Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

The anterior pituitary gland is comprised of a heterogeneous population of highly differentiated cell types that secrete distinct hormones, including lactotrophs that secrete prolactin (PRL), somatotrophs (GH), corticotrophs (ACTH), thyrotrophs (TSH), and gonadotrophs (LH and FSH). Within this heterogeneous cell population, gonadotrophs represent only about 5–15% of the total and are scattered throughout the anterior pituitary, making molecular and cellular characterization of this cell type a challenge (1). Dissection of the signaling pathways activated by GnRH, delineation of the subcellular routes of LH and FSH secretion, and determination of the sites and mechanisms of action of exogenous hormones in regulating gonadotroph function are all limited in this cell population. In a report in this issue of Endocrinology (2), an elegant new approach to the identification of individual live gonadotrophs for further study is presented. This approach opens new doors for studying the characteristics of this unique cell population. The development of murine gonadotroph-derived cell lines, first αT3-1 cells and subsequently LβT2 cells, which have been shown to possess many characteristics of mature gonadotrophs, have served as useful tools for analyzing the molecular and cellular events involved in such fundamental gonadotroph properties (3,4,5,6). LβT2 cells represent the most widely used cell model currently available for the study of mechanisms regulating LH and FSH subunit gene expression and have enabled significant advances in this field (7). However, immortalized cell lines may have characteristics distinct from those of mature gonadotrophs or may represent and reflect the response of only a subpopulation of gonadotrophs. Recently, Wu et al. (8) developed a novel strategy for identifying and purifying gonadotrophs. They generated a transgenic mouse model in which H-2Kk, a major histocompatibility protein, was targeted primarily to gonadotrophs using 4.7 kb of the ovine FSHβ promoter. This approach resulted in expression of H-2Kk in 3.2 ± 0.2% dispersed anterior pituitary cells. After immunopurification, 95% of the cells were gonadotrophs based on FSH immunostaining. The development of this murine tool provides a means for isolation of gonadotrophs for further study and for confirmation of findings obtained using gonadotroph-derived cell lines. For example, stimulation of purified gonadotrophs with activin increased FSH expression as expected, but the increase was not as great as in the presence of other anterior pituitary cells, suggesting that additional paracrine factors may be produced by nongonadotroph pituitary cells to further augment FSH production (8). An additional study comparing this purified cell population to the LβT2 cell line showed that LβT2 cells lack the TGFβ type II receptor, whereas the receptor is expressed in gonadotrophs; furthermore, TGFβ treatment reduced FSHβ mRNA levels in the purified gonadotrophs but had no effect in LβT2 cells. These data highlight the need to validate cell line findings in primary gonadotrophs (9). A limitation of this model is that LH immunostaining varied widely, indicating that this approach yields a cell population that preferentially expresses FSH but may not reflect the full gonadotroph population, as also suggested by the relatively small fraction of anterior pituitary cells positive for H-2Kk. In this context, a report in this issue of Endocrinology represents the next step, using a binary transgenic approach, that is, a two-tiered approach to achieve regulated transgene expression in transgenic mice, to identify, visualize, and manipulate gonadotrophs (2). In this approach, mice were first generated in which Cre recombinase was coexpressed with the GnRHR gene. This approach has the advantage over traditional transgenic approaches in that the Cre recombinase is produced under the control of the endogenous GnRHR promoter, with no concerns about influences on expression resulting from transgene copy number or from the site of genome insertion of the transgene, thereby increasing specificity of gene targeting (10,11,12). These mice were then bred to a ROSA 26-yellow fluorescent protein (YFP) mouse reporter strain, such that Cre recombinase induction leads to constitutive YFP expression in all tissues where Cre recombinase is expressed. Cre-mediated activation of YFP is not reversible, so the level of fluorescence is not influenced by temporal changes in the GnRHR promoter activity but rather reflects the history of activity of the GnRHR promoter in each cell in the mice. Immunofluorescence analysis demonstrated that 99.9% of cells staining positive for LH or FSH were labeled by YFP fluorescence in this model. YFP fluorescent cells negative for LH/FSH signal were detected with a frequency of 1.8%, indicating high specificity for gonadotrophs. These LH/FSH-negative fluorescent cells could represent gonadotrophs with gonadotropin expression below the immunodetection threshold, could be cells expressing the GnRHR that are not gonadotrophs, or could reflect low levels of nonspecific activation of either the Cre or the YFP gene. Fluorescent cells were found with a frequency of 15.4%, within the range of previous reports of percentages of gonadotrophs among the anterior pituitary cells and higher than those detected in the H-2Kk model, suggesting more complete detection of the gonadotroph population. The tagging of gonadotrophs with YFP provides a means for the identification, isolation, and characterization of live gonadotrophs for further study. Fluorescence-activated cell sorting could be performed on dissociated pituitary cells to produce enriched populations of gonadotrophs, enabling gonadotroph-specific studies such as gene expression profiling, electrophysiological characterization, signal transduction studies, and studies of hormonal regulation. Indeed, in this study, heterogeneity was demonstrated among gonadotrophs in terms of their resting electrophysiological activity, responses to GnRH, effects of GnRH on intracellular calcium concentrations, and resting and GnRH-stimulated LH and FSH secretion, consistent with other reports of heterogeneity of responses in gonadotroph-derived cell lines (13). A similar approach was used by Naik et al. (14) with the added component of temporal control of expression of Cre recombinase. In this case, a fragment of the bovine α-subunit (αGSU) promoter was used to direct expression of a transgenic cassette to the anterior pituitary, in this case containing the Cre expression vector with additional regulatory elements such that Cre expression is activated only in the presence of doxycycline. This transgenic mouse model was crossed with a ROSA26-β-galactosidase mouse reporter strain. This model resulted in expression of β-galactosidase in gonadotrophs (as identified by LHβ staining) in doxycycline-treated mice, thereby also allowing identification of gonadotrophs, although expression in other pituitary cell types was not determined (14). In this study, 10% of the anterior pituitary cell population stained for LHβ and β-galactosidase, somewhat lower than the cell population detected in the study by Wen et al. (2), perhaps reflecting the staining only for LHβ, therefore excluding gonadotrophs that express FSH only. The development of genetically labeled gonadotrophs provides a tool to overcome the previous challenges of studying primary gonadotroph populations among the mixed anterior pituitary cell population and expands our armamentarium for studies of gonadotroph biology. It is now feasible to prepare purified, more homogeneous populations of gonadotrophs for study. A caveat would be the loss of associations with surrounding pituitary cells and potential disruption of paracrine interactions; studies of purified cells would require careful consideration of the impact of this loss. The use of a binary transgenic model allows other genes to be targeted to gonadotrophs by breeding with other ROSA26 mouse strains. In this manner, for example, gonadotroph cell death could be selectively achieved by targeting the diphtheria A toxin (DTA) using the ROSA26-DTA mouse strain, previously reported using a more conventional αGSU-DTA transgene (15). An important consideration in this case would be expression of GnRHR in nonpituitary tissues, not studied in the current report (2). Gonadotroph-specific deletions of any target floxed gene can be similarly achieved. The addition of temporal regulation of Cre recombinase gene activation would further augment the utility of this model by allowing temporal as well as tissue-specific expression of specific genes or gene deletions in gonadotrophs. The advent of these new tools heralds a new era of opportunities to better understand gonadotroph biology, enabling us to cover the spectrum from the reductionist, single-cell, molecular approach through to the integrated, whole-animal model.