Gonadotrope plasticity at cellular and population levels

Abstract

Hormone-secreting cells within the anterior pituitary gland may form organized and interdigitated networks that adapt to changing endocrine conditions in different physiological contexts. For gonadotropes, this might reflect a strategy to cope with acute changes throughout different female reproductive stages. The current study examined gonadotropes in female mice at characteristically different hormonal stages: prepubertal, postpubertal, and lactating. Gonadotrope plasticity was examined at the level of the whole population and single cells at different stages by imaging both fixed and live pituitary slices. The use of a model animal providing for the identification of selectively fluorescent gonadotropes allowed the particular advantage of defining cellular plasticity specifically for gonadotropes. In vivo analyses of gonadotropes relative to vasculature showed significantly different gonadotrope distributions across physiological states. Video microscopy studies using live slices ex vivo demonstrated pituitary cell plasticity in the form of movements and protrusions in response to GnRH. As positive feedback from rising estradiol levels is important for priming the anterior pituitary gland for the LH surge, experiments provide evidence of estradiol effects on GnRH signaling in gonadotropes. The experiments presented herein provide new insight into potential plasticity of gonadotropes within the anterior pituitary glands of female mice.

Fig. 1.

Gonadotropes were homogeneously distributed in the anterior pituitary before puberty. Ventral view of a three-dimensional reconstruction (68 μm z-stack) of a whole-mount preparation of an entire pituitary gland prepared from a female 3-wk-old GRIC/eR26-τGFP mouse is shown. A, Vascular architecture was visualized with rhodamine-coupled gelatin (red). Note the hypophyseal portal vessels located in the pars tuberalis (Pt) and the capillary bed of the AP and PP. B, Gonadotropes (green) were visualized homogeneously distributed throughout the AP but were absent from the PP. C, Merge. Gonadotropes were homogeneously distributed throughout the rostral (D), lateral (E), and caudal (F) areas of the AP. G, Quantification of gonadotrope cells. Scale bars, 200 μm (A–C), 50 μm (D–F).

Fig. 2.

Gonadotropes were concentrated at the pars tuberalis in the anterior pituitary after puberty. Ventral view of a three-dimensional reconstruction (68 μm z-stack) of a postpubertal female pituitary gland prepared from a 14-wk-old GRIC/eR26-τGFP mouse at diestrus (A, C, E, G, H, and I) and proestrus (B, D, and F) is shown. Vascular architecture was visualized with rhodamine-coupled gelatin (red; A and B). Gonadotropes (green) were concentrated at the pars tuberalis (Pt) (C and D). E, and F, Merge. The overall distribution of gonadotropes within the AP did not change during the different stages of estrus. J, Increased number of gonadotropes in the Pt in postpubertal females. K, The total number of gonadotropes per animal increased during reproductive maturation. Scale bars, 300 μm (A–C), 50 μm (D–F).

Fig. 3.

Redistribution of gonadotropes during lactation. Ventral view of a three-dimensional reconstruction of an adult GRIC/eR26-τGFP pituitary after 1 wk of lactation and after perfusion with rhodamine-coupled gelatin (red; A, C, and D) is shown. The arrows highlight clusters of gonadotropes (green; B, C, and D) that have formed in the AP of a lactating GRIC/eR26-τGFP mouse. Scale bar, 300 μm (A, B, and C). D, The box in panel C at higher magnification. The scale bar (D), 30 μm.

Fig. 4.

Confocal time-lapse imaging. Individual image frames from a 60-min confocal time-lapse video of an acute 200-μm pituitary slice prepared from a GRIC/R26-YFP mouse. Arrows mark a new protrusion forming in the direction of a blood vessel (bv).

Fig. 5.

Gonadotropes contact multiple blood vessels. Three-dimensional reconstruction (20 μm z-stack) of the caudal area of postpubertal female pituitary gland prepared from a 14-wk-old GRIC/eR26-τGFP mouse at diestrus after perfusion with rhodamine-coupled gelatin is shown. The arrows mark gonadotrope protrusions extending in the direction of blood vessels (red). B, A three-dimensional surface rendering (Imaris) of A for better visualization of blood vessels. Scale bars, 15 μm.

Fig. 6.

Gonadotrope process extension with long-term E₂ and GnRH exposure. A, Organotypic pituitary gland slices were exposed to E₂ for 14 h. After 100 nm GnRH, processes extended from several of the YFP+ gonadotropes (B; white arrows).

Fig. 7.

Long-term estradiol treatment increased GnRH-induced plasticity in live pituitary slices. Adult female GRIC/R26-YFP mice in diestrus 1 with YFP expression in gonadotropes were used for live time-lapse video microscopy of organotypic pituitary gland slices. A, The graph depicts estradiol or vehicle exposure before and after GnRH treatment (100 nm). There was no effect of estradiol treatment alone on the percentage of gonadotropes with process extensions in a given region of interest. Long-term estradiol treatment (14 h+) significantly increased GnRH-induced cell process extension compared with vehicle or 1.5 h E₂ (10 nm). There were no discernible effects of short-term estradiol treatment (1.5 h) on GnRH-treated gonadotropes. Veh: Vehicle (n = 8 pituitary slices); 1.5 h: 1.5 h of E₂ treatment exposure started at start of video acquisition (n = 9 pituitary slices); 14 h: 14 h+ of estradiol treatment before start of video acquisition (n = 11 pituitary slices); *, P < 0.01. B, There were no differential effects of estradiol on the process extensions over time. Time-lapse video images were captured from live murine pituitary slices treated with GnRH for 90 min. Video acquisition began at the start of GnRH treatment and was analyzed in 30-min segments to quantify time-dependent cell process extension (note: some cells had process extension for more than one 30 min segment and were counted in each time period they had a process extension.) Estradiol treatment (10 nm) for the short term (1.5 h) and long term (14 h+) was analyzed compared with vehicle (1 μl per 1 ml of 100% ethanol). C, There was no effect of estradiol pretreatment on the length of cell process extensions after GnRH treatment. The lengths of the prominent process extensions from five gonadotropes per slice after GnRH treatment were analyzed to quantify the average length of the process extension. There was no difference between estradiol treatment groups and vehicle. Length is reported in micrometers from the edge of the cell to the edge of the process extension.