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

and either ACTH [54–56] or GH [57, 58]. Early studies of gonadotropes purified by centrifugal elutriation reported a fraction that contained 91-93% immunolabeled LH-FSH cells (a 9-fold enrichment). This group of cells were enriched based on their response to GnRH. The stimulated secretion caused them to enlarge. Which allowed them to be separated and enriched in a fraction containing larger cells. The fraction also contained gonadotropes that immunolabeled for other hormones. In the female gonadotrope fraction, we detected: 29.2% GH cells, 4% prolactin cells, 6.8% adrenocorticotropin (ACTH) cells, and 2.8% thyroid stimulating hormone cells (TSH [59]).

More recently we bred a Cre-reporter gene into our Cre-LH line to purify gonadotropes by fluorescence activated cell sorting mice [60]. Floxed tdTomato (red fluorescence) was expressed in all pituitary cells. However, in cells bearing Cre-recombinase (Cre-Lhb), the tdTomato was ablated promoting the expression of eGFP (green fluorescence). Thus, all non-gonadotropes (not producing Cre-*Lhb*) fluoresced red and all gonadotropes bearing LH expressed the green eGFP fluorescence. The red and green fractions were then separated by Fluorescence Activated Cell Sorting (FACS).

### **Figure 4.**

*FACS experiment comparing non-fluorescent cells (4A) with those bearing tdTomato-eGFP, but no Crerecombinase (4B) and those bearing Cre-recombinase, which ablates the tdTomato, allowing expression of eGFP (4C). Only the profile in 4C shows the presence of eGFP cells. (D–F) show that >90% of LH, FSH, and GnRHR is assayed in the eGFP fraction and barely detectable in the tdTomato fraction. Immunolabeling in (G) shows that >90% of the eGFP cells are labeled for LH (cyan shows blue label over eGFP green). (H–K) detect 70-90% of other pituitary hormones in the tdTomato fraction, but 10-30% of these hormones are found in the pure gonadotrope fraction. These data have never been published elsewhere and are original to this chapter.*

The FACS and assay methods are identical to those used for the Cre-GH line, as described by Odle et al. [60].

**Figure 4** shows the FACS separation profiles for non-fluorescent pituitary cells (**Figure 4A**); fluorescent Cre-negative populations bearing only tdTomato (**Figure 4B**) and Cre-positive populations, which contain the eGFP cells (**Figure 4C**). Assays for content of gonadotropins and GnRH receptors show that over 95% of the total content is in the eGFP fraction (**Figure 4D**–**F**). Over 90% of eGFP cells were immunolabeled for LH (**Figure 4G**). However, multihormonal expression is evident as shown in **Figure 4H**–**K**. The eGFP fraction contains 30% of the GH and TSH content and 10% of the ACTH and prolactin content. In contrast, the non-gonadotrope, tdTomato fraction (red bars) contain over 70% of the content of GH and TSH and 90% of the content of ACTH and prolactin.

When mRNA levels were assayed by qPCR, similar results were seen. **Figure 5A** shows the 72-88% enrichment in gonadotropin and *Gnrhr* mRNAs in the eGFP fractions, with little evidence for expression in the tdTomato fraction. **Figure 5B** shows that 10-20% of the levels of *Gh, Tsh*, and *Pomc* mRNA were also found in the gonadotrope fraction with the remaining tdTomato fraction containing the bulk of these RNAs (80%). It is also interesting to note that *Pou1f1* (also known as *Pit1*) mRNA, a transcription factor important in the production of *Gh, Tshb* and *Prl* is also found in the eGFP-gonadotrope fraction at about the same levels as that of *Gh* and *Tshb*. This expression would be important as Pou1f1 would be available to support the transcription of *Gh, Tsh,* and *Prl.*

FACS fractions from male mice from this line were also analyzed for mRNA content and similar enrichment of gonadotropins and *Gnrhr* mRNA levels was evident as well as similar levels of *Gh*, *Prl* and *Tshb* mRNAs in the eGFP-gonadotrope fractions (data not shown). Also, it is interesting to compare the expression profile of these purified gonadotrope fractions with that of the purified fractions produced in mice bearing Cre-GH. As previously reported, the Cre-GH/eGFP fraction contains most of the GH but very little LH, FSH or ACTH (see Figure 5 in [60]). The pure somatotropes contain significant amounts of Pou1f1, prolactin and TSH proteins and mRNA. Thus, somatotropes also include a multihormonal subset, but the expression profile is different.

More recently, multihormonal pituitary cell populations have been detected by single cell RNA-sequencing, especially in the study by Ho et al. [61], which investigated

### **Figure 5.**

*(A) qPCR assays show enrichment in Lhb, Fshb, and Gnrhr mRNA in eGFP fraction. These data have not been published elsewhere and are original. (B) Shows enrichment of Gh, Prl, and Pit1 mRNA levels in tdTomato fractions, but expression of about 20% of the total in the gonadotrope fraction. (C) Shows Tshb and Pomc mRNA levels and about 20% are in the gonadotrope fraction and 80% in the tdTomato fraction. These data have not been published elsewhere and are original to this chapter.*

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

changes in multihormonal cell transcriptome patterns in mice subjected to different physiological stresses. As we have outlined in a recent review [62], these pools of multihormonal cells may serve to support pituitary plasticity and add to the functions of pituitary populations as physiological needs arise. We hypothesize that these cells may include progenitor cells. Our data showing that gonadotrope LEPR-null mice have deficiencies in other hormones suggest that leptin may regulate multihormonal expression from this group of cells. The fact that secretion of a particular hormone is reduced in animals with LEPR-null gonadotropes highlights the importance of leptin to multihormonal function and suggests a role for leptin in the regulation of pituitary plasticity.
