0013-7227/92/1311-0029$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 131, No. 1

Printed

Society

in U.S.A.

Maturation of Follicle-Stimulating Hormone Gonadotropes during the Rat Estrous Cycle* GWEN

V. CHILD&

Department

of Anatomy

GEDA UNABIA, and Neurosciences,

BYUNG

LAN LEE,

University

AND

JONATHAN

of Texas Medical

Branch,

LLOYD Galweston, Texas 77551

ABSTRACT FSH mRNA is transcribed after the onset of high FSH secretion during proestrus and e&us. Pituitary cell fractions separated by size and density were studied to determine if expression of FSH mRNA activity was predominantly in one subset during the estrous cycle and to determine the source and significance of “silent FSH” cells that secrete FSH, but store too little for detection. Pituitary cells were separated by centrifugal elutriation, plated, and then exposed to 0.1-l nM [n-Lys6]GnRH for 3 h. Media were assayed for FSH by RIA, and the cells were fixed for immunocytochemistry or in situ hybridization. The percentages of immunoreactive FSH cells in unseparated populations increased from 8% at metestrus to 12% during proestrus. Percentages of cells with FSHP mRNA showed the same rise; however, peak levels were higher (17%) during proestrus and estrus. Small cells with FSHp mRNA were more frequent than those with antigens early in the cycle. The largest cell fractions contained 38-44% immunore-

active cells. Only 8-21% of these cells had FSH@ mRNA, except during the morning of proestrus (33%). The distribution analyses showed that the increment in immunoreactive FSH cells during diestrus initially stemmed from smaller subsets; however, over half of immunoreactive FSH cells were large by the evening of proestrus. During the time of active transcription of FSH mRNA, more than half of the cells with FSHp mRNA were small or medium-sized. Thus, early in the cycle, FSHp mRNA is transcribed in the smaller cells, which may be the source of the silent FSH cells reported in previous studies. During proestrus, smaller FSH cells also secreted as well if not better than those in the unseparated population or large fractions. When they secreted more than expected from their percentages of FSH cells, this response was interpreted to be due to either the presence of cells that are immunoreactively silent or the possible removal of autocrine or paracrine regulatory factors. (Endocrinology 131: 29-36, 1992)

F

(7). Gonadotropes that were bihormonal increasedfrom 57% to 74% (7). Since this shift occurred without an increase in the percentage of gonadotropes, we concluded that the bihormonal gonadotropes were not derived de nova and that monohormonal cells were multipotential. Perhaps, small monohormonal gonadotropes are in a reserve or transitional state, ready to be activated to produce the other gonadotropin. In later studies, Lloyd and Childs (8) compared responses in different sized subsets of gonadotropes and found that the GnRH-mediated shift from monohormonal to bihormonal gonadotropes occurred primarily in the smallest subsets(average diameter, 8.2 pm). Thus, GnRH appeared to be one of the mediators of the change in storage patterns. It also increased the secretory potential of some subsets.For example, when FSH’secretion was studied via the reverse hemolytic plaque assay, GnRH increased the percentages of plaques from the smallestcells (8.2 pm average diameter) over those found by immunolabeling. This suggestedthe presenceof “silent FSH” cells that may be secretory, but store too little FSH for detection by immunolabeling. In contrast, two thirds of the largest FSH gonadotropes (averaging 14.5 pm in diameter) did not secrete unless stimulated by GnRH. Furthermore, some of the medium-sized gonadotropes (11.2 pm average diameter) did not secrete FSH, even in the presence of GnRH. These heterogeneous secretory responses may indicate either different stagesof maturation or receptivity in the FSH cell population or, possibly, a division of labor among FSH cells (9). To answer this question, one would need to use a more homogeneous population of rats. The previous studies (6-8) had used randomly cycling female rats; thus, it was not

SH is found by immunolabeling in 65-95% of all gonadotropes, depending on the age of the animal or the stage of the estrous cycle (1). The remaining gonadotropes show immunolabeling for only LHP. During the first week of neonatal development (2) or the transition from diestrous day 2 (diestrous II) to proestrus (l), there is an increase in the proportion of gonadotropes that store both FSH and LH (bihormonal) to 70-75%, with a corresponding decrease in the percentage of cells that store only FSH (monohormonal FSH). After castration, there is a further increase in the proportion of bihormonal gonadotropes to 92% of all gonadotropes (3-5). Therefore, it appears that the gonadotrope population undergoesinternal changes. As the need for both gonadotropins arises, more gonadotropes appear. Furthermore, there are more bihormonal gonadotropes. During the past decade, we conducted in vitro studies to learn about the mechanisms behind these internal changes. In 1983, we studied gonadotropes separated by size from randomly cycling female rats (6) and reported that monohormonal cells tended to be smaller (8-10 pm in diameter) and store less hormone. Bihormonal gonadotropes were large (12-15 pm in diameter) and contained abundant storage granules. When 2- to 3-day monolayers of pituitary cells from randomly cycling female rats were stimulated with GnRH for l-4 h, there was a significant decreasein the proportion of monohormonal cells Received January 7, 1992. Address all correspondence and requests for reprints Childs, Ph.D., Department of Anatomy and Neurosciences, of Texas Medical Branch, Galveston, Texas 77551. * This work was supported by NIH Grant ROl-HD-15472.

to: Gwen V. University

29

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30

MATURATION

possible

to correlate

the particular

OF FSH GONADOTROPES

activity

of small or large

subsetswith a stageof the cycle. Recently, we introduced an elutriation technique that separates cells from a single rat pituitary by size and density (10, 11). In these reports (10,

11) the protocol was used to study changes in LH/3 storage, LH secretion, subpopulations

and expression of LH@ mRNA of gonadotropes throughout

in the different the cycle. The

present report describesparallel studies of FSH synthesis and release from the same cells. This study tests the hypothesis

that the silent

FSH cells

seen in the small cell populations by the reverse hemolytic plaque during

assay may be in a transitional stage of maturation the early part of the cycle. They may be identifiable

DURING

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CYCLE

Endo. Voll31.

1992 No 1

concentrations reported previously (9). No labeling was seen if the probe was omitted

from

the hvbridization

buffer

or if biotinvlated

was substituted for the’antisense oligonucleotide

sense mRNA

p&be.

RIA Media were assayed for FSH with the rat FSH RIA kit from the National Pituitary Agency and Dr. A. Parlowe (RI’-2 was the FSH standard).

The

method

has been

described

in previous

reports

(12).

Inter- and intraassay variabilities were less than 10%. In a given experiment, each concentration or treatment was run on three or four wells, and the experiments were repeated five to seven times. The data were normalized

to nanograms

per 20,000

cells to correct

for differences

in

plating density.

by their content of FSH@mRNA. Furthermore, the report

Analysis

describes changes in the size and density of cells bearing FSHP mRNA or FSHP antigens that point to stages of mat-

The labeled cells were analyzed by counting 500 labeled and unlabeled cells per treatment group/rat. The percentage of labeled cells in each population (either unseparated or small, medium, or large fractions)

uration designed to support proestrous and estrous secretory activity.

Experimental

analysis

and Methods

design

and unseparated

populations).

in 2% glutaraldehyde

and washed

4 times in 0.1

M phosphate buffer (pH 7.4) containing 4.5% sucrose. They were prepared for either immunolabeling for FSHP or in situ hybridization with a complementary oligonucleotide probe to F’SH@mRNA. The immuno-

and in situ hybridization

protocols

were

described

in previous

reports (9, 10). For immunolabeling, the antisera were directed against either human or rat FSHP (National Pituitary Agency and Dr. A. Parlowe) by the

and

diluted

1:30,000. peroxidase

The primary

antibodies

were

detected

avidin-biotin complex method (Vector Laboratories, Burlingame, CA) and nickel-intensified diaminobenzidine. For the in situ hybridization, the oligonucleotide probe was comple-

mentary

(3-4

of variance,

followed

coverslips/data

by Duncan’s

point.

rat). The final

to the sense DNA

encoding

amino

multiple

range

test at the

Results Valid&m

of the

elutriation

technique

In the previous report we showed that the new elutriation method separated by size (10). However, a cell could change to another sized pool by either enlarging or changing density of hormone stores. There was no correlation between the numbers of cells loaded or the cell yield and the distribution

of cells in each fraction (10). There were also no significant changes in the overall cell numbers with the stage of the estrous

cycle. For example,

acids 24-36

the average

yield

from

(*SD)

dissociated pituitaries from some of these rats is listed as follows: 5 estrous rats, 2.2 f 0.8 million; 6 metestrous rats, 2.7 + 0.15 million; 5 diestrous rats (morning), 2.65 + 0.6 million; 6 proestrous rats (morning), 2.5 f 0.8 million; and 6 proestrous rats (evening), As reported previously

experiments

The cells were fixed

labeling

rat was averaged

5% level to identify significantly different groups. Comparisons were made between the unseparated populations and the small, medium, and large cell populations.

The same populations of cells used in fhe previous studies (10, 11) were used in these experiments (animal care and use protocols were approved; Animal Care and Use Committee no. 89-10-220). Normal cycling rats were killed by guillotine, and their pituitaries were removed and dissociated. The cells were immediately separated by centrifugal elutriation into three major fractions. As described in the previous report (6,10), small cells (average diameter, 8.2 hrn) were collected at a flow rate of 15 ml/min (or less). Medium-sized cells (average diameter, 11.2 am) were collected at 25 ml/min, and large cells were collected at 35 ml/min or in the chamber (14.5 pm average diameter). Viability tests showed more than 98% living cells (by trypan dye exclusion) after elutriation. The cells were counted and plated on glass coverslips in 24-well trays for 1 h. They were then stimulated for 3 h in 0.1-5 nM [o-Ly&]GnRH. Media were collected and assayed by RIA for FSH. In separate tests of secretion from the entire cell population, the cells were not separated and were plated immediately after the dissociation. The stages of the cycle included 1000 h on mete&us, diestrous II, e&us, and proestrus, and 1400 h on proestrus. There were four to seven rats collected per stage of the cycle for each of the two groups (separated

Labeling

a given

data point is the average of data from 4-7 different rats (or cell populations) and the analysis of 2000-3500 cells. The data were analyzed by Materials

fractions

from

and statistics

2.4 f 0.5 million. (lo), there was a change

in the

distribution of total cells among the three fractions with the stage of the cycle. During metestrus, a higher percentage of the total cells eluted

in the smallest

fraction.

There

was an

increase in the total number of cells in the medium-sized pool during diestrous II. Finally, there was an increase in the total number

of cells eluted

in the largest cell fraction

during

proestrus and estrus. The previous report showed that some of this shift was caused by enlargement or increases in density

of LH gonadotropes

(see Table

1, Ref. 10).

of FSH (11). Its

sequence is 5’-CAG-ATC-CCT-GGT-GTA-GCA-GTA-GCC-CTCACA-CCA-AGT-GGT-3’. It was photobiotinylated with the Vector Laboratories kit (Photoprobe) according to our previous protocol (5). It was

Changes in percentages

detected

percentages of FSH cells in the unseparated pituitary cell

by the avidin-biotin

peroxidase

complex

kit and nickel-inten-

sified diaminobenzidine. Tests of time of hybridization, optimal hybridization temperature, and saturation concentrations were run as described in previous studies (5, 9). The complementary oligonucleotide probe hybridized

optimally

at 37 C. Densitometric

studies

showed

that satu-

ration of binding sites was reached at lo-100 rig/ml, which is similar to

The first phase

of

FSH cells during

of the study

focused

the cycle on changes

populations. Counts of FSH cells in the unseparated cell populations showed that there was a significant

in the pituitary increase

in the percentages of cells labeled for FSHP antigens from the morning

of diestrous

II to the morning

of proestrus

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(Fig.

MATURATION

OF FSH

GONADOTROPES

1). The lowest percentages (8%) occurred at estrus and metestrus. The highest percentages (12%) were found late in diestrus and on proestrous morning. The changes in the percentage of cells bearing FSHP mRNA paralleled those in the antigen-bearing cells, except that significantly more cells were identified by their content of FSHP mRNA during proestrous morning and estrus (17%). Recall that in the previous section no changes in overall numbers of pituitary cells were detected when different stages of the cycle were compared. Therefore, the changes in percentages of FSH cells may be interpreted to reflect changes in actual numbers of FSH cells. The next phase of the study focused on changes in the percentages of FSH cells in each of the fractions. Data on percentages of immunoreactive or secretory FSH cells in different elutriation fractions have been reported previously (6,8). However, these cells were from randomly cycling rats. Table 1 shows the percentages of FSH cells in different sized elutriation fractions taken from rats in different stagesof the estrouscycle. Early in the cycle (metes&us), there was a significantly higher concentration (percentage) of cells with FSH/3 mRNA than with antigen in the small cell fractions. The mediumsized fraction showed a significant increase in the percentage of cells with FSH antigens from metestrus to the morning of proestrus, followed by a significant decline at estrus. There was also a significant increase in the percentage of FSH mRNA-bearing cells in medium-sized pools from diestrous II to proestrous morning. No changes in overall percentages of cells bearing FSHP antigens were seen in the largest cell pool. Compared with antigen-bearing cells, fewer of the largest cells contained FSH/3 mRNA, except during proestrous morning, when there was an increase to percentages that match those of the immunolabeled FSH cells. There was w z 0 16%’ Z -e-.-

FSH FSH

Antigens mRNA

5 6 e z E :: 5 P

12%

8%. 1 4%’ 096 ’ Y*t

O-AM O-PM Stage of the

Pro-AU estrous

Pro-w cycle

sat

FIG. 1. Illustrates cytochemical studies of the unseparated pituitary cell population in five to seven rats per stage of the estrous cycle. There was a significant increase (star) in the percentage of cells that were labeled for FSH@ antigens or FSHp mRNA when groups from diestrous II evening (D-PM) and proestrous morning (Pro-AM) were compared with groups taken from metestrous (Met) rats. During Pro-AM, the percentage of cells with FSHp mRNA was greater than that seen when cells with FSH@ antigens were counted. After proestrous evening [estrus (Est)], the percentage of cells with FSHp antigens declined to levels seen during metestrus. This is probably due to exhaustion of stores of FSH after the estrous secretory activity. However, the percentage of cells that express FSHp mRNA remained high on the morning of estrus. Star, Significantly different from metestrous group, by Duncan’s multiple range test, 5% level.

DURING

ESTROUS

CYCLE

31

TABLE 1. Percentage of cells in each fraction that store FSH or express FSH/3 mRNA Fraction Small

Medium

Metestrus % Ag 11 + 1 18 + 5 35 t- 7 % mRNA 20 2 4” 20 f 1 21 f 4” Diestrous II % Ag 9+3 23 + 6 44 + 8 % mRNA 15 f 2” 14 iz 2* 14 + 1’ Proestrous AM % Ag 13 + 3 30 f 7 35 + 2 % mRNA 22 & 5 25 f 5 33 + 3* Proestrous PM % Ag 7f lb 25 + 5 33 + 6 9 + 2b 12 + 2”s” 14 * 5-* % mRNA Estrus % An 17 + 2b 21 zk 3 38 f 2 8 f la,* % mRNA 13 + 2 12 * 3”sb % Ag, Percentage of cells storing FSHp antigens + SE; % mRNA, percentage of cells containing label for FSHp mRNA * SE; AM, morning; PM, evening. a Significantly different from % Ag. * Significantly different from metestrous value.

a decline in the percentage of large gonadotropes bearing FSH/3 mRNA at estrus compared to those at all other stages. Changes in distribution cycle

of

FSH gonadotropes

throughout

the

The third phase of the study focused on the dynamics of the changes in distribution of FSH cells among the different fractions. This was performed using the percentages given in Table 1 with the data on total cell numbers recovered to calculate the absolute numbers of FSH cells in each fraction. These numbers were added to determine the total numbers of FSH cells collected. Then, the numbers of FSH cells in each fraction were divided by the total to determine what percentage of the total was small, medium, or large. The reason for this type of analysis is as follows. A change in the distribution of FSH cells may not be seen clearly in the counts listed in Table 1, because other cell types could be changing size or density and entering or leaving a given pool. This change might dilute or concentrate the gonadotropes and mask some of the changes in that population. Figures 2 and 3 illustrate the changes in the proportion of small, medium, or large FSH cells from metestrus to diestrous II. Among cells expressing FSHP antigens, there was an increase in those that were eluted with medium-sized cells. When cells bearing FSH/3 mRNA were analyzed in metestrous populations, 34% of cells were small, compared with 25% that express antigens. By diestrous II, the proportion of small cellsbearing FSH/3mRNA had increasedto 61%. Recall that this period had the lowest percentagesof FSH cells (Fig. 1). Becausethere was no difference in the absolute numbers of cells from metestrus to diestrus, this is the period during which the lowest numbers of cells express FSH activity. However, as the percentages (numbers) of FSH cells increased from diestrus to proestrus, there were significant changes in their distribution in the different fractions (Figs. 4 and 5). By the morning of proestrus, cells with FSHP

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32

MATURATION METESTRUS SCAntlgens

0

OF FSH GONADOTROPES

DURING

ESTROUS

CYCLE

Endo. Vol131.

1992 No 1

DIESTRUS %mRNA

%Antlgens

KmRNA

Lug0

Large

PROESTROUS WAntlgens

AM %mRNA

PROESTROUS KAntlgens

Medium

PM KmRNA

mall

Q Lug*

ESTROUS KAntlgens

AM KmRNA

LWgO

Mbdi

. 0 Large

2-6. Figures 2 and 3, Pie graphs illustrating the study of the distribution of FSH gonadotropes among the three fractions collected from metestrous and diestrous II rats. The percentages of gonadotropes labeled for FSHp mRNA or antigens found in Table 1 were used with the counts of total cells in each fraction to determine the number of FSH gonadotropes in each fraction. These three numbers were then added, and the number of FSH gonadotropes in each fraction was divided by this total. In cell populations from metestrous rats (Fig. 2), over 60% of the immunoreactive FSH gonadotropes and nearly half of cells bearing FSH/3 mRNA were eluted in the large cell pool. Recall from the data in Fig. 1 that cells from this stage have the fewest detectable FSH cells. In the cell population from rats on diestrous II (morning; Fig. 3), there has been a shift in the distribution, so that over 60% of cells bearing FSHP mRNA are small. A slight increase in the proportion of medium-sized immunoreactive FSH cells is evident. Figure 1 shows that diestrus is the stage during which the number of FSH cells begins to increase. The distribution analysis suggests that small and medium cells contribute to the increment. Figures 4 and 5, Pie graphs illustrating the distribution study of FSH gonadotropes (see Figs. 2 and 3) in populations from rats on proestrus. The rise in the percentage of immunoreactive FSH cells shown in Fig. 1 again appears to be caused by an influx of small and medium cells, because the distribution analysis in Fig. 4 shows an increase in the proportions of both of these subtypes, with a corresponding decrease in the proportion of large FSH cells. However, by the evening of proestrus (Fig. 5), the FSH cells may have enlarged or become more dense, because the larger subtype predominates. There is no further increment in the overall number of FSH cells (Fig. l), which indicates that this is an internal change in the cell population. The percentages of cells bearing FSH@ mRNA are at a peak on the morning of proestrus (Fig. l), and the distribution analysis indicates that half of these cells are large (Fig. 4). However, the small cells must be activated to transcribe the mRNA during proestrus because over half of the population bearing FSH mRNA are small during the evening. Figure 6, Pie graph showing the distribution study of FSH cells from estrous rats (see Figs. 2 and 3). As shown in Fig. 1, the percentages of immunoreactive FSH cells are low. The distribution analysis in Fig. 6 shows that over half of them are large. The increase in the proportion of small subtypes could be due to a loss in density as a result of secretion of FSH stores during the early morning of estrus. The predominance of small cells bearing FSHP mRNA on proestrous evening (Fig. 5) persists during the morning of estrus. The overall percentages of cells bearing mRNA are at a peak at this time (Fig. 1). FIGS.

antigens were evenly distributed among the three fractions. When this change is correlated with the increase in overall percentages of FSH cells shown in Fig. 1, these data provide clues as to the source of the new FSH cells. First, the fact that there are no overall changes in cell numbers suggest that they are not derived from mitosis. The increase in the numbers of cells in small and medium pools during this time (see Table 1, Ref. 10) also correlates with these data. Collec-

tively, the data suggest that small and medium FSH gonadotropes dilute the population during early proestrus and are the source of the new gonadotropes seen in Fig. 1. By the evening of proestrus, however, there was another shift in the distribution, suggesting maturation in preparation for secretion. Large cells bearing FSH/3 antigens predominated in the population, The proportion of small immunoreactive cells declined to 14% of FSH cells.

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MATURATION

OF FSH GONADOTROPES

Among cells with FSHP mRNA, there was a shift from diestrous II to proestrous morning, so that half of the cells were large. However, by the evening of proestrus, small FSH cells that express mRNA again represented half of the population. Finally, Fig. 6 shows that over half of the cells with FSHP antigens were large on the morning of estrus. However, over half with FSHP mRNA were small. Figures 7-9 illustrate the labeling for FSHP mRNA in populations from proestrous morning or estrous rats. The labeling is in patches, lines, or whorls in the cells. In proestrous rats (from the morning), the labeling intensity on the cells was very strong (Fig. 7). It was reduced to three to four patches in the large cells from the estrous populations (Fig. 8). However, intensely labeled cells were seen in the small fractions from these estrous rats (Fig. 9). Previous electron microscopic studies have shown that the labeling sites cor-

FIG. 7. Field showing gonadotropes from the largest cell fraction collected from rats on the morning of proestrus and labeled for FSHp mRNA. The arrowheads ooint to the dense label in lines or natches in these large gonadotropes: This is the only time when the percentages of large cells labeled for mRNA equaled those labeled for the antigen (Table 1). The inset shows another example where the label was mainly in a cellular process. The cell just below the inset has a process projecting up from the focal plane. Since the field was photographed in the plane that displays most of the labeled regions. this cell is partially out of focus. Magnification, x998.

DURING

ESTROUS

CYCLE

33

respond to the surface of sacsof rough endoplasmic reticulum (9). Secretion

from

FSH

cells

The fourth phase of these studiesfocused on FSH secretion from individual fractions (assayed in the medium with and without GnRH stimulation). Table 2 compares basal and GnRH-stimulated levels of FSH from unseparated cultures and each of the 3 fractions. To conserve space, only data from cultures exposed to 0.5 nM GnRH are reported. The data were normalized to nanograms per 20,000 cells to allow comparisons between the groups. Studies of secretion from unseparated cells showed that the highest levels of FSH came from cells in late diestrus and proestrus (Table 2). This pattern is similar to that found by other workers (13-17). When secretion from the smallestcell fractions was analyzed, the cells secreted well, in spite of their smaller size. FSH levels were comparable to those of the unseparated population. However, in cells from rats on the evening of proestrus, the basal secretory levels were significantly greater than those of unseparated cells (by Duncan’s multiple range test, 5% level). Recall that the data in Table 1 showed that small cell fractions from rats on the evening of proestrus have fewer detectable FSH gonadotropes (7&l%) than unseparated cultures (12%; Fig. 1). This was magnified when the percentages were translated to absolute numbers of gonadotropes. Table 1 in Ref. 10 shows that more of the overall pituitary cell population from proestrous rats is large. Therefore, the secretion from small cells is higher than might be predicted from the absolute numbers of FSH cells. Medium-sized fractions are 1.5-2X more enriched in immunoreactive FSH cells than the unseparated or small cell populations (compare data in Fig. 1 with Table 1). Absolute numbers of medium-sized cells increased only in early diestrus (Table 1, Ref. 10). However, their secretory activity was often higher than one might expect from their enrichment or concentration in the population. For example, medium-sized cells from rats on the evening of proestrus, estrus, and metestrus secreted 2.5-4X more FSH than unseparated populations in testsof basal secretion. GnRH stimulated even higher levels of FSH from medium-sized populations on

FIG. 8. Field showing

gonadotropes from the largest cell fraction collected from rats on the morning of estrous and labeled for FSHp mRNA. This figure illustrates that the density of the label has decreased in the large cells to the point where it is difficult to detect. Only two of the cells show definite dense grayblack patches of label (arrowheads). Magnification, x953.

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34

MATURATION

OF FSH GONADOTROPES

DURING

ESTROUS

CYCLE

Endo. Vol131.

1992 No 1

FIG. 9. Field showing gonadotropes from the smallest cell fraction collected from rats in estrous and labeled for FSHp mRNA. Unlike that of their counterparts in the largest cell fraction, the labeling of these cells is more intense and shows as dense gray-black patches (arrowheads). Insets illustrate more examples of labeled cells from this group. The labeled “cell” depicted in the center of the field may actually be a process that has broken from a gonadotrope. However, cells this small (as small as a red blood cell) can also be seen among gonadotropes and PRL cells. Analysis of the field with xl00 oil, phase contrast is used to detect nuclei in these small cells. The focal plane was chosen to depict the label; therefore, some parts of the cells may be flattened and below the plane. Magnification, ~862. TABLE 2. FSH secretion from unseparated and separated cell populations (nanograms per 20,000 cells) stage

Unseparated

Fraction

Small Medium Large Metestrus Control 19 + 3 30 AZ7 59 + I” 54 f 6” +GnRH 35 + 3b 30 zk 6 89 f 12”~~ 87 + 12”1* Diestrus’ Control 33 + 7 22 5 4 55 + 7” 67 + 6” +GnRH 45 + 6 31 f 3b 65 + 7” 16 + 7”ab Proestrous AM Control 20 f 4 24 + 5 50 + 7” 39 * 7” +GnRH 46 C 12b 39 + 8b 44 + 8 67 k lo”,* Proestrous PM Control 24 f 6 45 + 3” 80 c 6” 66 f 6” +GnRH 48 t 5’ 52 + 7 117 f gas* 96 + Vb Estrus Control 21 + 7 24 zk 2 70 iz 6” 40 + 9 +GnRH 32 t 5 35 + 3b 108 + 8’s* 61 k 4”,b Dat,a are expressed as nanograms of FSH per 20,000 cell + SEM; the GnRH level was 0.5 nM for 3 h. AM. Morning: PM. evenine. a Significantly different from unseparated calues, P < 0.05. * Significantly different from control, P < 0.05. ‘In the unseparated cultures, secretion from cells from diestrous PM was also assayed (taken at 1400 h). It was 35 + 7 ng/20,000 cells in control culture and 76 f 15 rig/ml after 0.5 nM GnRH.

proestrous evening, estrus, and metestrus. Large fractions contain 3-3.6X more immunoreactive FSH cells than unseparated cultures (Table 1). Overall numbers of large cells are highest in populations from proestrous and estrousrats. However, in spite of their abundance, these cells did not always secrete3 times more FSH. The highest levels came from large cells on proestrous evening and metestrus. These values were comparable to those from medium-sized cells (which had fewer FSH cells) and were 2.5-2.8 times higher than those from unseparated populations. Large cells were responsive to GnRH at all stages.However, an analysis of estrous populations showed that the levels of FSH from large cells were lower than those from medium-sized cells (by Duncan’s multiple range test, 5% level).

Discussion This study was designed to determine the significance of different populations of FSH cells. We were particularly interested in those that appeared to be functionally silent to a given test in our earlier studies (8). For example, some small FSH cells from a randomly cycling population could be identified by secretion in a plaque assay, but not by immunolabeling (8). Some large FSH cells could be identified by immunolabeling, but did not appear to be secreting in the plaque assay (8). Table 1 and the distribution studies in Figures 2-6 provided clues about the source of the silent FSH cells found in the small fractions from randomly cycling rats (8). As predicted, they could be identified by their content of FSH/3 mRNA (Table 1 and Fig. 9) and they appeared early in the cycle (Figs. 2 and 6). In fact, the percentages of FSH cells detected by in situ hybridization matched those detected by the plaque assays after GnRH stimulation (8). Thus, these silent cells detected in the earlier studies (8) could have been derived from rats in estrus or metestrus. In the earliest electron microscopic studies of small FSH cells (6), we showed that they had few secretory granules arranged in a row at the cell periphery. This may have rendered them difficult to identify by immunolabeling. They may be in a stageof maturation at which FSH storesare low, or they may have depleted their stores after the high estrous secretory activity. This loss in density may also have caused them to move to a small cell pool. Cells that expressLH also appear to shift to a smaller pool after estrus (10). Recall that our earlier reports showed that most unstimulated small gonadotropes are monohormonal (6, 8). Perhaps these cells were rendered monohormonal after exhaustion of their storesof antigens during proestrous or estroussecretion of LH and FSH. Their potential to synthesize and store the other gonadotropin was also recognized in the earlier studies (7, 8). In this and the recent study of LH cells, we show that they can also be recognized by their content of mRNA for

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MATURATION

OF FSH GONADOTROPES

the P-subunits. Thus, they may be in a state of readiness awaiting stimulation during diestrus to translate the gonadotropin mRNAs and prepare for the next proestrus. It is important to note that in spite of their low storage levels, small FSH cells did secrete as well as the FSH cells in the unseparated populations. In fact, populations from rats on proestrus evening that had only 7% detectable FSH cells secreted 1.9 times more FSH basally than unseparated cells that had 14% FSH cells. This correlates with the previous plaque assaydata (8) and suggeststhat small FSH cells may have a rapid dynamic secretory cycle during which FSH is secreted as rapidly as it is packaged. The presence of these silent immunoreactive FSH cells precluded expression of the assaydata as nanograms of FSH per 1000 FSH cells, as was done in the study of LH cells (11). Maturation

of small gonadotropes

When the overall percentages of FSH cells were determined in the unseparated populations, there was a steady increase in the percentage of cells with FSH mRNA, with a peak during proestrus. This response is similar to that seen by cells bearing LHP mRNA (lo), but the rise in percentages of LH cells during diestrus and early proestrus was much slower. The percentages of cells with LHP mRNA did not match those of immunolabeled LH cells until estrus. The changes in FSHP mRNA during proestrus also mirror the changes reported by Shupnik et al. (18), who assayed basal transcriptional rates of FSHP mRNA. Ortolano et al. (19) also assayed an increase in FSHP mRNA with dot blot assaysduring proestrus, followed by a decline at 0800 h on the morning of estrus. Attardi and Fitzgerald (20) found similar profiles in immature females treated with estradiol. In all of the studies (18-20) the peak in FSH/3 mRNA content or transcriptional rates occurred after the onset of high FSH secretion, However, in studies by Shupnik et al. (18) and the present studies expression of FSHP mRNA also remained high throughout the morning of estrus. The labeling shows that it is weaker in the largest FSH cells and stronger in the smallestcells during that time. The distribution analysis demonstrates that, early in the cycle, the expression of FSHP mRNA is mostly in small FSH cells. As the overall percentages of FSH cells begin to rise on diestrous II, there is a continued increase in the proportion of small cells bearing FSHP mRNA. This change is similar to that seenin previous studies of pituitary cells from male rats (9). Over 79% of cells bearing FSHP mRNA in normal intact male rats were small (cl50 pm’). During the first 7 days after stimulation by castration there was a rapid increase in cell area, so that over 80% became 200 pm2 or larger. The new FSH cellsseen 14 days after castration alsowere derived from small subsets(9). In the female, as the percentages of FSH cells continue to rise after diestrus, there is an increase in the proportion of medium and large cells bearing FSH mRNA and antigens, with a corresponding reduction in the proportion of small cells bearing FSH antigens. These data suggestthat the small immunoreactive silent FSH cells that expressed the mRNA early in the cycle may enlarge or become more dense and,

DURING

ESTROUS

CYCLE

35

thus, populate the larger cell fractions later in the cycle. The pattern of changes in cells with LHP mRNA is different (10). The new LH/3 mRNA that is produced during the LH surge appears first in the largest cells. Then, as the population involutes after the surge, cells bearing LH@ mRNA may become smaller or lighter and thereby join the small cell pool. This suggests a division of labor in the gonadotrope population that may allow for different times of onset of transcriptional activity for the LH and FSH psubunits. By the evening of proestrus, the predominant FSH/3 antigen-bearing cells are the large or more dense subsets.These data fit with the earlier studies that showed an increase in average area of immunoreactive FSH cells from 144 + 10 pm2 at 1400 h on diestrous II to 271 + 17 pm2 at 0800 h on estrus (1). The increased area probably reflects the increase in surface membranes added during exocytosis of the granules. Changes in patterns

of secretion

with the cycle

In most cases,FSH secretory responsesfrom the fractions were consistently equal to or greater than those of their counterparts in the unseparated cultures, which suggeststhat the elutriation protocol did not damage their responses.The patterns of FSH secretion were similar to those from previous reports of unseparated pituitary cells (14-17). The higher basal secretion from medium or large gonadotropes reflected the enrichment in FSH cells only in certain stages. Sometimes the basal levels from medium-sized cells were higher than expected from simple counts of their numbers and analysis of their percentagesin the population. This was especially true in populations from rats taken during proestrous evening or estrus. Furthermore, the largest gonadotropes did not secreteas expected from an analysis of their enrichment (compared to unseparated populations), except in populations from rats on proestrous evening or metestrus. In the analyses of separated fractions, one must recognize that full control of FSH secretion may require steroids and polypeptide hormones. FSH secretion is stimulated by activin (21, 22) or inhibited by inhibin (23, 24) or follistatin (FS) (25-27). Thesepeptides are found in the pituitary and stored in subsetsof pituitary cells, including gonadotropes (28, 29). They may be autocrine or paracrine regulatory factors for certain phasesof FSH secretion and synthesis. These new data on regulatory peptides can be used in the interpretation of data from this and previous studies (8, 11) which showed that smaller gonadotropes secretedbetter than expected and larger gonadotropes sometimesdid not secrete as well. The separation of gonadotropes into different sized subsets changed the content of the medium. Perhaps this change removed an important sourceof activin from its target cell, produced abnormally high concentrations of activin or the inhibitory polypeptides in a given fraction, or both. Summary

and conclusions

The present studies have identified stages of maturation during the estrous cycle that provide cells for the rise in

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36

MATURATION

OF FSH

GONADOTROPES

serum FSH during proestrus and estrus. After estrous secretory activity, the cells may be depleted of FSHP stores and be identified by their content of FSHP mRNA in small or medium subsets. The smaller or lighter cells may be the monohormonal cells identified previously that have the potential to produce and store both FSH and LH. They appear to be the predominant sites for the initial transcription of FSH@mRNA during estrus. During diestrus, these small cells may translate the mRNA and enlarge or become more dense and contribute to the rise in percentages of cells with FSH antigens, which reaches a peak during proestrus. The potential for highest secretory activity is evident in medium-sized FSH cells during proestrus and estrus. Large FSH cells express highest activity during metes&us, diestrus, and proestrous evening. It is possible that this reflects a division of labor in the FSH cell population that provides different subsets of cells for each phase of secretion. Further studies of secretion from these separated fractions are needed to elucidate steroid and autocrine factor(s) that regulate FSH secretion during each phase.

DURING

The authors wish to thank Dr. T. J. Collins for help with the RIAs. We also thank Diana Rougeau for help with the dissociation and elutriation. We greatly appreciate the typing assistance of Ms. Betty Williams.

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FSH mRNA is transcribed after the onset of high FSH secretion during proestrus and estrus. Pituitary cell fractions separated by size and density were...
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