Transgenic Research8: 191–202, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Lactotroph hyperplasia in the pituitaries of female mice expressing highlevels of bovine growth hormone

Sergio Vidal1, Lucia Stefaneanu1,∗, Kamal Thapar1, Roya Aminyar1, Kalman Kovacs1 &Andrzej Bartke21Department of Laboratory Medicine, St. Michael’s Hospital, University of Toronto, 30 Bond Street, Toronto,Ontario, M5B 1W8, Canada;2Department of Physiology, Southern Illinois University, School of Medicine, Carbondale, Illinois, 62901, USA

Received 9 September 1998; revised 28 January 1999; accepted 11 February 1999

Key words:bovine growth hormone, electron microscopy, immunocytochemistry,in situ hybridization, pituitary,transgenic mice

Abstract

PEPCK/bGH transgenic mice have very high blood levels of foreign GH, and prominent reproductive disturbances,especially in females. To obtain a deeper insight into the causes of these abnormalities, pituitaries of PEPCK/bGHtransgenics were studied by immunocytochemistry, electron microscopy andin situhybridization (ISH) techniques.Pituitary weights were significantly reduced (P < 0.05) in transgenic males, while in transgenic females theywere increased without reaching significance compared to nontransgenic controls. In both sexes, GH cells wereinhibited, as previously described in other lines of GH transgenic mice. In females, PRL cells were increased by37% compared to controls. Ultrastructurally, the lactotrophs had characteristics of stimulation and PRL mRNAwas increased by 35%. In males the increase in the number of PRL immunoreactive cells was not significant, thePRL mRNA signal did not differ from controls, and there were no changes in their ultrastructure. Only in femalesACTH cells were significantly reduced (P < 0.05) in number and unchanged in males; however, POMC mRNAsignal was increased in both genders and reached significance (P < 0.05) in males. In females, but not in males,the percentage of LH cells was lower than in control mice. In conclusion, the high blood bGH levels induced sexrelated changes in transgenic mice from the present line. The infertility of PEPCK/bGH transgenic females maybe attributed to lactotroph hyperplasia and marked reduction in number of gonadotrophs.

Introduction

Since the first transgenic mice for growth hormone(GH) have been generated (Palmiter, et al., 1982), adiversity of hybrid genes coding for heterologous GHhave been introduced into the mouse genome, includ-ing rat (r), human (h), bovine (b) and ovine (o) GH(Palmiter, et al., 1983; Shea et al., 1987; Orian, etal., 1989). The ectopic expression of GH transgenes inmice causes gigantism and a variety of functional andpathomorphologic alterations in many organs, includ-ing pituitary. The inhibition of pituitary GH cells has

∗ Author for correspondence (E-mail:L.Stefaneanu@utoronto.ca)

been indicated by Palmiter et al. (1983) in mice trans-genic for mouse metallothionein-1 (mMT)/hGH fu-sion gene, in which a decrease in the number of acido-phils based on histologic staining was reported. By im-munohistochemistry, electron microscopy andin situhybridization (ISH), we have demonstrated changes inseveral adenohypophysial cell types in different linesof transgenic mice overexpressing GH (Stefaneanu etal., 1990, 1993a,b; Terada et al., 1994). The morpho-logic changes are related to properties of the foreignGH and/or its blood levels. Pituitary morphology dif-fers between MT/bGH and MT/hGH transgenic mice(Stefaneanu et al., 1990, 1993a,b) since in rodentsbGH exhibits purely somatotropic activity (Aguilar

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et al., 1992) whereas hGH has in addition prolactin(PRL) like actions (Hartree et al., 1965). Since bGHhas no PRL-like activity, bGH transgenic mice providea useful model for assessing the effects of GH over-secretion on the interrelated endogenous hormones,hormone-releasing factors, and growth factors (Mc-Grane et al., 1988). Transgenic mice expressing bGHgene have been produced by fusion of bGH structuralgene with mMT (Palmiter, et al., 1982, 1983; Naaret al., 1991) or rat phosphoenol-pyruvate carboxy-kinase promoter (PEPCK) (McGrane et al., 1988).In our previous studies, we have used one line ofMT/bGH and three lines of PEPCK/bGH transgenicmice. Transgenic animals from these lines of trans-genic mice show different growth and reproductivepatterns related to the fact that bGH levels are signi-ficantly higher in PEPCK/bGH than in MT/bGH mice(Naar et al., 1991). Body weights are also higherin PEPCK/bGH than in MT/bGH mice from theselines (Hurley et al., 1994). Regardless of the promoterused, and plasma bGH levels achieved, transgenicbGH males are mostly fertile (Bartke et al., 1992),whereas the females show varying degrees of infertil-ity which appear to correlate with circulating levels ofbGH (Bartke et al., 1988; Naar et al., 1991; Cecimet al., 1995). It has been suggested that infertility ofPEPCK/bGH females may be due to luteal failure sim-ilar to that of MT/hGH females (Bartke et al., 1988;Naar et al., 1991). However, the mechanism respons-ible for the luteal failure in the PEPCK/bGH femalesmay not be the same as in the MT/hGH females.While hGH interferes with endogenous PRL produc-tion (Hartree et al., 1965) bGH does not have such aneffect (Aguilar et al., 1992). Most probably, alterationsin the hypothalamic control of PRL secretion lead-ing to suppression of PRL surges of early pregnancyare responsible for the infertility of PEPCK/bGH mice(Cecim et al., 1995). Other possibility is that ovarianfunction could be adversely affected by the very highplasma bGH levels found in this line of PEPCK/bGHanimals.

In order to obtain a deeper insight into the causesof neuroendocrine and reproductive disturbances oc-curring in mice with high blood levels of bGH,we investigated the morphologic alterations in thepituitaries of PEPCK/bGH transgenic mice by histo-logy, immunohistochemistry, ISH and electron micro-scopy.

Material and methods

Mice transgenic for rat PEPCK/bGH structural genewere produced as previously described (McGrane etal., 1988). The line used in the present study wasderived from a single founder male containing onecopy of the transgene (line designation: PEPCK/bGH-1) and was propagated by mating transgenic males(donated by Dr. TE Wagner and JS Yun, Edison Bi-otechnology Center, Ohio University, Athens, OH,USA) with normal C57BL/6J×C3H/J F1 hybrid fe-males (The Jackson Laboratory, Bar Harbor, ME,USA). The animals were housed in a room with con-trolled photoperiod and temperature with constant ac-cess to a laboratory rodent pellet diet and tap water.Two-month-old transgenic mice (eight virgin femalesand seven males) and nontransgenic siblings (seven fe-males and seven males) were anesthetized with etherand killed by decapitation. Body weights and pituit-ary weights in transgenic versus control groups werecompared using Student’st-test. Primary antibodiesraised against mouse PRL, rat GH, human ACTHandβ-subunits of rat TSH, FSH and LH were kindlydonated by Dr. Parlow (National Institute of Diabetesand Digestive and Kidney Diseases, Bethesda, MD,USA).

For light microscopy, pituitaries were fixedovernight in 10% buffered formalin and embedded inparaffin. Sections of 5µm thickness were stained withhematoxylin-eosin (H&E) and the periodic acid-Schiff(PAS) technique.

For immunocytochemical demonstration of adeno-hypophysial hormones (GH, PRL, ACTH, TSH, LH,and FSH), the streptavidin-biotin-peroxidase complextechnique (Hsu et al., 1981) was applied on 5µmparaffin-embedded sections. The morphometry wasperformed using an analysis image program (CellAnalysis System, INC, Lombard, IL, USA). The ab-solute cell-number was calculated from the formula(Brolin and Theander, 1945):

C = NVG

whereC is approximately the absolute cell-number ofcells per pituitary,NV is the numerical density andit represents the number of counted nuclei per unitvolume of pituitary and G is the absolute weight ofthe anterior lobe in mg.NV was calculated using theFloderus (1944) equation:

NV = NA/(T +D − 2h)

whereNA is the total number of nuclei present in 100fields that represent 360,000µm2,D is the average

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nuclear diameter andT is the average thickness of thesections (5µm). The sections were always made bythe same operator using the same microtome, anyhow,even though there could be small variations in sectionthickness this would not affect significantly the res-ults. h is the height of the smallest recognizable capsection that was estimated to be≈ 10% of the nucleardiameter. Statistical comparisons of the data were per-formed by Student’st-test. Statistical significance wasaccepted forp < 0.05.

ISH was carried out on 5µm deparaffinizedsections. Oligonucleotide probes for GH, PRLand POMC mRNAs were complementary to mGHresidues 145–151, mPRL 64–70, and rPOMC 99–108.The GH and PRL probes were produced by GenosysBiotechnologies, Inc. (The Woodlands, TX, USA).The POMC probe (NEP-20/4) was purchased fromDu Pont Inc., Canada (Mississauga, Ont. Canada).The 3′-end method with [35S]-dATPαS was appliedfor probe labeling, using a kit (NEP-100, Du PontInc., Canada). After labeling, the probes were pur-ified with the NENSORB-TM20 cartridge providedwith the kit. The details of hybridization signal de-velopment and controls were described in detail in aprevious paper (Stefaneanu et al., 1992). Silver grainssignaling GH, PRL and POMC mRNAs were countedexamining 200 cells per slide with×100 objective,from two male and two female transgenics as wellas four sex matched controls. Nonspecific hybridiza-tion was obtained from the number of silver grains onposterior lobes, and the mean number was subtractedfrom the mean number of silver grains/cell/pituitarysections.

For transmission electron microscopy, pieces fromeach pituitary were post-fixed in 1% osmium tetroxidein Millonig’s buffer, dehydrated in graded ethanol, andembedded in Epon-Araldite mixture. Semi-thin sec-tions were stained with toluidine blue. Ultra-thin sec-tions were stained with lead citrate and uranyl acetateand investigated with a Philips 410-LS transmissionelectron microscope.

For ultrastructural immunocytochemistry, pituitarypieces fixed in glutaraldehyde were processed and em-bedded as described above. The ultra-thin sectionswere mounted on nickel grids, and double labelingfor GH-PRL with IgG-28nm gold particles (PRL) andprotein A-15 nm gold complex (GH) was applied. Thedetails of this procedure were described previously(Felix et al., 1986).

Results

Gross findings

In both sexes of transgenic mice, the body weightswere significantly increased (p < 0.05) when com-pared to normal control mice. In transgenic males, thepituitaries were smaller and weighed significantly lessthan those of normal animals (p < 0.05). In contrast,in transgenic females, the pituitaries were enlarged,however, the apparent weight increase in comparisonwith normal mice did not reach significance (Table 1).

Immunocytochemical, in situ hybridization andultrastructural findings

On hematoxylin and eosin stained sections, in bothmale and female mice, the anterior lobe contained areduced number of acidophils. No histologic changeswere noticed in intermediate and posterior lobes oftransgenic mice.

Growth hormone (GH) cellsImmunocytochemistry for GH revealed a marked de-crease in number and size of GH immunoreactive cellsin transgenic mice (Figures 1a and b). In contrastto the normal control pituitaries, GH immunoreactivecells of transgenic mice had an angular or elongatedcontour, and only rarely an ovoid or round shape.Quantitative assessment of GH cells revealed that theirdecrease was greater in males (approximately 3-folddifference between the normal and the transgenics)than in females (approximately 2-fold difference). ByISH, the signal for GH mRNA was decreased by86% in transgenic pituitaries compared to normal ones(Figures 2a and b).

Table 1. Summary of body and pituitary weights (mean±SEM) inPEPCK/bGH transgenic mice and nontransgenic siblings

Body weight (g) Pituitary weight (mg)

Transgenic 49.4± 5.1∗ 1.40± 0.1∗males

Control 31.2± 1.3 2.10± 0.2

males

Transgenic 43.6± 1.5∗ 3.68± 0.9

females

Controls 25.3± 1.6 2.70± 0.2

females

∗ Significantly different from controls (p < 0.05.)

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Figure 1. Histograms of absolute number of hormone immunoreactive cells/pituitary in transgenic males (a) and transgenic females (b) incomparison with wild-type mice. Significant differences (p < 0.05) are marked with asterisk.

Ultrastructurally, in both sexes of transgenic anim-als the somatotrophs were smaller than in nontrans-genic siblings due to the reduction of cytoplasmicarea. The nuclei were heterochromatic and sometimesa small nucleolus was evident. Typical somatotrophswere easily recognized. The secretory granules werespherical with high electron density and they weredisposed at the periphery of the cytoplasm and oc-casionally accumulated at one pole of the cell. Thesize of secretory granules was smaller than in controls(200–300nm vs. 300–500nm). The Golgi complexwas poorly developed and rarely contained few smallsecretory granules (Figures 3a and b). Approximatelyhalf of the somatotrophs had small, spherical secretorygranules of 100–200nm and with moderate electrondensity; they could be reliably identified only by ap-plying the immunogold technique for GH (Figures 4aand b).

Prolactin (PRL) cellsIn the pituitaries of transgenic males the slight in-crease in number of PRL immunoreactive cells didnot reach significance compared to control pituitar-ies (Figures 5c and d). The intensity of the hybrid-ization signal for PRL mRNA did not differ fromthat found in normal glands. By electron microscopy,the lactotrophs were similar to those seen in controlpituitaries.

In pituitaries of transgenic females, the number ofPRL immunoreactive cells were numerous with veryprominent Golgi pattern (Figures 5a and b) and theirquantification indicated that they were increased by37% compared to control females (Figure 1b). Theintensity of hybridization signal for PRL mRNA wasincreased by 35% (Figure 6a and b). Ultrastructurally,the lactotrophs with typical pleomorphic secretorygranules were hypertrophied (Figure 7a and b). The

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Figure 2. By in situ hybridization, in a transgenic female mouse(a) the signal for GH mRNA is markedly decreased and found in areduced number of cells compared to that found in a nontransgenicsibling (b). Original magnification×400.

euchromatic nuclei contained 1–2 middle-sized nuc-leoli. The RER was well developed and composedof parallel cisternae or of concentric whorls resem-bling fingerprints. Golgi apparatus was prominentand contained numerous forming secretory granules.Lactotrophs with small secretory granules were alsoidentified due to the presence of extruded secretorygranules and of an euchromatic nucleus. Immuno-gold method for PRL was helpful to distinguish themfrom somatotrophs with small secretory granules (Fig-ure 4b).

In one transgenic female, a well demarcated smallPRL cell adenoma protruding into the Rathke’s cleftwas found. The PRL mRNA signal was stronger in theadenoma cells than in the surrounding nontumorouspituitary.

Adrenocorticotropin (ACTH) cells

In transgenic males, no change in the number ofACTH immunoreactive cells was evident. The hybrid-

ization signal for POMC RNA was significantly in-creased by 37%. However, no ultrastructural changesof corticotrophs were found. In female transgenics thepercentage of ACTH immunoreactive cells was halfof that found in nontransgenic siblings (Figure 1b).The increase of POMC mRNA signal by 15% was notsignificant. By electron microscopy, occasional corti-cotrophs showed signs of stimulation, such as euchro-matic nucleus with prominent nucleolus, abundantcytoplasm, and increased number of secretory gran-ules. The majority of corticotrophs did not differ fromthose seen in nontransgenic siblings.

Thyrotropin (TSH) CellsNo changes in the number, size or ultrastructural fea-tures of this cell type were noticed in either sex oftransgenic mice (Figures 1a and b).

Gonadotropin (FSH/LH) cellsNo changes were apparent in the gonadotrophs ofmale transgenic mice (Figure 1a). In female transgenicmice, the pituitaries showed a significant decrease inthe number of LH immunoreactive cells (one quarterof the number found in controls) (Figure 1b).

Discussion

Mice transgenic for PEPCK/bGH fusion gene de-velop gigantism secondary to high blood levels offoreign GH, which is produced ectopically in severalorgans especially liver and kidney (McGrane et al.,1988). As already described in other lines of trans-genic mice with overexpression of heterologous GH(Stefaneanu et al., 1990, 1993; Terada et al., 1994),the pituitaries of transgenic mice from the present linecontain a decreased number of GH immunoreactivecells. The decrease is more pronounced in males thanin females. This is in contrast to previous findingsin MT/bGH, MT/hGH and PEPCK/hGH transgenics,in which the decrease in GH cell number was lesspronounced in males than in females. The ultrastruc-tural features indicating inhibition of somatotrophsare similar to those described in MT/hGH, MT/bGHand PEPCK/hGH transgenics. When comparing thedecrease of GH mRNA signal in PEPCK/bGH withMT/bGH transgenics, it is evident that the signalreduction is stronger in the former (86%) than inthe latter group (50%). The percentage decrease ofGH mRNA signal is also greater in PEPCK/hGHversus MT/hGH transgenic mice (about 80% and

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Figure 3. Ultrastructurally, in a pituitary of a transgenic male (a) the somatotrophs (gh) exhibit a decreased cytoplasmic area, and smallersecretory granules lined up under the plasma membrane, compared to those seen in a control pituitary (b). Magnification×4,400.

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Figure 4. Double immunogold technique depicts in a transgenic female mouse: a) somatotrophs with small (full arrow) and medium (emptyarrows) sized secretory granules, labeled with small gold particles, and a typical lactotroph (prl), labeled with large gold particles; b) asomatotroph (gh) with tiny secretory granules labeled with small gold particles, surrounded by lactotrophs with small secretory granules(prl1) or pleomorphic secretory granules (prl2), both labeled with large gold particles. Magnification×9,000.

60%). It can be concluded that the level of circu-lating GH (100–700ng/ml in PEPCK/bGH mice and3–4 ng/ml in the employed MT/bGH mice) (Stageret al., 1994) influences the level of inhibition ofthe endogenous (mouse) GH gene transcription. Themorphologic changes of somatotrophs in the adeno-

hypophyses of mice transgenic for PEPCK/bGH areregarded as results of multiple abnormalities in neur-oendocrine regulation due to actions of high bloodbGH levels. The extent of these changes is related tothe concentration of heterologous GH in the circula-tion.

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Figure 5. Pituitary of a transgenic female mouse (a) contains an increased number of PRL immunoreactive cells, compared to a control gland(b); in contrast, in a transgenic male (c) only an apparent increase of PRL immunoreactive cells is seen relative to a control mouse (d). Originalmagnification×400.

The unique change limited only to PEPCK/bGHtransgenic female mice is the marked increase in PRLcell number (35%), causing a higher pituitary weightcompared to controls. In all lines of GH transgenicmice so far studied, pituitary weights have been de-creased in both sexes. The increase in the numberof PRL cells, together with the ultrastructural fea-tures of lactotrophs, and the increased PRL mRNAsignal point to the stimulation of PRL production infemales with high levels of blood bGH. These mor-phologic changes of lactotrophs are in agreement withthe reported elevated basal plasma PRL levels in thisline of transgenics compared to nontransgenic siblings(Steger et al., 1993). Plasma PRL levels have alsobeen increased in transgenic PEPCK/bGH-1 femalesin early phases of pregnancy, but mating-induced PRLsurges were absent or markedly attenuated (Cecimet al., 1995). The mechanism(s) by which excessivebGH induces stimulation of lactotrophs in females isspeculative. One possibility is that the extremely high

plasma bGH levels encountered in PEPCK/bGH fe-male mice interfere with hypothalamic regulation ofpituitary PRL secretion. Alterations in serotoninergictransmission in the hypothalamus of this line of trans-genic mice may offer a plausible explanation (Cecimet al., 1995). It has been reported (Mathiasen et al.,1992; Sagrillo and Voogt, 1992) that serotonin is re-sponsible for the inhibition of dopamine, which, inturn, results in the PRL surge. GH may influence PRLsecretion also indirectly via circulating insulin-likegrowth factor I (IGF-I) and that produced by hypothal-amus and the pituitary itself. Liver expression of IGF-Iand serum IGF-I levels are elevated in GH transgenicmice (Mathews et al., 1988) and IGF-I might playan important role in regulating PRL secretion throughseveral different mechanisms. However, results con-cerning PRL regulation by IGF-I are contradictory.Thus, in vitro treatment of cultured rat pituitary cellswith semipurified preparation of IGF-I has been re-ported to have an inhibitory (Goodyer et al., 1984,

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Figure 6. In situ hybridization for PRL mRNA reveals in thepituitary of a transgenic female (a) increased hybridization signalcompared to a control one (b). Original magnification×400.

1986), or no effect on basal or stimulated PRL release(Yamashita and Melmed, 1986, 1987). However highconcentrations of recombinant IGF-I stimulate PRLproduction by cultured rat pituitary cells (Lambertset al., 1989) and tumorous human pituitaries (Atkinet al., 1994). In IGF-I knockout mice we reportedmarked inhibition of lactotrophs supporting a role forIGF-I in stimulation of PRL production (Stefaneanuet al., 1999). We have also found that treatment for2 weeks of male and female mice with physiologicdaily doses of IGF-I elevates significantly plasma PRLand increases pituitary PRL mRNA (Stefaneanu et al.,1999). The role of IGF-I in the stimulation of PRLsecretion was also supported by the findings in dwarfrats with isolated GH deficiency, in which pituitaryand serum PRL levels, PRL mRNA and the numberof lactotrophs are markedly reduced (Charlton et al.,1988; Nogami and Takeuchi, 1993). From our resultsin normal and IGF-I knockout mice, we speculate thatIGF-I interacts with estrogen in order to stimulate PRL

cells. We assume that the gender related differencesin the response of PRL cells in the present line oftransgenics, may be due to testosterone and estrogencirculating levels. Plasma testosterone level is normalin PEPCK/bGH mice, while in females the estrouscycle is significantly longer, and in those females thatare fertile, the number of ova shed is significantlygreater than in normal females (Cecim et al., 1995).

Another change that occurred only in the pituit-aries of transgenic females consists of reduction inthe number of gonadotrophs. One explanation for thisfinding could be the suppression of the magnitude ofLHRH effect on gonadotrophs by the high blood PRLlevels. However, a paracrine inhibitory effect of PRLon gonadotrophs can not be excluded, as well (Denefand Andries, 1983). The marked decrease in num-ber of gonadotrophs may explain in part the reductionof plasma FSH levels in transgenic females from thepresent line (Steger et al., 1993). Our morpholo-gic findings suggest that the luteal failure responsiblefor infertility of female PEPCK/bGH transgenic micecould be due to alterations in both lactotrophs and gon-adotrophs, although a defect in PRL surge of earlypregnancy appears to be of primary importance. An-ovulatory sterility in middle-aged PEPCK/bGH trans-genic mice (Cecim et al., 1995) may be related togonadotroph deficiency. These results support the sug-gestion of Bartke et al. (1994) that defects in ovarianfunction in PEPCK/bGH females are not due to bGHaction at the ovarian level.

The pituitaries of PEPCK/bGH males showed nochange in the number of gonadotrophs. The biochem-ical data of lower blood FSH levels and lower pituitarycontent of FSHβ and LHβ mRNA indicate suppressionof gonadotrophs.In vitro, the LH content and basaland stimulated LH release by pituitary explants are notaltered in transgenic males from this line (Tang et al.,1993).

All lines of transgenic mice overexpressing GHhave high plasma corticosterone levels (Cecim etal., 1991). Our findings of a significantly increasedPOMC RNA signal in transgenic males and a lesspronounced increase in transgenic females suggesthigher rate of gene transcription compared to non-transgenic littermates. This is in agreement with theelevated plasma ACTH levels reported in bGH trans-genic males (Cecim et al., 1996). These findings addsupport to the suggestion that the increase in circulat-ing ACTH is the result of GH action on hypothalamic–pituitary axis rather than a direct effect on adrenalcorticosterone production.

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Figure 7. Ultrastructurally, the lactotrophs (prl) of a transgenic female mouse (a) are larger and the RER and Golgi areas are more prominentthan in a control pituitary (b). Magnification×7,000 (a and b).

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In conclusion, the present investigation providesevidence that high blood bGH induces different pituit-ary responses in male and female PEPCK/bGH trans-genic mice. Lactotroph hyperplasia and marked re-duction in gonadotrophs may explain the reproductiveimpairments in PEPCK/bGH female mice.

Acknowledgements

The authors are indebted to Mr. Fabio Rotondo, Dr.Zi Cheng, Mrs. Anca Popescu and Mrs. Elisabeth Mc-Dermott for the excellent technical assistance and toNIDDK for the kind donation of antibodies againstpituitary hormones. This work would not have beenpossible without the generosity of Dr. TE Wagnerand JS Yun who provided transgenic mice to start ourbreeding colony. This study was supported in part bygrant MT-11279 awarded by Medical Research Coun-cil of Canada (L.S. and K.K.), St. Michael’s ResearchCouncil (L.S.) and NIH (HD20033 and DK42137)(A.B.). Dr. Sergio Vidal was supported by a re-search grant from Conselleria de Educacion (Xunta deGalicia) Spain.

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