Developmental Changes in Ovine Corticotrophs in Vitro*

FRANK M. PEREZ, JEFFREY SCHWARTZ, AND JAMES C. ROSE

The Perinatal Research Laboratory (F.M.P., J.S., J.C.R.), Department of Physiology and Pharmacology(F.M.P., J.S., J.C.R.), and Department of Obstetrics and Gynecology (J.S., J.C.R.), The Bowman GraySchool of Medicine, Winston-Salem, North Carolina 27157

ABSTRACTWe recently reported that fetal sheep corticotrophs (ACTH-pro-

ducing cells) at 108 6 5 d (days) of gestation are relatively moreresponsive to CRH than to AVP, whereas those at 139 6 0 d (term 5145 d) and in the adult are more responsive to AVP. To furthercharacterize these developmental changes, we used immunocyto-chemical, RIA, and cell immunoblotting techniques to examine pop-ulations of corticotrophs and individual cells. Immunocytochemicalstudies revealed that corticotroph frequency decreased from 22 6 1%of all pituitary cells at 100 d of gestation to 14 6 1% at 135 d and 9 60% in the adult. RIA measurements of ACTH secretion by cell pop-ulations showed that the response of corticotrophs to CRH dimin-ished, whereas that to AVP increased during gestation and intoadulthood.

Cell blot analysis of individual corticotrophs identified two types ofsecretory responses (increases in the number of secreting cells andaverage amount of ACTH released per cell) to CRH or AVP thatchanged during fetal development. At 100 d of gestation, CRH in-

creased the proportion of secreting cells from 65 6 3% (no test agent)to approximately 90%; AVP exerted a negligible effect on the relativeabundance of secreting cells. At 120 d of gestation, both secreta-gogues, alone or in combination, increased the proportion of secretingcorticotrophs from 49 6 6% to about 85%. At 135 d of gestation andin the adult, AVP, alone or in combination with CRH, increasedsecreting corticotrophs from about 53 6 6% to about 80%. CRH aloneexerted a nominal effect on the proportion of secreting cells. Addi-tional analyses showed that, at 100 or 120 d of gestation, the averageamount of ACTH secreted by individual corticotrophs did not changein response to CRH or AVP. However, near term and into adulthood,the average quantity of ACTH released from individual cells in-creased in response to these agents.

Our findings suggest that maturational changes in fetal cortico-trophs dictate whether their secretory response to CRH or AVP re-sults from an increase in the proportion of cells secreting ACTH and(or) an increase in the average amount of hormone secreted by indi-vidual cells. (Endocrinology 138: 916–921, 1997)

SUCCESSFUL preparation of the mammalian fetus forbirth depends, in part, on timely activation of the hy-

pothalamo-pituitary-adrenal (HPA) axis. As an integral partof this axis, corticotrophs (ACTH-producing cells) undergoboth morphological and functional changes during HPAmaturation. At least two types of cells (designated fetal andadult) are histologically distinguishable at 90 days (d) ofgestation; beyond 130 d and into adulthood, the latter cellpredominates (1, 2). During these periods, corticotroph fre-quency decreases in the adenohypophysis (3).These developmental changes in corticotrophmorphology

are accompanied by alterations in their response to hypo-thalamic regulatory factors. In this regard, we recently re-ported (4) that corticotrophs at 108 d of gestation are rela-tively more responsive to CRH than to AVP, whereas thoseat 135 d and in the adult are more responsive to AVP.These findings were obtained by measuring cumulative

responses of cell populations to CRH and AVP in vitro. Tofurther investigate corticotrophs during development, weused an immunoblotting technique to study individual cells.Other investigators (5–9) have used this method to charac-terize secretion of hormones and small peptides by singlepituitary cells. We used cell immunoblotting in the presentstudy to determine whether corticotrophs are heterogeneous

in terms of basal ACTH secretion and response to CRH andAVP.

Materials and MethodsAnterior pituitaries

Anterior pituitary glands were obtained from adult pregnant ewesand their fetuses at 101 6 1, 121 6 1, and 135 6 4 d of gestation. Aminimum of six animals was used for each group. Each ewe was deeplyanesthetized with ketamine and pentobarbital before removal of thefetus by cesarean section. Each fetus was given a lethal injection ofpentobarbital (iv) before removal of the pituitary. After this procedure,the ewe was given a lethal injection of potassium chloride (iv) beforeremoving its pituitary. All procedures involving the animals were ap-proved by the institutional animal care and use committee.

Preparation of pituitary cells

Individual cells were prepared from adult and fetal anterior pitu-itaries according to a method described elsewhere (4). Minced tissuefragments were placed in HEPES-buffer containing 0.4% collagenase(Worthington Biochemical Corp., Freehold, NJ) and DNAase I (SigmaChemicals, St. Louis, MO) and incubated for 2h at 37 C with gyratoryshaking. After incubation, the fragments were passed gently through aflame-tapered glass pipet and the dispersed cells were washed inDMEM/Ham’s F12 containing 0.2% polypep. Cells were filtered toeliminate clumps and reaggregates and treated as described below.

Cell culture

Adult and fetal sheep pituitary cells were divided into three groups:those cultured on glass cover slips, coated plastic surfaces, or a protein-capturing membrane (Immobilon, Millipore, Bedford, MA). Cells of thefirst group were used to measure the proportion of corticotrophs in thetotal cell population. Cells were cultured according to a modifiedmethod of Wilfinger (10). This procedure consisted of incubating 3.75 3104 cells in a 50-ml droplet of medium on a glass cover slip (9 3 9 mm2;

Received September 9, 1996.Address all correspondence and requests for reprints to: Dr. FrankM.

Perez, Assistant Professor, Department of Physiology and Pharmacol-ogy, The Bowman Gray School of Medicine, Medical Center Boulevard,Winston-Salem, North Carolina 27157. E-mail: fperez@bgsm.edu.

* This work was supported by NIH Grant HD-11210.

0013-7227/97/$03.00/0 Vol. 138, No. 3Endocrinology Printed in U.S.A.Copyright © 1997 by The Endocrine Society

916

Bellco Glass, Inc., Vineland, NJ) at the bottom of a well (24-well tissueculture plate; Corning, Corning, NY). After 1 h for the cells to attach tothe glass, the well was filled with medium (1.0 ml) and the cells wereincubated for an additional 1 h. After this step, the cells were fixed for1 h, as described below, and processed by immunocytochemistry forACTH.

Cells of the second groupwere used tomeasure cumulative responsesto hypothalamic stimulatory factors and cultured at a high cell density(1.53 105 cells/28mm2) onpoly-l-lysine-coatedplastic surfaces (24-welltissue culture plate) according to the method of Perez (11). Cells wereincubated for 2 h, washed once, and then treated with fresh mediumcontaining vehicle (control), CRH (10 nm), AVP (100 nm), or both agentstogether. After 2 h, the medium was collected and centrifuged, and thesupernatant solutions were stored at 220 C for ACTH assay.

Cells of the third group were used to measure the response of indi-vidual corticotrophs to hypothalamic stimulatory factors. Cells werecultured on Immobilon; a droplet (100 ml) of medium containing cells(1.53 104)was applied to themembrane. After 15min, CRH (10 nm, finalconcentration), AVP (100 nm), or both agents together (10 ml each) wereadded directly into the droplet. Medium alone (10 ml) was added to thecontrol droplets. After 2 h, the cells, attached to the membrane, werefixed (1 h) with paraformaldehyde (4.0%) and processed by immuno-cytochemistry for ACTH.

ACTH immunocytochemistry

Pituitary cells, attached to cover slips, were stained for ACTH ac-cording to a modified method of Denef et al. (12). Cells were fixed for1 h with Bouin’s solution (1.0 ml), washed, and incubated overnight inTRIS/NaCl buffer containing primary antiserum (rabbit antisheepACTH; 1:3,000). After this step, they were washed and incubated for 2 hwith secondary antiserum (goat antirabbit IgG/HRP; 1:500). Immuno-reaction product was developed with DAB and 0.006% H2O2; cells werecounterstained with hematoxylin.

Pituitary cells, attached to Immobilon, were immunostained forACTH according to a modified method of Denef et al. (12). Cells werefixed with glutaraldehyde (2.5%), incubated with primary (1:1500) andsecondary (1:500) antisera, reactedwith DAB/H2O2, and counterstainedwith hematoxylin.

Controls for all immunocytochemical staining procedures included:1) replacement of the primary antiserumwith nonimmune rabbit serum;2) preadsorption of the primary antiserum with purified ACTH beforeits use; and 3) immunolocalization of purifiedACTHon Immobilon. TheACTH antiserum that was used in these studies was prepared anddescribed elsewhere (13).

ACTH cell analysis

Aminimum of 500 cells on cover slips or Immobilon membranes wasincluded for analysis of each treatment by using a 40 3 objective andbright-field illumination. A serpentine surveying pattern was used toinsure that each cell was counted only once. Cells on cover slips andImmobilon were characterized as either ACTH immunopositive orimmunonegative.

After determining that analysis of cells on cover slips or Immobilonyielded similar results, we used the former condition to measure theproportion of corticotrophs in the total cell population. Cells on Immo-bilon were used to determine the proportion of corticotrophs that se-cretedACTHonto themembrane. Cells thatwere associatedwithACTHon the membrane were counted as secretors. ACTH release by individ-ual cells (see Fig. 7) was calculated by dividing the amount of hormonesecreted by cell populations (measured by RIA) by the number of se-cretors (determined by immunoblotting).

ACTH RIA and statistical tests

Concentrations of ACTH were measured in duplicate by RIA asdescribed elsewhere (13). The interassay and intraassay coefficients ofvariation were typically 8.0% or less. All data (shown as mean 6 sem)were analyzed by ANOVA and Fisher’s least-significant-difference testsand evaluated at the P# 0.05 level of significance. Synergismwas testedby statistically comparing the net response of cells with CRH and AVP

together with the arithmetic sum of the net responses to each peptidealone.

ResultsCorticotroph frequency

The percentage of corticotrophs in the fetal adenohypoph-ysis decreased during gestation and into adulthood (Fig. 1).Corticotroph frequency was 22 6 1%, 18 6 1%, and 14 6 1%at 100, 120, and 135 d of gestation, respectively. The fre-quency (9 6 0%) of corticotrophs in the adult did not differfrom that of late gestation.

Cell immunoblotting

Figure 2 shows a representative immunoblot of enzymat-ically-dispersed sheep pituitary cells. Cells that containedand released ACTH were readily identifiable on the basis ofACTH immunoreactivity. Individual corticotrophs stainedintensely dark-brown and the amount of ACTH that wassecreted on the membrane varied (as determined by com-puterized densitometry; data not shown). Immunonegativecells were stained only light-blue by hematoxylin. All cellsthat stained positively for secreted ACTH also were immu-nopositive for content. Immunoreactivity was not observedwhen the primary antiserumwas replaced with nonimmuneserumorwhen the primary antiserumwas preadsorbedwithACTH (data not shown).

Secretory profiles of individual corticotrophs

Studies of ACTH secretion by cell populations and indi-vidual cells showed that responses of corticotrophs to CRHand AVP changed during fetal development. In cells fromfetuses at 100 d of gestation, CRH (10 nm) increased ACTHrelease in cell populations by 54 6 3% over control value;AVP-induced ACTH release (13 6 2%) was statistically in-distinguishable from the control (Fig. 3, upper panel). Theseagents together increased ACTH release by 144 6 6% abovethe control value.These responses of 100-d cell populations toCRHandAVP

were paralleled by changes in the proportion of individualcorticotrophs that secreted ACTH. Approximately 65% of allcorticotrophs secreted ACTH under basal (unstimulated)conditions (Fig. 3, lower panel). This percentagewas increased

FIG. 1. Effect of development on corticotroph frequency. Enzymati-cally dispersed cells were incubated on glass cover slips, fixed andprocessed by immunocytochemistry for ACTH. Data are from six toeight experimental replicates. Letters associated with individual barssummarize statistical analyses; bars with no letters in common arestatistically different from each other.

ACTH SECRETION AND FETAL DEVELOPMENT 917

to 92 6 4% by CRH; in contrast, the proportion of cortico-trophs responding to AVP (76 6 5%) was not different fromthe basal value. The combined effect of these agents on theproportion of secreting corticotrophs was similar to that ofCRH alone.In cells from120 d of gestation, CRHorAVPby themselves

each increased ACTH release in cell populations by about80% above the control value (Fig. 4, upper panel). The com-bined effect of these agents on ACTH release was similar toeither one alone. Studies of individual corticotrophs showedsimilar responses to CRH andAVP (lower panel). Both agents,alone or in combination, increased the proportion of secret-ing corticotrophs from 49 6 6% (control value) to about 85%of the total ACTH cell population.In cell populations at 135 d of gestation, CRH did not

significantly increase ACTH release; however, AVP in-creased ACTH release by 200 6 50% over the control value(Fig. 5, upper panel). The response of cells to these agents incombination (226 6 43%) resembled that of AVP alone. Sin-gle-cell studies showed a parallel secretory profile (lowerpanel): 53% 6 6% of all corticotrophs secreted ACTH underbasal conditions. CRH had no effect on the percentage ofsecreting corticotrophs; AVP increased it to 82 6 4% Theseagents in combination increased the percentage of secretingcells to a value (77 6 6%) that was similar to that of AVPalone.In the adult, CRH increased ACTH release in cell popu-

lations by 144 6 7% over control value; AVP increased it by210 6 7% (Fig. 6, upper panel). The combined effect of theseagents on ACTH release was 554 6 9%. Single-cell studiesshowed that 56 6 4% of all corticotrophs secreted ACTHunder basal conditions (lower panel). CRH alone had no effect

on the proportion of secreting cells; AVP increased it to 82 64%. Therewas no difference between the response of the cellsto these agents in combination and that to AVP alone.Figure 7 shows the calculated average of ACTH release by

individual corticotrophs as determined by expressing hor-mone secretion by cell populations per individual secretingcorticotroph. This analysis showed that, at 100 and 120 d ofgestation, CRH and (or) AVP did not alter ACTH secretionper secreting corticotroph. However, at 135 d of gestation,AVP alone increased ACTH release per secreting cortico-troph by 240 6 80% over the control value and by 1606 40%in combination with CRH. In the adult, CRH or AVP in-creased ACTH release by 121 6 10% and 107 6 10%, re-spectively; these agents were additive (292 6 10%) together.

Discussion

The present studies show, for the first time, that the meansbywhich corticotrophs respond toCRHorAVP, i.e. increasesin the number of cells that secrete ACTH (Figs. 3–6, lowerpanels) or increases in the amount of ACTH released byindividual cells (Fig. 7), change during fetal developmentand in adulthood. Thus, maturation of the response of cor-ticotrophs to CRH or AVP depends, in part, on changesamong the population of ACTH-secreting cells that occur asa function of gestational age. This finding is consistent withother in vitro ontological studies (4) reporting that fetal sheepcorticotrophs at 108 6 5 d of gestation respond to CRH orAVP, but by 139 d, they respond significantly only to AVP.Both types of secretory responses of sheep corticotrophs to

FIG. 2. ACTH secretion by individual sheep pituitary cells of an adultewe. Enzymatically dispersed cells were incubated on Immobilon for2 h. After incubation, the membrane and attached cells were stainedby immunocytochemistry for ACTH and counterstained with hema-toxylin The area defined by the white boundary is enlarged in theupper left hand corner. Large arrow, secreted ACTH; small arrow,corticotroph. (1200 3 magnification).

FIG. 3. Secretory profile of corticotrophs at 100 d of gestation. En-zymatically dispersed cells were incubated (2 h) on plastic (upperpanel) or Immobilon (lower panel). Conditioned medium from cells onplastic was assayed by RIA for ACTH. Cells on Immobilon wereimmunostained for ACTH, counterstained and counted microscopi-cally. Data are expressed as a percentage of the total ACTH cellpopulation from six experimental replicates. Letters associated withindividual bars summarize statistical analyses; barswith no letters incommon are statistically different from each other.

918 ACTH SECRETION AND FETAL DEVELOPMENT Endo • 1997Vol 138 • No 3

CRH (i.e. increases in numbers of secreting cells or secretoryoutput per cell) have been reported also for adult rat pituitarycells. Leong and colleagues (14–16) found that CRH altersboth the number of ACTH-secreting cells and the amount ofACTH secreted by a fixed number of corticotrophs. Childs

and Burke (17) reported that CRH increases the percentageof secreting corticotrophs and speculated that the secreta-gogue recruits a subpopulation of quiescent cells to releaseACTH. Our immunoblotting studies support this idea byidentifying a subset of corticotrophs that had no associatedsecretory product on the membrane. Although it is possiblethat these quiescent cells released ACTH in amounts that fallbelow the detection limit of the cell blot assay, our resultssuggest that CRH stimulates a subpopulation of cortico-trophs to release ACTH. This type of secretory responseoccurs at 100 and 120 d of gestation (Figs. 3 and 4). As thefetus matures, the secretory response changes to one thatinvolves increased output of ACTH from already-secretingcells (Fig. 7).Developmental responses of sheep corticotrophs to AVP

involved changes in both the proportion of secreting cells(Figs. 4–6) and amount of ACTH released per cell (Fig. 7).The former secretory response has not been observed in ratpituitary cells and might reflect species differences in theregulation ofACTH secretion (16). Increases inACTHoutputby individual cells responding to AVP have been reportedfor rat corticotrophs (16).Developmental changes in secretory responses of cortico-

trophs to CRH and AVP reported herein coincide with in-creases in the total number of anterior pituitary cells, de-creases in cell frequency (as shown in Fig. 1 and reported byothers), and alterations in cell size (1–3). Whether these mor-phological changes correspond to differences in functionalsubtypes of corticotrophs remains unknown. However, ourfindings raise the possibility that maturation of the HPA axisinvolves alterations in subpopulations of secreting cortico-trophs and amount of ACTH released by individual cells.Several mechanisms might be involved in changing the

FIG. 4. Secretory profile of corticotrophs at 120 d of gestation. Cellswere treated and analyzed as described in Fig. 3. Data are from sixexperimental replicates. Letters associated with individual bars sum-marize statistical analyses; bars with no letters in common are sta-tistically different from each other.

FIG. 5. Secretory profile of corticotrophs at 135 d of gestation. Cellswere treated and analyzed as described in Fig. 3. Data are from sevenexperimental replicates. Letters associated with individual bars sum-marize statistical analyses; bars with no letters in common are sta-tistically different from each other.

FIG. 6. Secretory profile of corticotrophs from the adult ewe. Cellswere treated and analyzed as described in Fig. 3. Data are from eightexperimental replicates. Letters associated with individual bars sum-marize statistical analyses; bars with no letters in common are sta-tistically different from each other.

ACTH SECRETION AND FETAL DEVELOPMENT 919

response of ovine corticotrophs to CRH and AVP duringdevelopment. For example, the increased responsiveness oflate gestational fetal and adult corticotrophs to AVP com-plements radioreceptor bindings studies of Shen et al. (18).These investigators reported that twice as many AVP recep-tors are present on sheep, compared with rat, anteriorpituitary membranes, with both receptors showing similaraffinities for the hormone. Similarly, diminishing respon-siveness of late gestational corticotrophs to CRH (Fig. 5) isconsistent with decreases in the number of CRH receptorsnear term (19).These findings raise the possibility that developmental

changes in corticotroph responses, in part, are caused byexpression of CRH and AVP receptors. The mechanism(s)controlling expression of these receptors during develop-ment of the fetal sheep is unclear but may involve cortisoland other steroids (20–23). Additional mechanisms thatmight change corticotroph responses to CRHorAVP includealterations in signal transduction, cell-to-cell interactions,and differential release of these and other trophic factors bythe hypothalamus (15, 24–26).Concentrations of CRH and AVP used in these studies

were selected on the basis of in vitro experiments with adultsheep anterior pituitary cells and in vivo measurements ofconcentrations in portal blood. We used concentrations thatwould produce measurable responses and be within a phys-iological range. In adult sheep, CRH (10 nm) and AVP (100nm) induce the maximum secretory response from cortico-trophs in vitro (27). In portal blood, AVP is usually found inexcess to CRH at rest and in response to stress (28). We usedthe same concentrations of CRHandAVP, at all ages studied,to enable comparisons across ages. It remains possible that

higher concentrations of CRH could provoke responses fromcorticotrophs at ages where none were observed at the con-centration used in this study. It is also possible that cortico-trophs of pregnant ewes may respond to CRH and AVPdifferently from those of adult nonpregnant female or malesheep, but experience to date with tests of in vitro ACTHsecretion suggests that this is unlikely.In summary, we have identified at least two modes by

which sheep corticotrophs responded to CRH and AVP: in-creases in the number of secreting cells and quantity ofACTH released by single cells. The type of secretory responsechanged during fetal development and in adulthood. Ourfindings suggest that maturation of the HPA axis involvesalterations in responses of corticotrophs to CRH and AVP.

Acknowledgment

The authors thank Ms. Regina Parker for her expert technicalassistance.

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