Changes in leptin expression with the reproductive state
We had previously reported changes in leptin proteins (McDuffie et al. 2004
) with the stage of the cycle. The continuing analysis in this study added more data detecting both mRNA and proteins, including that from rats taken at 2 PM on the day of proestrus and a new graph has been constructed (). All data in the following paragraphs are averages ± sem.
Figure 2 Expression of pituitary leptin proteins and mRNA in different stages of the cycle. 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), (more ...)
There is a gradual rise in percentages of AP cells with leptin proteins from a low of 21.0 ± 4.0% on the AM of estrus to a peak of 55.0 ± 3.0% of AP cells on the afternoon of proestrus. illustrate fields labeled for leptin proteins, comparing labeling on the morning and evening of proestrus and showing reduced labeling on the morning of estrus. The peak expression of leptin during proestrus is higher than that in all other groups (p<0.001), including male rats, which had 39.6 ± 1.0% of AP cells with leptin proteins.
Leptin mRNA is expressed in 32–37% of pituitary cells in male or in estrous, metestrous, or diestrous female rats (). Thus, early in the cycle (estrus and metestrus), there are more cells with leptin mRNA than leptin proteins. There are no significant differences among these groups. However, there is a significant decline (p<0.002) in the percentages of cells with leptin mRNA to 20.0 ± 3.0% on the morning of proestrus. This reduction on the AM of proestrus was confirmed by the QRT-PCR assays (), which showed a significant 86% decline from diestrous PM to proestrous AM (p<0.007). The QRT-PCR assays show that leptin mRNA is maintained at relatively low levels through estrus. Then, levels rise during metestrus and diestrus. The 2—fold rise in diestrus AM and PM is to values higher than all other groups (p<0.015). The two diestrous values are not different from one another.
The increase in mRNA from metestrus to diestrus detected by the QRT-PCR assays, was not detected by a change in percentages of leptin-bearing cells. However, densitometric analysis showed that the total area of label for leptin mRNA increased from 844.0 ± 114.0 μm2 to 1246.0 ± 15.0 μm2 (p<0.01) during this period.
To learn if other reproductive states were associated with changes in pituitary leptin, cell populations from pregnant or lactating rats were studied. shows that populations from pregnant rats contained relatively the highest percentages of AP cells with leptin proteins (60.0 ± 2.0%) or mRNA (44.0 ± 2.0%), when compared with all other groups. The percentages of AP cells with leptin proteins are higher than those from males and all cycling groups except females taken on the afternoon of proestrus. The values for mRNA-bearing cells are higher than those from all other groups (p<0.001).
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. (more ...)
AP populations from females taken on the 3rd day of lactation had midrange levels of leptin proteins, when compared with other physiological states. There were 32.0 ± 3.0% cells with leptin proteins and 40.0 ± 2.0% cells with leptin mRNA (). The percentages of AP cells with leptin proteins in lactating rats are higher than those from estrous rats (p<0.02) and lower than those from proestrous (p<0.047) or pregnant rats (p<0.001). The percentages of AP cells with leptin mRNA are higher than all groups, but pregnant rats. Photographs of fields from these rats are also illustrated in .
Cell types that express co-leptin proteins
Our previous study had reported that most leptin-bearing cells were somatotropes. However, the timing and direction of changes in expression during the cycle () suggested that gonadotropes might be involved. Dual immunolabeling was therefore done on some of these experimental groups to test this hypothesis. The main question was focused on whether or not somatotropes or gonadotropes, or both, contributed to the rise in the percentage of leptin-bearing cells during proestrus and pregnancy.
When co-expression of leptin proteins and GH proteins was tested, there was a significant increase (p<0.001) in the percentages of dual-labeled AP cells from 25.0 ± 3.0% on the morning of diestrus to 37.0 ± 4.0% of the population on the morning of proestrus (). This increment (12 percentage points) matches that seen when the total percentages of AP cells with leptin were calculated (). The overall percentage of AP cells with GH proteins did not change significantly. It was 36.0 ± 6.0% on the AM of diestrus and 41.0 ± 4.0% on the morning of proestrus.
Figure 4 The top graph () 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 (more ...)
In contrast, the percentage of AP cells that co-expressed leptin and gonadotropins did not change from diestrus to proestrus. Diestrous populations had 6.5 ± 1.0% or 7.0 ± 1.0% cells with leptin and LH or FSH, respectively and proestrous populations had 8.0 ± 2.0% AP cells with leptin and LH or FSH (). Previous studies have shown that bihormonal (cells with both LH and FSH) gonadotropes predominate in the population of diestrous and proestrous rats, representing over 70% of the gonadotrope population (Childs et al, 1987
), which suggests that leptin is being expressed by cells that are mostly bihormonal.
Cultures from male rats exhibited a profile similar to that of diestrous rats with 26.0 ± 2.0% of AP cells co-expressing leptin and GH. Only 4.0% of AP cells co-expressed leptin and LH or FSH in the male, values which were also not different from diestrous rats. Those for AP cells co-expressing leptin and LH were significantly lower than percentages seen on the AM of proestrus (p=0.045).
Significant plasticity in expression was seen in populations from pregnant or lactating rats () as leptin-bearing cells shifted from being predominantly somatotropes to one that is predominantly gonadotropes. There were no significant changes in the percentages of cells with GH proteins in pregnant or lactating rats. There was a significant reduction (p<0.001) in the percentages of AP cells that co-expressed GH and leptin proteins to 12.0 ± 2.0% in the populations from pregnant females and 6.6 ± 1.0% in those from lactating females. In pregnant rat cells, there was a significant rise in percentage of AP cells that co-expressed LH or FSH and leptin to 42.0 ± 1.0% or 36.0 ± 4.0%, respectively. Cells from lactating females also had significantly more AP cells with LH and leptin (18.7 ± 0.4%) or FSH and leptin (17.0 ± 1.0%) than all other groups, but the pregnant rats.
The analysis also focused on the proportion of leptin-bearing cells that expressed each of the pituitary hormones tested. These values were also used to predict if other cells contributed to leptin-bearing cells and to validate the data in . The validation was done by manually multiplying the percentages of leptin cells that contained each of the hormones by the overall percentages of AP cells that contained leptin, reported in and . These calculated percentages were within 0.5–1.0 percentage points of those derived from the cell counts in .
GH stores are found in 67–71% of the leptin protein-bearing cells in males, diestrous or proestrous females, values that are not significantly different from one another (). In contrast, only 19–20% of leptin cells co-express GH proteins in pregnant or lactating animals, which is significantly lower than values from the other groups. (p<0.001).
LH is found in 14–17% and FSH is found in 17–19% of leptin cells in diestrous and proestrous rat populations, values which are not different from one another. As stated above, it is likely that leptin may be expressed in gonadotropes, at least half of which are bihormonal (store both LH and FSH). In populations from pregnant rats, most leptin-bearing cells express LH (73.0 ± 2.0% of leptin cells) and/or FSH (62.0 ± 6.0% of leptin cells). These data show clear overlap in the percentages of leptin bearing cells with gonadotropins, which supports the hypothesis that leptin is expressed in part by bihormonal gonadotropes.
In male rats, only 10% of leptin cells co-express LHβ or FSHβ proteins. The LH values are significantly lower (p<0.006) than all female groups, except those from proestrus AM. The percentage of leptin cells with FSH in the male are lower than all female groups (Student’s T test).
also depicts the dual labeling for leptin proteins and GH () or LHβ proteins () in proestrous () or pregnant () rats. The fields dual-labeled for leptin and GH show the contrast between the numerous dual labeled cells in the proestrous female rat () with only one in the field from the pregnant rat (). Similarly, few LH cells co-express leptin in the field from the proestrous rat () and there are numerous dual labeled LH-leptin cells in the field from the pregnant rat ().
Cell types that co-express leptin mRNA
Dual labeling for leptin mRNA and LH or GH proteins was also done on some of these animals, focusing on diestrus because it was a peak time of expression of mRNA. had shown that, during most stages of the cycle, but diestrus, leptin mRNA was found in 32–36% of pituitary cells. In diestrous cell populations, 19.0 ± 2.0% of AP cells co-expressed leptin mRNA and GH, which is 51% of leptin mRNA bearing cells (). Among LHβ gonadotropes, the percentages were similar to those seen when leptin protein-bearing cells were counted (7.25 ± 1.0% of AP with leptin mRNA and LHβ). Similar results were seen for FSHβ labeling (data not shown).
Figure 5 The top graph () 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 (more ...)
The loss in cells co-expressing GH and leptin mRNA was again evident in pregnant and lactating females; values are significantly lower than values in diestrous animals (p<0.001) (). In contrast, more AP cells co-expressed leptin mRNA and LHβ proteins (16–17%), values which were significantly higher than those from diestrous rats (p<0.001).
In cells from male rats, 26.0 ± 1.0% of AP cells express leptin mRNA and GH proteins, which is comparable to the levels seen in the dual immunolabeled fields and greater than values in the diestrous female or those from pregnant or lactating animals (p<0.001) (). When LH gonadotropes were analyzed, the percentages of AP cells with leptin mRNA and LHβ in the male were comparable to those seen after dual immunolabeling.
The percentages of leptin mRNA bearing cells that contain GH are higher in the male then all of the female groups (73.0 ± 2.0%; p<0.001) (). Percentages of leptin-mRNA bearing cells that contain LHβ are only 10.0 ± 2.0% in cells from male rats and 20.0 ± 2.0% in those from diestrous females, which is significantly higher than values in the male (p<0.01, Student’s T test).
As in the case of the dual immunolabeling, pregnant and lactating rats had more leptin-bearing cells with LHβ proteins (36.0 ± 1.0 or 44.0 ± 9.0% of leptin cells in pregnant or lactating groups, respectively). These values were not different from one another, but they were significantly higher than those from all other groups. also illustrates the dual labeling for leptin mRNA and GH or LHβ in pregnant rats, showing the low expression of leptin mRNA in GH cells () and the high expression in cells with LHβ antigens ().
GnRH and estrogen effects on leptin expression
The timing of the rise in leptin protein expression during the cycle from diestrus to proestrus coincided with the rise in estrogen, which stimulates the production of GnRH receptors by gonadotropes (Lloyd et al, 1988
) and somatotropes (Childs et al, 1994a
). We hypothesized that leptin expression might be regulated in vivo by GnRH pulses. To determine if GnRH could bind directly to leptin-bearing cells, we exposed freshly dispersed pituitary cells from 3 groups of diestrous rats (3 rats/group) to 1 nM biotinylated GnRH for 10 min. After biotinylated GnRH was detected by avidin-biotin complexes (McDuffie et al, 2004
), dual labeling was used to identify leptin in these cells. illustrate dual labeling for Bio-GnRH and leptin proteins. The biotinylated neuropeptide is seen as dark patches on or near the surface of the cell and the leptin labeling is seen inside the cells. In diestrous rats, cells with GnRH receptors and leptin proteins are 11.5 ± 2.0%. This represented 30.0 ± 3.0% of leptin-bearing cells and 73.0 ± 3.0% of GnRH target cells.
Figure 6 The top photograph depicts dual labeling for biotinylated analog of GnRH (black) followed by immunolabeling for leptin proteins (orange). Arrows in indicated leptin-bearing cells with GnRH receptors. Bar=20 μm. shows a cluster (more ...)
Estrogen is a well established modulator of GnRH receptors and our previous studies have shown that 100 pM increases the percentage of GnRH-target cells, when given overnight to diestrous rats (Lloyd and Childs 1988
). These studies used this experimental approach on an additional 3 groups of diestrous rats to learn if estrogen increased the number of GnRH receptors on leptin-bearing cells. shows that, whereas estrogen does increase the overall percentage of GnRH-target cells as in our previous studies (Lloyd et al. 1988
), it does not significantly increase the number of leptin-bearing cells that bind GnRH, which remain at 13.25 ± 2.0% of AP cells.
The next study was designed to learn if estrogen and GnRH could increase leptin expression by gonadotropes. Three groups of cells pooled from 3 diestrous rats/group were treated with and without 100 pM estradiol overnight and then given vehicle or 1 nM GnRH for 1 h the next morning. They were then fixed and labeled for leptin mRNA followed by immunolabeling for LHβ or FSHβ. Neither estrogen nor GnRH alone stimulated more gonadotropes to express leptin mRNA (data not shown). However, shows that when added together, estrogen and GnRH stimulated a significant increase in AP cells that co-express LHβ and leptin mRNA from 7.0 ± 2.0% to 11.0 ± 3.0% (p<0.03) of AP cells. Similarly, estrogen and GnRH together stimulated an even greater increase in the percentages of cells with leptin mRNA and FSHβ from 8.0 ± 3.0% to 15.0 ± 6.0% (p<0.02).
To test if the overnight incubation in estrogen may have caused losses in expression of leptin mRNA, these data were compared with those from freshly dispersed cultures (7.0 ± 1.0% of AP cells from diestrous rats express leptin mRNA and LHβ, as shown in ). Similarly, 7.25 ± 2.8% of AP cells co-express leptin mRNA and FSHβ in these same cultures. Both sets of findings point to no losses in leptin mRNA during the overnight incubation in estrogen.
GnRH stimulation of cellular and secreted leptin
Because GnRH receptors are at a peak late in diestrus, extending to the morning of proestrus (Lloyd and Childs 1988
; Childs et al. 1994b
) cells from proestrous AM female rats were studied to learn more about the specific effects of GnRH on leptin mRNA and protein expression.
illustrates the study of freshly dispersed cells from rats taken on the AM of proestrus. There is a significant increase in the average IOD of labeling after 1 h in 100 pM GnRH (p<0.001), which plateaus at 500 pM. However, there is a significant decrease in IOD of labeling for leptin proteins after 1 nM GnRH (p<0.03), when compared with that following 500 pM. The value for 1 nM GnRH is still higher than the IOD for the vehicle treated group (p=0.009). Similar studies were done of cells treated for 3 h with GnRH and there were no differences in expression of leptin proteins or mRNA.
Figure 7 Cellular leptin proteins () and mRNA () 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 (more ...)
shows a similar response to GnRH when IOD of label for mRNA was detected. Note that the average IOD in the vehicle control is about 5—fold lower than that for the proteins (). This reflects the lower expression of mRNA on the AM of proestrus seen in . The IOD for leptin mRNA label is significantly increased in all three concentrations of GnRH; the increase with 100 pM is significant by Student’s T test (p<0.001) and there is a further increase to reach a peak with 500 pM (p<0.008). The IOD following 1 nM is not different from that with 500 pM.
GnRH also stimulated secretion of leptin from cultures of pituitary cells taken from diestrus, proestrus or pregnant females. compares basal and GnRH stimulated secretion in these groups, Basal secretion is significantly higher when one compares media from pregnant rat AP cells with that from diestrous rats (p=0.012). GnRH- stimulated secretion is increased over basal in each of the groups (diestrous p=0.016; proestrus p<0.015 and pregnant p<0.01). The cells from pregnant rats show the highest responses to GnRH, when compared with all others.
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 (more ...)