**4. The importance of leptin to gonadotrope function**

The next series of studies investigated leptin's importance to gonadotrope function by selectively ablating LEPR in gonadotropes with Cre-LoxP technology. This work fills a critical knowledge gap, because, as summarized in our recent review [5], much of the research surrounding leptin's role in reproduction has been focused in the hypothalamus. There was a growing body of evidence showing that leptin's regulatory actions were mediated through its stimulation of Kisspeptin neuronal pathways that regulate GnRH neurons (reviewed in [5]).

Our first set of studies used Cre-recombinase driven by the *rLhb* promoter to delete either the JAK binding site (floxed LEPR exon 17) [1] or the signaling peptide of the LEPR (floxed LEPR exon 1). Deletion of LEPR exon 17 resulted in a nonsignaling receptor. Deletion of exon 1 resulted in ablation of all isoforms of LEPR. because the deletion removed the signal peptide thereby preventing the protein from entering the rough endoplasmic reticulum. Ongoing studies are using Crerecombinase driven by the *Gnrhr* promoter to delete LEPR exon 1.

The first question to be addressed related to the impact of loss of LEPR in gonadotropes on pubertal development, growth, and fertility of the mice [1]. When LEPR exon 17 was deleted in gonadotropes with the Cre-*Lhb* driver, mice showed no evidence of delayed puberty or growth. Mutant males showed no evidence of impaired fertility. However, mutant females exhibited a 36% increase in time to first pregnancy and the litters contained significantly fewer pups. Pup survival was 98% from either parent and there was no evidence of growth defects in weanlings from mutant females. Therefore, mutant females appeared to lactate normally.

We analyzed hormone levels in mice lacking LEPR exon 17 and reported that loss of LEPR resulted in several deficits [1]. In mutant diestrous females, serum levels of LH, FSH, and GH were reduced. In contrast, mutant males showed reductions in GH, prolactin (Prl), and thyroid stimulating hormone (TSH), but no reductions in gonadotropins. The loss of LEPR resulted in reduced *Fshb* mRNA levels in both males and females but no reductions in *Lhb, Cga* (in females) or *Gnrhr* mRNA levels. In addition, there was a reduction in inhibin/activin beta subunit mRNA (*Actßa* and *Actßb*) in females. The most striking reduction, however, was in GnRHR proteins, as detected by immunolabeling or binding to a biotinylated analog of GnRH [1]. The reduced binding was most severe in diestrous females, a stage where GnRHR expression is normally at the highest levels.

### *Leptin: A Metabolic Signal for the Differentiation of Pituitary Cells DOI: http://dx.doi.org/10.5772/intechopen.100922*

However, during this phase of the study, we detected Cre-recombinase in the testes and therefore continued these studies focusing only on mutant females [7] bearing Cre-*Lhb* and floxed LEPR exon 1. The deletion of this exon was impactful because it results in loss of all isoforms of the receptor. Tests of fertility showed normal litter numbers from three breeding cages of F2 generation heterozygous females (bearing only one deleted allele of LEPR exon 1). However, the study showed severe subfertility/infertility in F3 generation mutant homozygous and heterozygous females [7]. Out of the five F3 generation homozygous females, only two were fertile, producing litters more slowly than control females (one every 30-45 days). One of the litters did not survive. In addition, three F3 homozygous females and two F3 heterozygous females were completely infertile in breeding tests that lasted from 65 to 281 days with a fertile male [7].

We were able to study cyclicity in the progeny from the two F3 fertile females. These mutant F4 female progeny cycled irregularly. Two of them remained in diestrus and the remaining females spent more time in diestrus than normal females. Collectively, these breeding studies showed that ablation of all isoforms of LEPR in gonadotropes had a profound impact on a subset of females; less than half could cycle and were fertile [7]. This highlighted the importance of leptin to gonadotrope functions. However, because of the infertility issues in the line expressing Cre-LH X LEPR exon 1, our ongoing studies have now switched to mice bearing Cre-recombinase driven by the *Gnrhr* promoter. Whereas the mutant females in this line are still subfertile, they produce sufficient progeny for our ongoing and continuing studies of this line.
