary may account for some adverse effects of obesity on ovarian function.
Firstly, according to our previous study that Y123F mice had low E2 level, no estrous cycle and anovulation [
10], we fully tested sex hormone levels of Y123F mice at the age of 12 weeks and found that the hypothalamic hypogonadism existed. In accordance with this result, previous studies demonstrated that leptin null (ob/ob) and leptin receptor null (db/db) mice exhibited low gonadotrophin concentrations, immature reproductive organs, and impaired sexual maturation [
21]. Also, leptin and leptin receptor gene defects result in human obesity and delayed puberty, oligo-anovulation, or subfertility in most human populations [
22]. Since leptin receptor is expressed abundantly within the hypothalamus and pituitary [
7,
23,
24], Watanobe
, et al. showed that leptin acted within the hypothalamus of animals to stimulate the release of GnRH within the median eminence-arcuate nucleus in fasted animals [
25]. Nicole Bellefontaine
, et al. reported that leptin plays a central role in regulating the HPG axis to increase circulating LH levels in vivo through the activation of neuronal NO synthase in neurons of the preoptic region [
26], in addition to through neuropeptides such as NPY [
27] or kisspeptin [
28]. Wen H. Yu
, et al. also confirmed that, in vitro pituitary tissue culture; leptin induces a dose-related increase in LH, FSH, and prolactin release [
20] via nitric oxide synthase activation in the gonadotropes [
29]. Female fertility relies on the regulation of sex hormones and positive/ negative feedback exists in HPG axis [
30,
31]. Due to the direct stimulatory effects of leptin on the HPG axis, thus for Y123F mice, abnormal lepR in central nervous systems resulted in low gonadotropin secretion, subsequently contributing to the low E2 levels.
Considering clinical patients undergoing assisted reproductive technology (ART), classic controlled ovarian hyperstimulation was performed using GnRH-agonist long protocol with exogenous recombinant FSH to stimulate oocyte development as well as increase E2 levels [
2,
32]. We tried to elevate E2 levels in Y123F mice by supplementing exogenous hFSH. Surprisingly, low serum E2 levels were only partly restored due to the active response of the ovary to FSH, still remained a significant lower level compared with WT mice. So we hypothesized that low E2 levels of Y123F mouse were not only due to low gonadotropin levels, but also probably due to the local factors in the ovary directly relating to leptin and lepR, such as steroidogenisis disorder,
It is well established that a series of ovary steroidogenic enzymes modulate hormone biosynthesis, which subsequently regulates reproductive functions such as follicle development and ovulation [
13]. Here, we found that ovary steroidogenic enzymes changed in 12-week-old Y123F mice, with a remarkable decrease of STAR, CYP11A1, HSD17B7, as well as an increase of CYP19A1, fully confirmed our hypothesis. Several studies have reported direct local leptin effects on ovarian functions. Zachow RJ
, et al. indicated that leptin antagonizes the stimulatory effects of transforming growth factor-β and insulin-like growth factor-I on FSH-dependent estrogen production by a mechanism involving the leptin-induced attenuation of P450arom activity and mRNA expression in rat ovarian granulosa cells (GC) in vitro [
33,
34]. Ghizzoni L
, et al. found that 30% decrease in E2 production was caused by the certain Leptin concentration; while P4 levels were not influenced in the culture media of human granulosa-lutein cells obtained from follicular fluid of women undergoing in vitro fertilization. They concluded that leptin suppresses E2 secretion by interfering with either the translational or post-translational steps of the baseline CYP17 and/or aromatase synthesis and/or the activation of the enzymes [
35]. In other species, such as sheep or bovine, leptin also inflicted a negative effect on ovarian steroidogenesis in vitro and in vivo experiments, which were corresponding with our results [
36,
37]. However, some studies showed stimulatory effects of leptin on the ovary [
38,
39]. These discrepancies may stem from differences in the doses of leptin and species as well as experiment design. In our study, ovary steroidogenic enzymes expressed abnormally in local ovary of lepR-deficient Y123F mice, even if to supplement exogenous hFSH, the expression defects of ovary steroidogenic enzymes were just partly recovered. But WB results showed that FSHR had higher expression in Y123F mice than WT mice, which had no significant increase after hFSH stimulation. By means of IHC, we found that higher expression of FSHR overall in Y123F mouse ovaries dectected by WB was due to more antral follicles in Y123F ovaries than in WT ovaries. But its feedback regulation may be up to saturation, leading to limited improvement of ovary steroidgenesis via hFSH supplement. Overall, our results fully illustrated that leptin and lepR in the Y123F mouse ovary had a negative effect on local ovarian steroidogenesis, independently of its central actions in the hypothalamus and pituitary.
Apart from previous studies above, Clark BJ and Lin D, et al. confirmed that overexpression of mouse STAR in mouse leydig MA-10 cells increased their basal steroidogenic rate [
40], and co-transfection of expression vectors for both StAR and the P450scc system in nonsteroidogenic COS-1 cells augmented pregnenolone synthesis more than that obtained with the P450scc system alone [
41]. Thus, in this study, the insufficiency of STAR, CYP11A1 and HSD17B7 can directly disrupt estradiol biosynthesis, leading to the low E2 levels of Y123F mice. It’s worth mentioning that Y123F mouse ovaries had higher expression of CYP19A1 and higher level of testosterone. As the substrate of estrogen biosynthesis, we considered that testosterone induced a compensatory response of higher expression of CYP19A1 to facilitate more conversion from testosterone to estradiol [
13]. But IHC showed that CYP19A1 mainly distributed in cumulus granulosa cells of tertiary follicles of Y123F mouse ovaries; and while mostly in the granulosa cells of secondary follicle and corpora lutea of WT mouse ovaries. So we supposed that estradiol produced in granulosa cells of Y123F mouse ovaries could be less available to get into the blood circulation due to the lack of corpus luteum that had enriching blood vessels, ultimately resulting in low blood E2 levels.
Intriguingly, there was no significant difference in all 4-week-old mice. These findings suggested that lepR tyrosine site mutations led to ovary steroidogenic enzymes changes only in adult mice, which had correlations with metabolic abnormalities due to fatness. Before 4 weeks, on one hand, mice did not reach up to sexual maturity and most of steroidogenic enzymes remained inactive; on the other hand, Y123F mice still maintained regular body weight due to complete compensation for lepR deficiency by other signal pathway [
9]. Corresponding with our finding, Luba Sominsky and colleagues have shown that neonatal overfeeding increased circulating levels of leptin and reduced the number of ovarian follicles, specifically, the primordial follicle pool in adult rats. The adult rats had increased levels of ovarian leptin and its receptor, but diminished release of pituitary gonadotropins at ovulation and altered expression of ovarian markers, such as GDF9, important for follicular recruitment and survival [
42]. So our results also confirmed the negative effects of leptin/lepR related obesity on reproduction.
In view of the local effects of leptin on ovary, although estradiol production is mainly regulated by FSH acting via the cAMP-dependent protein kinase (PKA) pathway in ovary granulosa cells [
43,
44], lepR mediated JAK2-STAT3/STAT5 signaling pathway in local ovary was worthy of being determined. Leptin has been shown to primarily activate the JAK2-STAT3/STAT5 signaling pathway through three tyrosine sites on long isoform leptin receptor [
45‐
47]. The long isoform LepR, which contains an intact intracellular domain with necessary protein motifs, can activate the JAK2, further stimulating the phosphorylation of multiple residues (Tyrosine 985, Tyrosine 1138, and Tyrosine 1077) on the intracellular domain of LEPR [
48]. Phosphorylation of tyrosine residue 1138 mainly recruits STAT3 and it has been reported that hypothalamic leptin control to reproduction is regulated by signals independent of STAT3 signaling [
49]. Meanwhile, Tyrosine 1077, the major phosphorylation site mediating leptin’s effects on reproduction, promotes the recruitment and transcriptional activation of STAT5 [
50] and is required for ongoing appropriate function of the female reproductive function [
51]. As expected, we found that in 12-week-old ovaries, phosphorylation of JAK2, STAT3 and STAT5 changed in Y123F ovaries, but no significant differences in 4-week-old ovaries between two genotypes.
Previously, Ding
, et al. found that sileptin treatment decreased the secretion of E2 and the cell proliferation, but increased the secretion of progesterone and cell apoptosis in human ovarian granulosa cells in vitro. They confirmed that impaired activation of lepR mediating JAK2/STAT3 signal pathway contributed to ovarian granulosa cell apoptosis [
52]. Therefore, our results indicated that LepR was involved in adult ovary steroidogenesis by mediating JAK2-STAT3/STAT5 signaling pathway in local ovaries.