Pituitary gonadotropes in females are a fascinating subset of pituitary cells that must be remodeled every cycle to support a preovulatory LH surge and a postovulatory rise in FSH (109). Depending on the gonadotrope gene marker being detected and the stage of the cycle, these heterogeneous cells represent at least 15% of the pituitary population. Our studies over the past 42 years have shown that precise accounting of the gonadotrope population is complicated by their dynamic remodeling, such that they can be difficult to identify or detect when a marker gene product has been downregulated or secreted. At least two gonadotrope markers must be detected to identify the entire population, especially during periods of low gonadotropin storage (estrus or metestrus).

Identification of the structural and molecular mechanisms behind the remodeling of gonadotropes and the regulators that drive these changes has been the subject of decades of investigative studies (109–116). Figure 2 shows a cartoon depicting some of the molecular and cytological changes that occur during the remodeling process that produces an actively secreting gonadotrope. Included in this population would presumably be any progenitor cell that contributes to the secreting, GnRH-responsive gonadotrope population, such as the somatogonadotrope (117).

Thanks to cytoskeletal remodeling, gonadotropes become more structurally elongated and stellate during diestrus and proestrus before the LH surge (109, 114, 118, 119), sending processes to blood vessels to facilitate surge-level secretion. Because they have actively secreted their stores during the LH surge and FSH rise, gonadotropin storage is significantly reduced on the morning of estrus (after the surge), reducing numbers of detectable gonadotropes (109, 110, 114, 120). Gonadotropes also increase their content of LEPRs during proestrus (33).

Figure 2 shows that, early in the cycle (metestrus), the cells destined to support the estrous rise in FSH and proestrous LH surge begin to produce gonadotropin and Gnrhr mRNA, which is followed by translation of these proteins during diestrus (121–123). The transcription of Gnrhr mRNA is under the control of GnRH pulses and rising levels of estrogen from the ovarian follicles (which had been stimulated by FSH early in estrus).More rapid pulses of GnRH in proestrus will facilitate the actual LH surge.

A critical step in this gonadotrope remodeling is the increase in GnRHR proteins. The changes in GnRHR depicted in Figure 2 were first reported by early radioreceptor assays, which detected the timing of the cyclic increase in GnRHR (121, 123) in rodents. The reports showed that gonadotropes undergo an increase in numbers of GnRHR early in diestrus I (metestrus) to reach a peak in late diestrus or on the morning of proestrus. Just before the LH surge, GnRHR numbers fall precipitously to remain low throughout the remaining stages of the cycle. This renders the gonadotrope population relatively quiescent during the postovulatory period of the cycle. There are LH and FSH pulses during this quiescent period, but they are of lower frequency and amplitude than those seen during mid-cycle surge activity.

The complex mechanisms controlling the increase in GnRHR clearly precede the remodeling needed to increase stores of gonadotropins needed for the surge activity, and it is not surprising that this initial process is regulated by pulses of GnRH itself (109). We have observed that the increase in GnRHR reflects an increase in the percentages of living gonadotropes that bind a biotinylated analog of GnRH (111). Collectively, these changes culminate in an increased population of responsive gonadotropes, which could then respond in synchrony to the higher GnRH pulse amplitude and frequency seen at mid-cycle.

The foregoing review of gonadotrope remodeling sets the stage for Hypothesis 1, which states that the cyclic changes in pituitary GnRHR expression create a mechanism by which the gonadotropes are activated only when environmental conditions are optimal. This mechanism would constitute an ideal checkpoint for metabolic regulation by leptin.

This hypothesis originated when we discovered that GnRHR proteins and activin subunit mRNA levels were reduced in pituitaries lacking LEPRs in gonadotropes. Our studies of mice with LEPR ablated in gonadotropes discovered that both of these gene products were reduced (33). More recent studies determined if GnRHR and activin were direct targets of leptin. We assayed mRNA extracts from pituitary pieces that were stimulated for 3 h with 10 nM leptin. Methods describing our approach to leptin stimulation are detailed in previous studies (33, 52, 124, 125). Pituitary pieces or cells in 24-h culture are exposed to leptin for 3 h at 37°C, and then extracts of proteins and mRNA are produced, as described (126). Methods describing our RT-PCR assays are found in the legend to Figure 3 and in Ref. (33). Figure 3A shows leptin stimulation of pituitary activin (but not inhibin) subunit mRNA levels (see reference (33) for information on primer sets). Similarly, leptin stimulation for 3 h does not affect Gnrhr mRNA levels (Figure 3B). This correlates well with our previous study showing that lack of LEPR in gonadotropes does not affect Gnrhr mRNA (33).

Our in vitro studies also show that leptin stimulates GnRHR proteins in a dose-dependent manner, with 10 nM resulting in the highest levels of GnRHR proteins or percentages of cells that bind biotinylated analogs of GnRH (127). This study was recently expanded to determine if the gonadotropes were the target cells. We used our established fluorescence-activated cell sorting-purification protocol (126) to separate gonadotropes by their eGFP fluorescence (with mice bearing Lh-cre and floxed tdTomato-eGFP). Freshly purified gonadotrope fractions (66,000 cells) were split. Half of the population was stimulated for 3 h with 10 nM leptin, and the remaining half received vehicle. Immunolabeling showed that the eGFP fractions were 98% gonadotropes. EIAs showed that the fraction contained most of the GnRHR, LH, and FSH, with other hormones assayed in the non-eGFP fraction. Protein extracts from the leptin- or vehicle-treated gonadotropes were assayed for GnRHR as described (33). Figure 3C shows that the leptin stimulation resulted in an increase in GnRHR protein levels in this population of pure gonadotropes, which agrees with recently published evidence (127). This is the first evidence for leptin’s direct stimulation of gonadotropes.

Thus, collectively, our studies of gonadotrope-Lepr-null mice and in vitro responses to leptin highlight the importance of gonadotrope leptin-target cells to the HPG axis and support our hypothesis that gonadotrope GnRHR represents a metabolic checkpoint. However, we have expanded this hypothesis to include a novel leptin-mediated post-transcriptional pathway to control translation of the Gnrhr mRNA. This expansion is based on the following evidence: (1) GnRHR protein levels are reduced in gonadotrope Lepr-null mutants, but Gnrhr mRNA levels are unchanged (33); (2) leptin does not directly stimulate Gnrhr mRNA levels (Figure 3B), and (3) leptin directly stimulates GnRHR proteins in a population of purified gonadotropes (Figure 3C) or mixed pituitary cultures (127).

We propose that the levels of Gnrhr mRNA are normal in our gonadotrope-Lepr exon 17-null mutants likely because LEPR was not ablated in the hypothalamus, allowing GnRH secretion and the regulation of transcription of Gnrhr, Lh, and Fsh mRNA (33, 128, 129). LH and FSH stores are also normal in these mutant gonadotropes (33). However, the diestrous gonadotropes did not appear to secrete normally, as reported by low serum LH and FSH levels (33). Whereas we can explain the fact that Gnrhr mRNA is normal, the mechanism underlying leptin’s permissive modulation of GnRHR protein synthesis is unknown. As a first hypothesis, we, therefore, propose that leptin may stimulate translation by alleviating repression of Gnrhr mRNA by mRNA regulatory proteins and/or miRNA.

miRNAs are small (~22 nucleotides), single-stranded RNAs that interact with target sequences within cellular mRNAs and exert translational repression. A significant role for leptin signaling in regulation of miRNA-mediated translational control has been observed in adipocytes and hepatic cells (130). In ob/ob mice, miR-103 and miR-107 levels are increased in the absence of leptin, contributing directly to insulin resistance (130). Furthermore, leptin signaling involves JAK–STAT pathways and precedent for pSTAT3-dependent downregulation of target miRNAs has been reported in breast cancer (131).

For our study, we initially wanted to determine which miRNAs might target the Gnrhr mRNA 3′-untranslated region (UTR). Our in silico analysis [Targetscan 7.1 (132)] revealed that the Gnrhr mRNA 3′-UTR (ENSMUST00000031172.8) contained 16 potential miRNA binding sites, including two that are also conserved in humans: miR-581/669d and miR-3061-3p. We began assays to detect differences, if any, in expression of candidate miRNAs. RT-PCR assays of whole pituitaries from control and gonadotrope Lepr exon 17–null diestrous females (n = 4–5 mice/group) determined that miR-581/669d was increased in the absence of leptin signals to gonadotropes, consistent with increased repression of Gnrhr mRNA translation in the mutants (Figure 4B). Detailed methods of our RT-PCR assays for miRNA are in the Figure legend of Figure 4. The specific role of miR-581/669d and the remaining 14 candidate miRNAs are currently being investigated. Complementary to this candidate approach, ongoing miRNA sequence analyses will provide an unbiased global analysis of pituitary miRNA expression related to loss of LEPR.

We also identified three consensus binding elements for the translational regulatory protein Musashi (MSI) (MBEs) in the 3′-UTR of murine Gnrhr mRNA (Figure 4A). The two vertebrate members of the MSI family, Musashi1 (MSI1) and Musashi2 (MSI2) are highly related, sequence-specific RNA binding proteins. MSI typically functions as a repressor of target mRNA translation and is specifically implicated in promoting stem/progenitor cell self-renewal where it functions to oppose translation of mRNAs encoding pro-differentiation factors and inhibitors of cell cycle progression under both physiological and pathological conditions (133). While the mouse pituitary is reported to express Msi mRNA (134), the function of MSI in the pituitary has not been determined. Ongoing studies show promise as they demonstrate binding by MSI to the Gnrhr mRNA 3′UTR and MSI repression of reporter expression driven by Gnrhr 3′UTR. We also have evidence that leptin reduces Msi mRNA (127).

Therefore, at this point, the evidence points to the hypothesis that leptin may play an important role in de-repressing Gnrhr mRNA during the critical period of cyclic upregulation of these vital receptors. We propose that MSI1 as well as at least one miRNA may be candidate Gnrhr mRNA regulators. Specifically, we propose that if nutrition and energy stores are normal, the mid-cycle leptin surge opposes MSI1-dependent mRNA repression, allowing the continued translation of Gnrhr during diestrus to reach optimal levels needed for a full LH surge. We propose that our mice that lack all isoforms of LEPR in gonadotropes (Table 1; Figure 1) may have not been able to produce sufficient GnRHR to mount an effective LH surge. Also, based on previous data (33) and Figure 3A, we propose that activin levels might be reduced, which would compromise FSH secretion and the development of the follicles. This first hypothesis will now be integrated into our second hypothesis about the overall mechanisms by which leptin signals metabolic information to the HPG axis.

As stated in the introduction, early studies of leptin regulation of reproduction have emphasized the hypothalamus as a primary target site for leptin and suggested that other sites might be less important or even redundant. The pioneering studies by McMinn et al. were the first to note the diversity in the LEPR-responsive neurons and the fact that all must be receptor deficient to cause infertility (8). Two studies selectively restored LEPR in the hypothalamus. The first of these studies reported that obesity, diabetes, and infertility in Lepr-null db/db mice could be rescued completely by re-introducing neuron-specific LEPR-B transgenes (82) to restore LEPR function selectively in the neurons. One of the drivers that introduced LEPR into the LEPR-null neurons was Synapsin (SYN-1). The selectivity of the SYN-1 driver was shown by expression in the brain; however, weak expression was also reported in the pituitary. This pituitary expression of SYN-1 was recently confirmed in Lβ-T2 gonadotropes and pituitary explants (135). Thus, based on the most recent evidence, we hypothesize that the Syn-1 driver may have introduced LEPR-B transgenes into both neuronal and anterior pituitary cells. Specifically, the expression of Synapsin in Lβ-T2 gonadotropes suggests that gonadotropes or their progenitor cells would have been among the restored leptin-target cells. Thus, fertility in these mice may have been restored by leptin-target neurons regulating GnRH and by leptin-target gonadotropes expressing GnRHR.

The second study by Donato et al. used Flp/FRT recombination approaches and a strain of mice carrying a neomycin cassette flanked by FRT sites targeted to the Lepr locus (50), which rendered the mutant mice globally LEPR-null. They selectively restored LEPR in the ventral premammillary (PMV) neurons of these mice by injecting an adeno-associated virus vector expressing Flp recombinase. The virus-restored mutant female mice showed evidence of pubertal development and cyclicity. In addition, five of the six females became pregnant although fertility was not optimal as four of these females did not carry the pups to term and the pups from the one female who delivered did not survive and died with no milk spots evident. These responses may also be due to the fact that the females remained morbidly obese. Thus, whereas the restoration of LEPR in the PMV clearly and selectively confirmed the importance of these neurons in the regulation of GnRH and the production of young, it appears that other leptin-target cells are vital to ensure that the progeny survive.

Based on our recent studies of Lepr-null gonadotropes (33), we hypothesize that the LEPR-null pituitaries in the study by Donato et al. expressed sufficient GnRHR on gonadotropes to go through puberty, cycle, and become pregnant. Because GnRH is an important stimulator of Gnrhr mRNA transcription [(128, 129) and Figure 2], restoration of LEPR in the PMV may have resulted in sufficient GnRH secretion to induce functional levels of GnRHR in gonadotropes. The observation that none of the litters survived, however, indicates that extra-PMV, pituitary, and ovarian LEPR-target cells are needed to support full reproductive competence. Also, the morbid obesity is a confounding factor. Detecting levels of gonadotropins, growth hormone, prolactin, estrogen, and progesterone may determine elements of the HPG axis that might have been most affected.

The importance of the working partnership between the hypothalamus and the pituitary is further elucidated in a recent study in which Cre-LoxP technology was used to restore only pituitary gonadotrope LEPR (101). As stated in the introduction, fertility was not restored in these animals presumably because LEPR-target neurons stimulating GnRH secretion remained deficient and unable to induce functional GnRHR signaling in gonadotropes (128, 129). This study provides another important clue to a role for leptin in gonadotropes, as they reported that FSH was elevated in this gonadotrope-specific LEPR model (101). As reported in our previous study (33), female mice bearing Lepr-null gonadotropes have reduced activin mRNA in the absence of leptin signals. We also reported reduced Fsh mRNA in these mutant animals. As activin stimulates FSH synthesis, we suggest that when LEPR was restored in pituitary gonadotropes, activin production may have been rescued (33). In the present report, we add evidence that leptin directly stimulates levels of activin mRNA (Figure 3A), which further supports this hypothesis. Also, recent studies of leptin actions in monkey pituitary cells show that 4 h of leptin stimulation in vitro results in elevated FSH secretion (55). It is interesting to note that leptin did not stimulate LH secretion in vitro in these female monkeys, which were reported to be of mixed cycles. We have shown that LEPRs in LH cells are maximal during the preovulatory period (33), and perhaps leptin’s effects on LH secretion are dependent on the stage of the menstrual cycle.

Based on these findings and the studies described above, we hypothesize that leptin’s role in the permissive regulation of the reproductive cycle depends on timed events that involve multiple interactive target cells in the HPG axis. Figure 5 proposes a set of integrating pathways by which changing energy stores could allow leptin to signal metabolic information and permit, delay, or stop the next cycle. As shown in this figure, nutritional and fat level sufficiency will result in optimal leptin levels that in turn will signal target cells in the hypothalamus and pituitary gonadotropes. We hypothesize that leptin acts on hypothalamic and pituitary target cells to signal changing energy stores. The pathway designated in green shows how leptin may activate gonadotropes directly to effect transcription of activin subunits to raise local activin levels and stimulate synthesis of FSH. This would support the early estrous rise in FSH, which stimulates ovarian follicles to develop and secrete estradiol, which then exerts positive feedback on the hypothalamus and the pituitary. Estrogen-sensitive neuronal pathways stimulate GnRH neurons to increase secretion and pulse frequency. The pathway in red highlights the important role of leptin in stimulating the LEPR-sensitive neurons in the hypothalamus to ultimately regulate GnRH neurons. The red pathway also shows that GnRH pulses stimulate Gnrhr mRNA, as well as LH and FSH secretion. Most of the elements in the green and red pathways are well established, although the role of leptin in stimulating activin in the green pathway is relatively novel.

What is most novel is the hypothetical blue pathway. Based on our studies of gonadotrope Lepr-null mice, we propose that leptin sends a third signal directly to gonadotropes that de-represses the translation of Gnrhr mRNA. The timing of this gateway signal could be during the metestrous to diestrous increase in GnRHR proteins. Our studies of females that lack all isoforms of gonadotrope LEPR (Table 1; Figure 1) strongly emphasize the importance of this blue pathway for optimal reproductive success. As discussed for Hypothesis 1, we propose that Gnrhr mRNA translation may normally be inhibited by MSI1 and possibly miRNAs. Consequently, leptin signaling acts to de-repress the Gnrhr mRNA by blocking the inhibitory action of MSI1 and/or miRNA repressive activity. This ultimately would activate translation of Gnrhr mRNA and provide the full complement of receptors needed for a fully responsive gonadotrope population ready for the LH surge and estrous rise in FSH.

Our two hypotheses reconcile and integrate findings from several studies of leptin-target cells. First, with the use of the Syn-1-driver, de Luca et al. (82) restored LEPR in both the brain and pituitary of db/db mice, which allowed multiple target cells seen in Figure 5 to function in partnership. Donato et al. (83) restored LEPR in the PMV of global LEPR-null mice, which stimulated GnRH to produce sufficient GnRHR and improve gonadotrope functions, although LEPR-target cells in the pituitary were still deficient and full reproduction (defined by the production of living pups) was not successful. As shown by Allen et al. (101) and Donato et al. (83), the system diagrammed in red and green pathways in Figure 5 will function only if leptin signaling to the brain is normal and only if there are GnRH pulses to stimulate the gonadotropes to make Gnrhr mRNA. However, as shown by our studies [Figure 1; Table 1; Ref. (33)], there must also be leptin input to gonadotropes for optimal levels of GnRHR proteins as well as responses to GnRHR for successful reproduction. Without that input, gonadotrope Lepr exon 1-null females failed to reproduce or had impaired fertility (Table 1).

Thus, the collective findings from the selective ablation or restoration of LEPR have highlighted the importance of leptin and LEPR to regulate function of the reproductive axis. Most importantly, they show that leptin’s permissive actions are operating in both the brain and the pituitary. These studies have further identified important pituitary signaling molecules activated directly or indirectly by leptin. Our hypotheses are integrated into the model in Figure 5 to indicate where each signal is needed and to highlight the fact that they act in partnership to optimize gonadotrope function. We also include a novel regulatory pathway that may involve control of MSI1 and/or miRNAs. Leptin regulation of these post-transcriptional pathways mediates the rapid de-repression and translation of Gnrhr mRNA, allowing for sufficient GnRHR to respond in synchrony and produce the LH surge. Subsequently, MSI1 and/or miRs would re-repress the Gnrhr mRNA late in the cycle, resulting in lower GnRHR levels and rendering the gonadotropes less responsive to GnRH. Continued studies are clearly needed to fully elucidate the targets and molecular pathways for leptin control of the HPG axis.

This study was carried out under the guidelines of the Department of Lab Animal Medicine and the protocols were approved by the UAMS Animal Use and Care Committee.

These authors MM, AM and GC are all designated as Senior Authors on this manuscript as they supervised different elements of the studies that led to the manuscript.

AO performed the analysis of the estrous cycles in the gonadotrope LEPR-null females, all experiments involving leptin stimulation of gonadotropes and FACS separation of gonadotropes, all qPCR assays for mRNA and miRNAs. She also helped with the literature review and the development of the hypotheses. NA and MS performed the cytochemical labeling studies that are cited in this work showing leptin stimulation of GnRHR. MA-J helped with cyclicity studies and cytochemistry cited in the paper. HB, MC, and MM worked on studies of MSI binding that are cited in this paper. AM did the in silico analysis of the GnRHR 3′-UTR and, with MM, designed experiments to test MSI binding. GC set up and monitored all breeding studies and wrote the initial drafts of the manuscript. All authors helped with the development of the hypotheses and the editing. MM, AM and GC are Co-Senior authors on this study working as equal partners in the development of the hypotheses and monitoring the final writing and editing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.