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Endocrinology, doi:10.1210/en.2007-1502
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Endocrinology Vol. 149, No. 6 2701-2711
Copyright © 2008 by The Endocrine Society

Functional Characterization of Genetically Labeled Gonadotropes

Shuping Wen1, Jürgen R. Schwarz1, Dragos Niculescu, Crenguta Dinu, Christiane K. Bauer, Wiebke Hirdes and Ulrich Boehm

Institute for Neural Signal Transduction (S.W., J.R.S., D.N., C.D., W.H., U.B.), Center for Molecular Neurobiology, D-20253 Hamburg, Germany; and Institute for Vegetative Physiology and Pathophysiology (C.K.B.), University Hospital Hamburg-Eppendorf, D-20246 Hamburg, Germany

Address all correspondence and requests for reprints to: Dr. Ulrich Boehm, Institute for Neural Signal Transduction, Center for Molecular Neurobiology, Falkenried 94, D-20253 Hamburg, Germany. E-mail: ulrich.boehm{at}zmnh.uni-hamburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADOTROPES OF THE anterior pituitary gland are crucial in the control of reproductive function in mammals. They produce and secrete the two gonadotropins, LH and FSH, that regulate the ovaries and testes (1). Secretion of gonadotropins from gonadotropes is triggered by GnRH, a decapeptide produced by a small population of hypothalamic neurons [GnRH neurons (2, 3)]. GnRH is secreted into the hypophysial portal circulation and transported to the anterior pituitary where it binds to the GnRH receptor (GnRHR), a G protein-coupled receptor specifically expressed by gonadotropes (reviewed in Refs. 4 and 5).

Gonadotropes comprise a scattered population of cells within the anterior pituitary. Accurate electrophysiological analysis of live primary gonadotropes requires that they be identified and isolated from the general population. In previous analyses this has been accomplished using either cell lines (6, 7), cultured native pituitary cells (8, 9), or Percoll density gradient centrifugation of a pituitary cell suspension (10) to obtain a gonadotrope-enriched fraction. Gonadotropes within the cultured native pituitary cells are identified by plaque formation in the reverse hemolytic plaque assay (RHPA), indicating secretion of LH/FSH (8), or by morphological criteria along with positive response to the application of GnRH (9). However, cell lines likely represent only a subpopulation of the gonadotropes, and in the methods used for identification of gonadotropes, those that do not secrete LH/FSH or respond to GnRH, as well as those that are found outside of the Percoll fraction taken will escape detection, again resulting in only a subpopulation being used for the analysis. That this assessment is true is confirmed by immunocytochemical studies using antibodies against gonadotropins as well as morphological criteria suggesting considerable heterogeneity within the gonadotrope population (reviewed in Ref. 1), raising the possibility that a significant number of gonadotropes was previously undetected and, thus, excluded from functional analysis.

To overcome this shortcoming, we devised a binary genetic strategy to express yellow fluorescent protein (YFP) in gonadotropes. We used gene targeting to generate mice in which Cre recombinase is coexpressed with the GnRHR gene. We show that we can direct Cre-mediated recombination of a YFP reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. Using YFP fluorescent gonadotropes in primary culture from the pituitaries of young adult male mice, we performed patch-clamp experiments, calcium imaging, and the RHPA. We demonstrate significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. Approximately 50% of fluorescent gonadotropes formed plaques in RHPA, suggesting that 50% of gonadotropes do not secrete LH or FSH under these conditions. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular [Ca2+].


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of GnRHR-internal ribosome entry site-Cre (GRIC) mice
A clone containing exon 3 of the GnRHR gene (Fig. 1AGo) was isolated from a 129/SvJ mouse genomic library (Stratagene, La Jolla, CA) and subcloned into the pKO-V901 plasmid (Lexicon Pharmaceuticals, The Woodlands, TX) with a phosphoglycerate kinase (pgk) promoter-driven diphtheria toxin A cassette. An AscI restriction enzyme site was created 3' to the stop codon of the GnRHR gene using PCR. An internal ribosome entry site (IRES)-Cre-Flp recombination target (FRT)-neomycin (neo)-FRT (11) cassette containing a pgk promoter-driven neomycin resistance cassette flanked by FRT sites was inserted into the AscI site to generate the final targeting construct. The construct was transfected into R1 mouse embryonic stem cells by electroporation, and resistant clones were analyzed by Southern blotting using EcoRV and an external 500-bp probe (Fig. 1Go, A and B). Correctly targeted embryonic stem cells were injected into C57BL/6J blastocysts to generate chimeras that were backcrossed to C57BL/6J animals to give knock-in mice still carrying the neomycin resistance cassette (GRICneo+). For genotyping, genomic DNA from tail biopsies was prepared by proteinase K (Roche Diagnostics, Mannheim, Germany) digestion and subsequent purification with DNA Isolation Reagent for genomic DNA (AppliChem GmbH, Darmstadt, Germany). Mice were genotyped by Southern blotting as described previously.


Figure 1
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  		FIG. 1. Targeted integration of the IRES-Cre cassette into the GnRHR locus. A, Schematic representation of the targeting strategy used to express Cre recombinase under control of the GnRHR promoter. From top to bottom, the targeting vector, the GnRHR wild-type (wt) allele, and the targeted GnRHR allele before (neo+) and after (neo–) removal of the neomycin selection cassette are shown. Restriction sites for EcoRV and BspTI, as well as the location of the probe are indicated. Black boxes represent exons 2 and 3. The inserted cassette is composed of an IRES followed by the coding sequence for Cre recombinase (Cre), and a pgk promoter driven neomycin resistance flanked by Flp recombinase recognition sites (FRT). B, Southern blot analysis of DNA from wild-type and heterozygous mutant mice after digestion with EcoRV. The expected fragment sizes detected by the probe used for hybridization (shown in A) are indicated (wild type, 15.9 kb; mutant, 10.5 kb). Mice Nos. 3 and 6 carry the mutant GnRHR allele (GRICneo+). C, Southern blot analysis of DNA digestion with BspTI from wild-type, and heterozygous mutant mice before and after removal of the neomycin selection cassette. The expected fragment sizes detected by the probe shown in A are indicated (wild type, 7.4 kb; mutant allele I GRICneo+, 10.8 kb; mutant allele II GRICneo–, 9.2 kb). Mouse No.10 carries mutant allele I GRICneo+, and mice Nos. 8, 11, and 12 carry mutant allele II after Flp recombinase-mediated excision of the neomycin cassette (GRICneo–).

 
Mice
Animal care and experimental procedures were performed in accordance with the guidelines established by the animal welfare committee of the University of Hamburg. Mice were kept under a standard light/dark cycle with food and water ad libitum. To remove the FRT flanked neomycin resistance cassette, GRICneo+ animals were bred to Flp recombinase deleter mice (12) to result in GRICneo– (GRIC) mice. Flp mediated excision of the selection cassette was verified by Southern blot analysis using BspTI and an external 500-bp probe (Fig. 1Go, A and C). GRIC mice were kept in a mixed (129/SvJ and C57BL/6J) background. ROSA26-YFP mice were kindly provided by S. Srinivas (University of Oxford, Oxford, UK) and kept in a mixed (129/SvJ and C57BL/6) background (13). All animals analyzed in this study were young adult male GRIC/R26-YFP mice (3–4 months old), heterozygous for both the GRIC allele and the ROSA26-YFP reporter allele, respectively. GRIC/R26-YFP mice are viable and when interbred (GRIC/R26-YFP X GRIC/R26-YFP), produce litters with frequency and size indistinguishable from those of wild-type animals breeding in our colony (data not shown).

Immunofluorescence
GRIC/R26-YFP mice were perfused transcardially with 4% paraformaldehyde (fixative) under ketamine/xylazine (Bayer, Fernwald, Germany) anesthesia. Pituitaries were removed and soaked in fixative for 2 h, in 30% sucrose for 48 h, and then frozen in tissue-freezing compound optimal cutting temperature (OCT) (Leica Microsystems GmbH, Wetzlar, Germany) and cut into 14-µm sections with a cryostat (14). Pituitary sections were blocked in 1x PBS, 0.025% TX-100, 5% horse serum, and then treated with rabbit anti-LH-β and rabbit anti-FSH-β antibodies (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD; each at 1:5000) overnight at 4 C, followed by Cy3-donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Laboratories, Inc., West Grove PA) for 1 h at room temperature. Slides were treated with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Appropriate controls omitting primary antibodies were performed and did not yield any staining (data not shown). Endogenous YFP fluorescence was visualized using a YFP filter set (EX BP 500/20, BS FT 515, EM BP 535/30; Carl Zeiss, Inc., Thornwood, NY).

Primary cell culture
GRIC/R26-YFP mice were anesthetized with isoflurane (Abbot GmbH & Co. KG, Wiesbaden, Germany) and killed by decapitation. Pituitaries were quickly removed and transferred into dispersion medium containing Hanks’ F10 medium supplemented with 10 mM D-glucose, 10 mM HEPES, and 0.5 mg ml–1 BSA (pH adjusted to 7.3 with NaOH). Pituitaries were cut into small pieces and digested with collagenase type CLS II (Biochrom KG, Berlin, Germany; 678 U ml–1 dispersion medium) for 30 min at 37 C. Cells were gently triturated with a glass pipette, centrifuged for 15 min at 4 C, and resuspended in growth medium containing low-glucose DMEM (Life Technologies, Inc., Gaithersburg, MD) enriched with 10% fetal bovine serum (Biochrom KG; containing 11.39 pg ml–1 estradiol, 0.03 ng ml–1 progesterone, and 0.02 ng ml–1 testosterone as reported from the supplier), 1.78 mM L-glutamine (Life Technologies), penicillin (100 U ml–1), and streptomycin (1 mg ml–1). The cell suspension was then plated on five glass coverslips (coated with poly-L-lysine), placed in cell culture dishes (Nunc, Thermo Fisher Scientific Inc., Waltham, MA), and kept at 37 C in a humidified incubator with 5% CO2.

In some of the electrophysiological experiments, a modified protocol for preparing the cell culture was used. For dissociation the pituitary pieces were kept in a solution containing papain (20 U ml–1; Worthington Biochemical Corp., Lakewood, NJ) dissolved in Earle’s Balanced Salt Solution (Invitrogen Corp., Carlsbad, CA) for 30 min at 37 C. Thereafter, the cells were washed three times with plating medium containing MEM (Invitrogen), 0.6% glucose, and 10% horse serum (Invitrogen). After trituration the cells were plated on glass coverslips. We did not observe any difference between these two protocols in the appearance of pituitary cells and percentage of successful recordings (data not shown).

Calcium imaging
Cultured cells prepared from GRIC/R26-YFP mice were incubated in the dark with fura-2/AM at 37 C for 30 min in Ringer solution containing 135 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 5 mM glucose, and 10 mM HEPES (pH 7.3) (NaOH). Fura-2 fluorescence was elicited with a polychromator V (TILL Photonics GmbH, Gräfelfing, Germany; emission 510 nm, alternating excitations 340/380 nm) and recorded with a SensiCam camera (PCO imaging, Kelheim, Germany). Images were acquired every 260 msec with 100-msec exposure time using TILLvisION Software (TILL Photonics), and ratio images (340/380 nm) were calculated. The fluorescence ratios were converted to Ca2+ concentration by the equation:

Formula
with dissociation constant (Kd) for fura-2/Ca2+ [224 nM (15)], ratio at any pixel point (R), ratio values of fura-2 [at zero (Rmin) and saturating (Rmax) [Ca2+]i, respectively], and ratio (β) of fluorescence at 380 nm for the dye in saturating and zero [Ca2+]i. The calibration constants (Rmin, Rmax, and β) were empirically determined using ionomycin (1 µM) in the presence of either 2 mM EGTA or 20 mM Ca2+, added to the Ringer solution. The experiments were performed at room temperature using an Axioskop microscope (Zeiss) and the solutions bath perfused at 3 ml min–1 in an approximate 1 ml volume under a water-immersed objective. GnRH (10 nM; Sigma-Aldrich, St. Louis, MO) together with 0.2% BSA (Sigma-Aldrich) were added to the Ringer solution.

Electrophysiology
All electrophysiological experiments were performed on YFP-fluorescent cells after 1–6 d in culture. To detect the fluorescent cells, a YFP filter set (Zeiss) was used. Voltage clamp experiments were done with the conventional whole-cell patch-clamp configuration, and current clamp measurements were done using the nystatin-perforated-patch whole-cell configuration of the patch-clamp technique. For stimulation and data acquisition, the Pulse 8.65 software (HEKA Instruments, Inc., Port Washington, NY) in combination with an EPC-9 patch-clamp amplifier (HEKA Instruments) was used. Data were low-pass filtered at 3 kHz. The sampling rate was 10 kHz for Na+ currents, 2 kHz for K+ and Ca2+ currents as well as for current clamp recordings. Experiments were done at room temperature (21–23 C). Pipettes were made from borosilicate glass capillaries and coated with Sigmacote (Sigma-Aldrich). When filled with intracellular solution, the pipette resistance was 2–3 M{Omega}. Compensation for series resistance errors was performed (>70%). Compensation for fast and slow capacitances was performed before data acquisition. Data were analyzed with PulseFit (HEKA Instruments), Igor (WaveMetrics, Portland, OR), and Excel (Microsoft Corp., Redmond, WA). All data are presented as means ± SEM.

Current clamp (perforated-patch) experiments. The pipette solution contained 140 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 2.5 mM EGTA, 10 mM HEPES (47 nM free Ca2+) (pH adjusted to 7.3 with KOH), and 0.24 mg ml–1 nystatin. The extracellular solution contained 135 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 5 mM glucose (pH adjusted to 7.3 with NaOH). GnRH (1 or 10 nM) was added to the 0.2% BSA containing extracellular solution.

Voltage-clamp experiments (conventional whole cell)
Recording of K+ currents.
The same intracellular solution was used as in current clamp experiments, except that nystatin was removed and Mg-ATP (3 mM) and Na-GTP (0.3 mM) were added. The extracellular solution was also the same as in current clamp experiments, except that a concentration of 500 nM tetrodotoxin (TTX) was added.

Recording of Na+ currents.
The pipette solution contained 130 mM CsCl, 5 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 2 mM Mg-ATP, and 10 mM HEPES (pH 7.3 adjusted with CsOH). The extracellular solution was the same as in current clamp experiments, but in addition it contained 10 mM tetraethylammonium chloride (TEA-Cl) and 5 mM 4-AP.

Recording of Ca2+ channel currents.
The pipette solution contained 135 mM CsCl, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA, and 4 mM Mg-ATP (pH 7.3 adjusted with CsOH). The extracellular solution contained 130 mM NaCl, 1.2 mM MgCl2, 10 mM BaCl2, 10 mM HEPES, and 500 nM TTX. Ba2+ currents through Ca2+ channels were blocked by addition of CdCl2 (0.2 mM) to the extracellular solution.

Nystatin, nifedipine, and fura-2/AM were dissolved in dimethylsulfoxide, TTX was dissolved in sodium-acetate buffer (pH 5), and all other chemicals were dissolved in water. Except where indicated all chemicals were purchased from Sigma-Aldrich. Solutions were bath perfused at 2.5 ml min–1 in a 200 µl recording chamber using a multichannel peristaltic pump (Gilson, Inc., Middleton, WI).

The data shown have not been corrected for the liquid junction potential (<4 mV). Because the current clamp experiments were performed with the perforated-patch whole-cell configuration, a Donnan potential was present. The exact value of the Donnan potential is uncertain and could amount to about 10 mV. Because liquid junction potential and Donnan potential are additive, in the current clamp experiments, the membrane potential could be 14 mV more negative than the pipette potential. In addition, in the perforated-patch whole-cell configuration, the hypertonicity of the cells should induce a cell swelling. However, during the course of the experiments, which could last for up to 30 min, cell swelling of gonadotropes was not observed, similar to the initial observations in lacrimal gland cells (16).

RHPA
LH/FSH secretion of individual gonadotropes was studied with the RHPA (17). Freshly dissociated cells of pituitaries derived from one or two male GRIC/R26-YFP mice were mixed with protein A-coupled erythrocytes and infused into Cunningham chambers with a volume of approximately 20 µl. One pituitary yielded five to 10 Cunningham chambers. These chambers were prepared in poly-D-lysine-coated cell culture dishes using a round (12-mm diameter) coverslip as a roof and fishing line of 0.1-mm diameter as walls stabilized with drops of laboratory grease. The cells were allowed to settle for 1.5–3 h before they were subjected to the plaque assay. The cells were rinsed in the Cunningham chambers with DMEM plus 0.2% BSA and then incubated for 18 h in DMEM/BSA containing antibody against LH and FSH (National Institute of Diabetes and Digestive and Kidney Diseases; 1:50 dilution for both antibodies). After the incubation period with or without GnRH (200 nM; Sigma-Aldrich), guinea pig complement (1:50) was added for 20 min to develop the plaques. The cells were then rinsed with culture medium. All plaques of a Cunningham chamber were evaluated by measuring the diameter of the plaques.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Targeting the GNRHR
We prepared mice in which Cre recombinase was coexpressed with the GnRHR gene. Using gene targeting in embryonic stem cells, we modified the GnRHR gene by inserting, just after its coding region, an IRES sequence followed by a Cre recombinase cDNA (Fig. 1AGo). The IRES element results in production of a bicistronic mRNA, from which the GnRHR and Cre recombinase are independently translated (11). The altered stem cells were then used to generate GRICneo+ mice (Fig. 1BGo). To remove the neomycin resistance cassette, GRICneo+ animals were bred to Flp recombinase deleter mice (12), resulting in GRICneo– (GRIC) mice (Fig. 1CGo). GRIC mice are viable, fertile, and produce litters with frequency and size indistinguishable from those of wild-type animals breeding in our colony (data not shown).

Fluorescent visualization of gonadotropes
To monitor Cre recombinase activity, GRIC mice were bred to ROSA26-YFP (R26-YFP) mice. The R26-YFP reporter strain carries a targeted insertion of a YFP gene in the ubiquitously expressed ROSA26 locus (13, 18). Due to a loxP flanked (floxed) strong transcriptional termination sequence, the R26-YFP allele terminates transcription prematurely, but when the mice are crossed with Cre-expressing mice, the Cre-mediated excision of the floxed termination sequence leads to constitutive YFP expression (Fig. 2AGo).


Figure 2
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  		FIG. 2. Cre recombinase mediated YFP expression in gonadotropes. A, Breeding strategy to activate YFP expression in GnRHR expressing cells. Coexpression of Cre recombinase with GnRHR will lead to excision of the floxed stop cassette, which in turn will activate ROSA26 driven transcription of YFP in GRIC/R26-YFP double knock-in mice. B, Immunofluorescence analysis of pituitary sections from GRIC/R26-YFP mice using antibodies against LH and FSH. All gonadotropin-containing cells (red) display YFP fluorescence (green), demonstrating faithful activation of the ROSA26-YFP reporter gene in gonadotrope cells. Corresponding bright-field images are shown on the left. The merged images (right) show nuclei counterstained with 4',6-diamidino-2-phenylindole (blue). Scale bars, 50 µm.

 
Therefore, gonadotropes in GRIC/R26-YFP double-heterozygous mice should express YFP from the recombined R26-YFP allele and be identifiable by their fluorescence signal (Fig. 2AGo). Pituitary slices prepared from GRIC/R26-YFP mice contained brightly fluorescent cells in the anterior pituitary (Fig. 2BGo), but no fluorescent signal in the posterior pituitary, consistent with the distribution of gonadotrope cells in the mouse pituitary (19). Immunofluorescence analysis on pituitary sections from GRIC/R26-YFP mice using antibodies against LH and FSH showed that 99.9% of gonadotropin-containing cells are labeled by YFP fluorescence (4535 cells out of 4540 cells analyzed on 10 pituitary sections derived from three mice; Fig. 2BGo), demonstrating faithful activation of the ROSA26-YFP reporter gene in gonadotropes. YFP fluorescent cells without immunofluorescent LH/FSH signal were observed with a frequency of 1.8% (83 of 4623 cells analyzed on 10 pituitary sections derived from three mice).

Electrophysiological characterization of gonadotropes
Next, we prepared primary pituitary cell cultures from male GRIC/R26-YFP mice. YFP-positive cells were detected by their bright fluorescence using a YFP filter set (see Figs. 7Go and 8Go). Fluorescent cells were found with a mean frequency of 15.4% (three independent dispersions representing pituitaries obtained from three animals). For electrophysiological experiments only those cells that were not in contact with other cells were chosen. After selection of a particular cell, the experiment was continued in the bright-field mode of the microscope. Membrane capacitance varied between 6 and 11 pF (6.9 ± 0.2 pF; n = 72; cells were from pituitaries of 16 mice).


Figure 7
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  		FIG. 7. Calcium imaging reveals a large heterogeneity of GnRH responses. Pituitary cells from GRIC/R26-YFP mice held in culture were loaded with fura-2/AM, and the changes in [Ca2+]i were recorded. A, Shows the transmission (left) and the YFP fluorescence (right) images. Scale bar, 50 µm. B and C, Show examples of GnRH responses recorded in two different cells. Bars above the traces indicate the presence of GnRH.

 

Figure 8
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  		FIG. 8. LH/FSH secretion of single gonadotropes detected with the RHPA. The RHPA was performed on pituitary cells from GRIC/R26-YFP mice. A, Example of a fluorescent cell surrounded by a hemolytic plaque (top) and another fluorescent cell without plaque (bottom); scale bar, 50 µm. B, Percentage of plaque-forming cells in the absence (control) or presence of 200 nM GnRH during the incubation period.

 
Recording of membrane potential
Current clamp experiments were done in 28 gonadotropes (from eight mice) using the perforated-patch whole-cell configuration (see Materials and Methods). The resting potential varied between –13 and –75 mV (mean: –52 ± 3 mV). Due to the Donnan potential and the liquid junction potential, the exact value of the resting membrane potentials is more negative (see Materials and Methods). Six cells were spontaneously active, exhibiting a frequency of action potentials ranging from 0.3–1.2 Hz (mean: 0.7 ± 0.1 Hz). Action potential amplitudes varied between 53 and 93 mV (mean: 72 ± 7 mV), the overshoot being between 3 and 16 mV (mean: 12 ± 2 mV). Measured at –20 mV, the duration of action potentials was about 5 msec (mean: 5.4 ± 0.9 msec; Fig. 3Go, C and D).


Figure 3
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  		FIG. 3. GnRH induced different types of membrane potential responses. The membrane potential (E) was recorded with the nystatin-perforated-patch whole-cell configuration of the patch-clamp technique. Application of GnRH and its washout were performed with a multichannel peristaltic pump. Panels A–D demonstrate the large variability of membrane potential responses to application of GnRH. In all recordings the length of the bar above the traces indicates the presence of GnRH. A, GnRH (1 nM) induced a transient hyperpolarization. B, GnRH (10 nM) induced membrane potential oscillations. C, An initially silent cell responded to GnRH (10 nM) with the initiation of action potentials. D, Application of GnRH (10 nM) induced membrane potential waves. Application of apamin (1 µM) blocked the oscillations. Washout of both, apamin and GnRH, induced recovery from the GnRH response.

 
In the majority of cells (22 of 28), application of GnRH induced a change in the membrane potential that consisted of an initial hyperpolarization, followed by different types of depolarizations (Fig. 3Go). In eight of the 22 responding cells, the resting potential was more depolarized (between –13 and –40 mV). Three of these eight cells responded to GnRH with a large sustained hyperpolarization to potentials between –60 and –75 mV (Fig. 3AGo). The five other cells responded to GnRH with the production of slow oscillations of the membrane potential with frequencies of about 0.2 Hz (Fig. 3BGo). In the continued presence of GnRH, the membrane potential repolarized to the initial resting potential or to even more depolarized potentials after 1–2 min (Fig. 3Go, A and B). In 14 of the 22 responding cells, the resting potential was more negative (between –45 and –75 mV). Some of these cells responded to GnRH with transient hyperpolarizations, which then led to ongoing firing of action potentials (Fig. 3CGo) or with a sequence of transient hyperpolarizations, followed by depolarizing waves that initiated bursts of action potentials (Fig. 3DGo). Application of apamin (0.1 or 1 µM), a selective blocker of small conductance Ca2+-dependent K (SK) (20), stopped the oscillations in all cells tested (seven of seven). The effects of GnRH as well as of apamin could be washed out (Fig. 3DGo). In two cells tested, a second application of GnRH after a time interval of about 5 min elicited a similar membrane potential change as in the first response.

Increasing the GnRH concentration from 1 to 100 nM has been shown to increase the magnitude of the intracellular Ca2+ concentration (7), which then increases an outward current presumably mediated by SK channels (21, 22, 23). This K+ conductance increase tends to increase the membrane potential during the hyperpolarizing phase of the GnRH-induced membrane potential oscillations. This hyperpolarization was after application of 1 nM GnRH –62.5 ± 2.5 mV (n = 2), 10 nM GnRH –60 ± 3 mV (n = 7), 50 nM GnRH –65 ± 3 mV (n = 6), and 100 nM GnRH –72 ± 3 mV (n = 2). In addition, we found that 1 nM GnRH elicited a response in only 50% of the cells (five of 10), whereas with higher GnRH concentrations, almost all cells responded. The nonresponding cells had resting potentials between –35 and –75 mV, and were obtained from four animals.

Recording of membrane currents
Voltage-clamp experiments were performed with the conventional whole-cell configuration. The same major types of macroscopic membrane currents were found as previously reported for rat (8, 24, 25), female mouse (26), and ovine (10) gonadotropes. The Na+ current measured in 17 gonadotropes (from four mice) activated near –40 mV and exhibited a maximum inward current near –10 mV (Fig. 4AGo). Peak inward current amplitudes (varying between 0.2 and 2.5 nA) were converted into current densities using the corresponding cell capacitances (mean: –92.9 ± 19.7 pA/pF; n = 10). Na+ conductance was half-maximally activated at –20.8 ± 0.2 mV (slope factor k = 6.6 ± 0.5 mV; Fig. 4BGo) and half-maximally inactivated at –65.7 ± 0.18 mV (k = 11.5 ± 0.8 mV; n = 17; data not shown).


Figure 4
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  		FIG. 4. Na+ currents. Na+ currents were recorded with the conventional whole-cell configuration of the patch-clamp technique. The pulse protocol consisted of 10-msec (ms) depolarizing potential steps from –120 to 70 mV in steps of 10 mV. Each depolarization was preceded by a 50-msec pulse to –120 mV to remove steady-state Na+ current inactivation. A, Family of superimposed traces of Na+ currents. Na+ currents were corrected for leakage current. The leakage current was defined as the currents measured at the membrane potentials between –120 and –70 mV. This current was extrapolated to more positive potentials assuming a linear dependence of the leakage current from the membrane potential. B, Na+ conductance was calculated from the equation GNa = INa/(Em – ENa), with GNa, Na+ conductance, INa, peak Na+ current, Em, membrane potential, and ENa, Na+ current reversal potential. Means ± SEM of the normalized conductance values (n = 10) were plotted vs. membrane potential (E). The Boltzmann function was fitted to the mean values (E0.5 = –21.0 mV, k = 6.5 mV).

 
Currents through voltage-dependent Ca2+ channels were measured using Ba2+ as a charge carrier. The Ba2+ current was activated at potentials more positive than –50 mV, and the maximum inward current was measured near 0 mV (Fig. 5Go). The mean current density at 0 mV was –37.9 ± 3.4 pA/pF (n = 20 from seven mice). In our experiments, nifedipine blocked about 50% of the Ca2+ channel current amplitude measured at 0 mV (EC50 = 0.55 µM), indicating that in addition to L-type channels, other types of high-voltage-activated Ca2+ channels contribute to the recorded currents. We found no indication for the presence of T-type Ca2+ channels.


Figure 5
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  		FIG. 5. Ca2+ channel currents. Ba2+ currents (IBa) through Ca2+ channels were measured with the conventional whole-cell configuration of the patch-clamp technique. Currents were elicited with 200-msec (ms) potential steps between –100 and 40 mV in 10-mV increments from a holding potential of –70 mV (see pulse protocols in Aa and Ab). Aa, Current traces elicited with voltage steps from –100 to 0 mV; at 0 mV the maximum peak Ba2+ inward current was reached. Ab, Current traces elicited with voltage steps from 10 to 40 mV. Current traces have been low-pass filtered with 2 kHz off-line. B, Ba2+ current densities plotted vs. membrane potential (E) (means ± SEM, n = 20). Means were connected by straight lines.

 
In most gonadotropes the outward K+ currents consisted of a delayed rectifying K+ current and a transient A-type K+ current (Fig. 6Go). However, in about 30% of gonadotropes, the transient K+ current was absent or had a tiny amplitude. Control K+ currents were measured from a holding potential of –70 mV with 200-msec potential steps from –70 to 50 mV with 10 mV increments preceded by a hyperpolarizing pulse to –120 mV. The mean current density of the peak total outward K+ current at 50 mV was 221.0 ± 23.7 pA/pF (n = 17, from five mice). To isolate the transient K+ current from the total current, the delayed rectifying K+ current was blocked by 10 mM TEA (Fig. 6BGo). The transient TEA-resistant current had a peak current density of 120.0 ± 22.3 pA/pF, and its conductance was half-maximally activated at –11.0 ± 3.2 mV (k = 15.0 ± 2.5 mV, n = 6; Fig. 6CGo). The delayed rectifying K+ current component was obtained as the TEA-sensitive current (Fig. 6DGo). The activation kinetics became faster with stronger depolarizations, and at more positive potentials, a slight inactivation occurred during the 200-msec test pulses. The current density at 50 mV was 146.6 ± 26.2 pA/pF. The conductance of the delayed rectifying K+ current was half-maximally activated at –5.7 ± 1.1 mV (slope factor k = 6.9 ± 0.3 mV, n = 6; Fig. 6EGo).


Figure 6
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  		FIG. 6. K+ currents. K+ currents were recorded with the conventional whole-cell configuration of the patch-clamp technique. The pulse protocol consisted of 200-msec (ms) depolarizing pulses between –70 and 50 mV in potential steps of 10 mV. Depolarizing pulses were preceded by 50-msec pulses to –120 mV. A, Control currents. B, To separate the transient component of the K+ currents from the delayed rectifying K+ current, 10 mM TEA was applied. C, Activation curve of the transient K+ current obtained by calculating the conductance values from the peak current amplitudes (using EK = –86 mV). Ek, K equilibrium potential. Means ± SEM of the normalized conductance values (n = 6) were plotted vs. membrane potential (E) and fitted with the Boltzmann function (E0.5 = –11.9 mV, k = 13.6 mV). Gk, K conductance; Gk max, maximal K conductance. D, The delayed rectifying TEA-sensitive K+ current component was obtained by subtracting the currents shown in panel B from the control currents shown in panel A. E, Activation curve of the delayed rectifying K+ current. Conductance of the delayed rectifying K+ current was calculated, normalized to the maximal conductance, and plotted vs. membrane potential (means ± SEM; n = 6). The mean values were fitted with the Boltzmann function (E0.5 = –5.6 mV, and k = 7.0 mV).

 
Calcium imaging of gonadotropes
In the beginning of an experiment, we searched for YFP-positive cells using the YFP filter set. Primarily, those parts of the coverslip were selected for recording that showed at least two isolated YFP-positive cells (Fig. 7AGo). Fura-2 fluorescence was measured as an indicator for a change in the intracellular Ca2+ concentration ([Ca2+]i). Simultaneous electrophysiological measurements were not performed in the cells under investigation. After application of GnRH, we observed a broad range of response patterns. Of 25 cells (from four mice), 23 responded to GnRH (10 nM) with an increase in [Ca2+]i. Most cells (n = 15) exhibited oscillations in [Ca2+]i ranging between 0.08 and 0.25 Hz (0.17 ± 0.02 Hz; mean ± SEM); eight cells responded with a plateau-like increase. Figure 7Go shows two examples. One cell responded to application of 10 nM GnRH with a biphasic response, consisting of an initial transient and a subsequent plateau-like increase in [Ca2+]i (Fig. 7BGo). The other cell responded with slow oscillations in the [Ca2+]i of 0.12 Hz (Fig. 7CGo). The two fluorescent cells that did not respond to GnRH belonged to culture dishes obtained from different animals. We never observed a GnRH-induced increase in [Ca2+]i in a nonfluorescent cell.

RHPA
Previous electrophysiological and calcium-imaging work has been performed on gonadotropes identified by the RHPA. To determine whether all gonadotropes can be identified by their secretion of LH/FSH, we performed the RHPA on cells obtained from GRIC/R26-YFP mice. Dissociated cells from two pituitaries were pooled. GnRH (200 nM) was present during the incubation period in one part of the Cunningham chambers, whereas in the other chambers, no GnRH was applied. In both experimental conditions, we found that only about 50% of the fluorescent cells exhibited hemolytic plaques (without GnRH: 46.6%, with GnRH: 51.4%; 482 fluorescent cells evaluated). Figure 8AGo shows examples of fluorescent cells with and without plaque formation. The distribution of plaque areas was similar in both types of experiments with a mean plaque area of 1982 µm2 without GnRH (control) and 1767 µm2 with GnRH. An additional experiment with GnRH stimulation performed on dissociated cells from a single pituitary yielded comparable data: 52.4% of the fluorescent cells (185 cells evaluated) formed plaques, and the mean plaque area amounted to 1948 µm2. This result shows that by using the RHPA, even in the presence of GnRH, only about 50% of gonadotropes can be detected.

In all experiments, we observed that bigger cells tended to induce bigger hemolytic plaques. The plaque area weakly correlated (R2 = 0.16) with the cell size. In addition, the percentage of plaque-forming cells was not strictly independent of cell size because about 65% of the bigger cells (diameter > 15 µm) formed plaques, whereas the smallest cells (diameter < 10 µm) rarely induced plaques.

Preliminary current clamp experiments on gonadotropes forming plaques and those without plaques revealed no apparent differences between secreting and nonsecreting gonadotropes. This is also true for the responses of the [Ca2+]i to GnRH in secreting and nonsecreting gonadotropes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A binary genetic strategy to visualize and manipulate gonadotropes
We used gene targeting in embryonic stem cells and a binary genetic strategy rather than a conventional transgenic approach to visualize and manipulate gonadotrope cells in the anterior pituitary. By inserting an IRES-Cre cassette just downstream of the GnRHR coding region, a bicistronic message was produced under control of the endogenous GnRHR promoter, from which the GnRHR and Cre recombinase were independently translated (Fig. 1BGo). In contrast to transgenic approaches, we did not add additional copies of the GnRHR promoter (or parts thereof) to the genome. Similar strategies using IRES elements in gene targeting have faithfully directed expression of reporter genes to different cell populations of various tissues with high specificity (11, 27, 28). Because Cre-mediated activation is not reversible, YFP fluorescent cells report the history of activity of the GnRHR promoter in GRIC/R26-YFP double-heterozygous mice. Our results show that 99.9% of gonadotropes identified by antibodies against LH and FSH express YFP in GRIC/R26-YFP mice (Fig. 2BGo), demonstrating the validity of this approach. We have occasionally observed YFP+ cells negative for LH/FSH staining with a frequency of 1.8%. These cells could represent gonadotropes with gonadotropin expression levels below the detection threshold of our antibody staining protocol. Alternatively, they could be cells expressing the GnRHR, but not gonadotropins.

We decided to coexpress Cre recombinase rather than a fluorescent reporter gene such as YFP together with the GnRHR. Adopting this binary genetic strategy, we uncouple fluorescence intensity and activity of the GnRHR promoter. After Cre-mediated excision of the floxed Stop signal, transcription of the YFP reporter gene (and, thus, fluorescence intensity) depends on the ROSA26 promoter and not on the GnRHR promoter, which is highly regulated in gonadotropes (reviewed in Ref. 1). Therefore, gonadotropes should be easily identifiable by fluorescence, even if the GnRHR promoter is not particularly active at the time of analysis. This should for example facilitate experiments attempting to isolate and characterize gonadotropes at different stages of the female estrus cycle with reportedly high fluctuations in GnRHR expression (29).

Tagging gonadotropes with fluorescence provides a simple tool for their identification, isolation, and characterization without the necessity of additional manipulation. Using fluorescence-activated cell sorting on dissociated pituitary cells from GRIC/R26-YFP mice, we have been able to produce highly enriched populations of gonadotrope cells (Klein, T., and U. Boehm, unpublished observation). With these cell populations, gene expression profiling of primary gonadotropes will be possible. It will be interesting to compare these profiles to data obtained from cell lines derived from Sv40 transformed pituitary tumors (30). Using the same gonadotrope cell line, a recent study reported significant heterogeneity in the biosynthetic response to GnRH (31). With the availability of the GRIC/R-YFP mice, the GnRH signaling network could now be analyzed in primary gonadotropes. In addition, functional experiments using calcium-imaging and patch-clamp techniques can now be performed in cell culture as well as in acute pituitary slices prepared from GRIC/R26-YFP mice. This will also allow simultaneous analysis of populations of gonadotropes by calcium imaging, whereas events such as the recently reported plasticity in response to GnRH can be studied in great detail by time-lapse imaging (32).

Heterogeneity of gonadotropes
Fluorescent gonadotropes were found with a frequency of approximately 15% in primary pituitary cell cultures prepared from male GRIC/R26-YFP mice. This percentage is similar to the one reported for GnRH target cells in primary pituitary cell cultures prepared from male rats [16% (33)]. Fluorescent gonadotropes are easily identified using a YFP filter set, allowing potentially unbiased sampling from the gonadotrope population. To this end, we used electrophysiological experiments, calcium imaging, and the RHPA in these fluorescent cells to study the physiological properties of primary gonadotropes. We found that the resting potential of male mouse gonadotropes varies between –13 and –75 mV with a mean of –52 mV and that about 25% of gonadotropes were spontaneously active. Similar observations were made in ovine gonadotropes (10). The electrophysiological responses to GnRH consisted of a sequence of membrane potential hyperpolarizations and depolarizations. It has been well established that the GnRH-induced membrane potential changes are predominantly due to an interplay between the initial increase in the [Ca2+]i and the ensuing activation of apamin-sensitive, Ca2+-dependent SK-type K+ channels and the subsequent decrease in [Ca2+]i due to transport of Ca2+ into the endoplasmic reticulum (8, 9, 21, 22, 23). Therefore, after GnRH application in all responding cells, an initial hyperpolarization was observed that was followed by various types of membrane potential changes. Some cells initiated oscillations of the membrane potential of low frequency (~0.2 Hz), whereas cells that were initially silent responded to GnRH with ongoing firing of action potentials after initial transient hyperpolarizations. Gonadotropes with more depolarized resting membrane potentials responded to GnRH either with a large sustained hyperpolarization or with the development of slow membrane potential oscillations consisting of hyperpolarizing and depolarizing waves. In rat gonadotropes similar observations have been made (8, 9). Especially, in ovine gonadotropes a broad range of resting membrane potentials (from –20 to – 75 mV, with a mean of –43) and of responses of the membrane potential to GnRH has been reported (10). The responses of [Ca2+]i to GnRH application were also variable. GnRH could induce oscillations in [Ca2+]i with a frequency of about 0.2 Hz, which corresponded to the slow oscillating membrane potential waves. In addition, in mouse gonadotropes GnRH could induce plateau-like increases in [Ca2+]i. The dependency of the membrane potential response on the GnRH concentration in male mouse gonadotropes was similar to that observed in ovine gonadotropes (10).

In voltage-clamp recordings on gonadotropes from GRIC/R26-YFP mice, voltage-activated Na+ and K+ currents exhibited similar electrophysiological properties as has been reported in rat (24, 25, 34), female mouse (26), and ovine gonadotropes (10). Of the Ca2+ channel current, 50% was blocked by nifedipine, indicating the presence of L-type Ca2+ channels. The nifedipine-insensitive current was mediated by N-type Ca2+ channels in ovine gonadotropes (10). Because the concentrations of Ca2+ and Ba2+ in the extracellular solutions in the different publications vary, it is difficult to compare the density of Ca2+ currents between the gonadotropes of different species. We did not observe T-type Ca2+ channels in male mouse gonadotropes, similar to what has been described for ovine gonadotropes (10). In contrast, T-type Ca2+ currents are present in rat gonadotropes (24, 25). The total K+ current recorded in GRIC/R26-YFP mice has a current density similar to that found in ovariectomized adult female mice (26). The mean current density values obtained for the transient and the delayed rectifying K+ current component are also very close to the ones found in rat gonadotropes (25), except that in the present study, the transient K+ current had a smaller mean current density than the delayed-rectifying K+ current. In addition to voltage-dependent K+ currents, the apamin-sensitive Ca2+-dependent SK-type K+ current is present in all gonadotropes.

In previous studies the RHPA was used to identify rat gonadotropes for further functional analysis (8, 24). We show here that only about 50% of mouse gonadotropes produced plaques, indicating that not all gonadotropes released enough LH/FSH to form plaques. In the present study, antibodies against both hormones were applied simultaneously to enhance further the sensitivity of the assay, although this made a differentiated analysis of hormone secretion impossible. Even the presence of GnRH did not significantly enhance the percentage of plaque-forming gonadotropes. Our finding that a comparable percentage of nonsecretors was observed in experiments with and without application of GnRH might be due to the long incubation time. The incubation time was chosen to identify as many gonadotropes as possible by the RHPA rather than to study differences between basal and stimulated hormone secretion. Nonsecretors among RHPA-identified gonadotropes have been described in diestrus female rats in which only 50% of LH containing cells form plaques, whereas almost all LH containing cells form plaques if prepared from proestrus animals (17). Up till now, no comparable data sets exist for male mice. Our results, obtained in male mice, show that the RHPA used as a method for identification of gonadotrope cells yields only a subset of gonadotropes. Further investigation is required to determine whether the gonadotropes producing no plaques have different properties from those forming plaques.

Future experiments
Besides the questions that can be addressed in live YFP+ gonadotropes, our mouse model can be used for direct expression of other genes in gonadotropes simply by breeding GRIC mice with different members of the ROSA26 reporter zoo. For example, we have bred GRIC mice to the ROSA26-DTA mouse strain that carries a Cre recombinase-activated gene encoding diphtheria toxin chain A (DTA) (35). Activation of DTA in GRIC/R26-DTA mice leads to cell death, and pituitary sections prepared from these mice are completely devoid of LH/FSH containing cells, indicating efficient ablation of all gonadotropes (Wen, S., and U. Boehm, manuscript in preparation). Furthermore, we have generated a mouse model to ablate gonadotropes at any time point during development by breeding GRIC mice with the ROSA26- inducible diphtheria toxin receptor (DTR) mouse strain that carries a Cre-activated DTR (36). We are able to achieve efficient inducible ablation of gonadotropes in GRIC/R26-inducible DTR mice simply by ip injections of DTA (Wen, S., and U. Boehm, manuscript in preparation). This mouse model should also facilitate studies analyzing the functional dependence of different cell types within the anterior pituitary. In addition, GRIC mice can direct the knockout of any floxed gene to the gonadotrope population within the pituitary. Therefore, the GRIC mouse model represents an important new tool in the understanding of gonadotrope biology.


   Acknowledgments
 
We are indebted to Olaf Pongs for continuous support. We also thank Joseph Gogos (Columbia University, New York, NY) for providing the internal ribosome entry site-Cre cassette, Irm Hermans-Borgmeyer (Transgenic Service Unit, Zentrum für Molekulare Neurobiologie Hamburg, Hamburg, Germany) for help with embryonic stem cell work and blastocyst injections, Michaela Schweizer (Morphology Service Unit, Zentrum für Molekulare Neurobiologie Hamburg) for preparing the dissociated pituitary cell cultures, and Libby Guethlein for critical comments on the manuscript. We thank Christian Mayer for help with the calcium-imaging experiments. We also thank Sabine Wehrmann and Annett Hasse for expert technical assistance, and Dörte Clausen for help in figure preparation.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft Grant DFG BO1743/2-1 (to U.B.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2008

1 S.W. and J.R.S. contributed equally to this work. Back

Abbreviations: DTA, Diphtheria toxin chain A; DTR, diphtheria toxin receptor; FRT, Flp recombination target; GnRHR, GnRH receptor; GRIC, GnRH receptor-internal ribosome entry site-Cre; IRES, internal ribosome entry site; pgk, phosphoglycerate kinase; RHPA, reverse hemolytic plaque assay; TEA-Cl, tetraethylammonium chloride; SK, small conductance Ca2+-dependent K; TTX, tetrodotoxin; YFP, yellow fluorescent protein.

Received November 2, 2007.

Accepted for publication February 22, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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