Anterior pituitary leptin expression changes in different reproductive states: in vitro stimulation by gonadotropin-releasing hormone

Abstract

This study was designed to learn more about the changes in expression of rat anterior pituitary (AP) leptin during the estrous cycle. QRT-PCR assays of cycling rat AP leptin mRNA showed 2-fold increases from metestrus to diestrus followed by an 86% decrease on the morning of proestrus. Percentages of leptin cells increased in proestrus and pregnancy to 55-60% of AP cells. Dual labeling for leptin proteins and growth hormone (GH) or gonadotropins showed that the rise in leptin protein-bearing cells from diestrus to proestrus was mainly in GH cells. Only 10-20% of leptin cells in male or cycling female rats coexpress gonadotropins. In contrast, 50-73% of leptin cells from pregnant or lactating females coexpress gonadotropins and only 19% coexpress GH, indicating plasticity in the distribution of leptin. Leptin cells expressed GnRH receptors, and estrogen and GnRH together increased the coexpression of leptin mRNA and gonadotropins. GnRH increased cellular leptin proteins three to four times and mRNA 9.8 times in proestrous rats and stimulated leptin secretion in cultures from diestrous, proestrous, and pregnant rats. These regulatory influences, and the high expression of AP leptin during proestrus and pregnancy, suggest a supportive role for leptin during key events involved with reproduction.

Figure 1

illustrates controls for the detection of leptin mRNA. Figure 1a is a field from a male rat pituitary culture showing label for leptin mRNA with 100 ng/ml biotinylated antisense oligonucleotide probe complementary to leptin mRNA. Label is in irregular dense black patches in the cells (arrows). Figure 1b shows a field treated with 100 ng/ml biotinylated sense oligonucleotide leptin mRNA probe. No labeling is evident in this field. Bar=15 μm.

Figure 2

Expression of pituitary leptin proteins and mRNA in different stages of the cycle. Figure 2a is a graph showing the changes in percentage of AP cells with leptin proteins or mRNA with the stage of the cycle. Closed star=highest values in diestrus (die), or proestrus AM (ProAM) or PM (ProPM). Open stars=lowest values in estrus (est) or metestrus (met). Figure 2b shows a graph of the results from the QRT-PCR assays for leptin mRNA in extracts from pituitaries from cycling female rats. The rise in leptin mRNA from metestrus (met) to diestrus (die) is significant (p=0.014) as is the decline in expression from diestrus PM (diePM) to proestrus AM (p=0.002). Values remain low through estrus. Values for metestrous rat pituitaries are slightly higher than those for proestrous animals (p<0.022, proestrus AM and p<0.044, proestrus PM). Closed Star=highest levels; Open star=lowest levels. Figures 2c–e are light micrographs showing the increase in density of labeling from proestrous AM (Figure 2c) to proestrous PM (Figure 2d) and the loss of cellular leptin proteins on the AM of estrus (Figure 2e). Arrows=leptin bearing cell. Bar = 15 μm.

Figure 3

The graph shows the results of counts of leptin mRNA or protein-bearing cells in pregnant or lactating female rats. See text for statistics. The photographs depict immunolabeling for leptin in fields from pregnant (3b) or lactating (3c) rats. Bar=15 μm.

Figure 4

The top graph (Figure 4a) shows the counts of cells following dual immunolabeling for leptin proteins and LHβ, FSHβ or GH proteins and differences based on gender and reproductive state. The Y axis expresses the counts as percentages of total pituitary cells counted (% AP cells). See text for statistical details. Figure 4b shows the same counts expressed as percentages of leptin-bearing cells in these same experimental groups. See text for statistical differences. Filled stars=highest values and open stars=lowest values. Figure 4c and d illustrate fields from proestrous (Figure 4c) and pregnant (Figure 4c) dual labeled for leptin (black) and GH (orange). Numerous dual labeled cells (arrows) are evident only in the field from proestrous rats. In the pregnant rat, most of the gray, leptin-bearing cells do not contain GH. In contrast, Figures 4e and f are fields labeled for leptin (black) and LHβ (orange) from proestrous (Figure 4e) or pregnant (Figure 4f) animals. Only the field from the pregnant rat shows numerous dual labeled cells (arrows). In the proestrous rat, most of the gray, leptin bearing cells do not contain LHβ.

Figure 5

The top graph (Figure 5a) shows the counts of cells following dual labeling for leptin mRNA (with in situ hybridization) and LHβ or GH proteins and differences based on gender or reproductive state. The Y axis shows the counts as percentages of anterior pituitary cells. The bottom graph (Figure 5b) expresses these same counts as percentages of leptin-bearing cells. See text for statistical details. The colored photographs in Figure 5c depicts a dual labeled field from a pregnant rat showing a number of cells leptin mRNA (black patches, arrows) and GH (orange), but no dual-labeled cells. Bar=15 μm Figure 5d depicts a field from a pregnant rat dual labeled for leptin mRNA (black) and LHβ proteins (orange). Many dual labeled cells are shown in this field (hollow arrows) Bar=20 μm.

Figure 6

The top photograph depicts dual labeling for biotinylated analog of GnRH (black) followed by immunolabeling for leptin proteins (orange). Arrows in Figure 6a indicated leptin-bearing cells with GnRH receptors. Bar=20 μm. Figure 6b shows a cluster of cells that includes a leptin containing GnRH target cell (Gn-L, arrows show Biotinylated GnRH binding) and a leptin-bearing cell with no GnRH receptors (L) and unlabeled cells (U). Figure 6c shows the effect of overnight estrogen on the expression of biotinylated GnRH-labeled cells with and without leptin proteins. Star=significantly different from vehicle control. Figure 8d shows the effect of estrogen and GnRH overnight on the co-expression of leptin mRNA and LHβ or FSHβ proteins. The Y axis is the percentage of pituitary cells. Star=significantly different from vehicle control.

Figure 7

Cellular leptin proteins (Figure 7a) and mRNA (Figure 7b) were analyzed by automated image analysis and the Integrated Optical Density was calculated for each field. The Y axis is the average integrated optical density calculated over 20–25 randomly selected fields ± sem. Closed star= significantly different from vehicle control (see text for statistical details). Open star=significantly different from IOD with 500 pM. Photographs depict the labeling for leptin mRNA in the vehicle (Figure 7c), 100 pM GnRH treated (Figure 7d) or 1 nM GnRH treated (Figure 7e) cultures. Arrows show labeling for leptin mRNA. Bar=15 μm.

Figure 8

Cells from diestrous, proestrous AM and pregnant females were treated for 1 h with 1 nM GnRH. Media were assayed by EIA for leptin. Basal secretion from pregnant rat cultures is higher than that from diestrous rat cultures (open star). GnRH treatment stimulated higher media levels in all experimental groups (closed star). GnRH treated cultures from pregnant rats have higher media levels than all other groups. See text for statistics.