Proc. Natl. Acad. Sci. USAVol. 92, pp. 1322-1326, February 1995Medical Sciences

Targeted overexpression of luteinizing hormone in transgenicmice leads to infertility, polycystic ovaries, and ovarian tumorsKIMBERLY A. RISMA*, COLIN M. CLAYt, TERRY M. NETrT, THOMAS WAGNERt, JUN YUN4, AND JOHN H. NILSON*§*Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106; tAnimal Reproduction and BiotechnologyLaboratory, Department of Physiology, Colorado State University, Fort Collins, CO 80523; and *The Edison Biotechnology Center, Ohio University,Athens, OH 45701

Communicated by Anthony P. Mahowald, University of Chicago, Chicago, IL, November 23, 1994

ABSTRACT Hypersecretion of luteinizing hormone (LH)is implicated in infertility and miscarriages in women. A lackof animal models has limited progress in determining themechanisms ofLH toxicity. We have recently generated trans-genic mice expressing a chimeric LH j3 subunit (LH8) ingonadotropes. The LH,8 chimera contains the C-terminalpeptide of the human chorionic gonadotropin ,B subunit.Addition of this peptide to bovine LH,8 resulted in a hormonewith a longer half-life. Furthermore, targeted expression ofthe LHI8 chimera led to elevated LH levels and infertility infemale transgenics. These mice ovulated infrequently, main-tained a prolonged luteal phase, and developed pathologicovarian changes such as cyst formation, marked enlargementof ovaries, and granulosa cell tumors. Testosterone andestradiol levels were increased compared to nontransgeniclittermates. An unusual extragonadal phenotype was alsoobserved: transgenic females developed hydronephropathyand pyelonephritis. The pathology observed demonstrates adirect association between abnormal secretion of LH andinfertility and underscores the utility of the transgenic modelfor studying how excess LH leads to cyst formation, ovariantumorigenesis, and infertility.

Maturation of a fertilizable oocyte is a complex process thatdepends upon the proper development of the oocyte within thehighly defined environment of the ovarian follicle. Uponovulation, the mature follicle ruptures to release the oocyte,and the steroidogenic cells of the ruptured follicle terminallydifferentiate to form the corpus luteum. The corpus luteummust produce adequate progesterone to maintain pregnancy iffertilization has occurred. The control of these ovarian eventsis critically regulated by fluctuating levels of pituitary gona-dotropins: follicle stimulating hormone (FSH) and luteinizinghormone (LH). Both hormones are heterodimeric glycopro-teins containing a common a subunit and a unique 3 subunit.Heterodimers of LH and FSH are secreted by pituitarygonadotropes and bind to receptors in the gonads to stimulatesteroidogenesis and gametogenesis (1).

Several clinical studies have shown a correlation of hyper-secretion of LH and polycystic ovarian syndrome (PCOS),infertility, and miscarriage in women (2-4), suggesting thatchronically elevated LH impairs fertility. Unfortunately, thereare no studies showing a direct relationship between hyper-secreted LH and reproductive abnormalities. Since LH issecreted in regulated pulses (5, 6) and its serum ti12 is short[20-30 min (7)], it is difficult to devise protocols for chronicadministration of exogenous LH that mimic endogenous pulsepatterns of LH. To circumvent this limitation, we report atransgenic mouse model in which elevated hormone levels aremaintained chronically, without requiring multiple injections,

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supraphysiologic dosing, or dampening of the hypothalamic-pituitary-gonadal axis.We utilized a two-pronged approach to achieve elevated

levels of serum LH. (i) Increased secretion of hormone fromthe pituitary was achieved by expression of an LH(3 subunittransgene, targeted to the pituitary by a previously character-ized bovine a-subunit promoter (8, 9). (ii) The LH,B transgenewas modified to encode a peptide extension that we proposedwould slow the elimination ofLH from the serum. This peptideis normally found at the C terminus of the 3subunit of humanchorionic gonadotropin (hCG) (hCG,B) and is referred to asthe C-terminal peptide (CTP) (10). The CTP is thought to bea major determinant of the serum t,'2 of hCG and has beenshown to increase the til2 of FSH 2- to 3-fold when fused to its(3 subunit (11). Accordingly, we constructed a transgene withthe coding region of bovine (b) LH,B fused in-frame to thecoding region of CTP (bLH3-CTP).

MATERIALS AND METHODS

Construction of the bLH3-CTP Transgene. The bLH,B-CTP minigene was engineered by a two-step PCR. (i) A shortfragment containing the C terminus of bLH,B (30 bp) fusedin-frame to the CTP (last 87 bp of the hCGj3 gene) wasgenerated. (ii) This fragment was lengthened to contain theentire bLHf3 cDNA fused in-frame to the CTP. This fusiongene was utilized for transfection experiments but was modi-fied to contain the first intron of bLH3 for the transgeneconstruct. The resulting insert was ligated into a BSK- plasmidalready containing the bovine a-subunit promoter (positions-315 to +45) and the simian virus 40 late polyadenylylationsignal. Transgenic mice were generated by microinjecting thebLH3-CTP insert into fertilized oocytes as described (12).Mice were genotyped by PCR analysis of tail DNA usingprimers specific to the a-subunit promoter and (3-subunit gene.

Analysis of t,,2. Recombinant bLH and bLH-CTP het-erodimers were generated by stably cotransfecting CHO cellswith viral expression vectors containing the bovine a-subunitgene and either the bLH( or bLH,3-CTP genes as described(13). Serum-free medium was collected and ammonium sul-fate-precipitated or concentrated with an Amicon ultrafiltra-tion cell. Female rats were pretreated with 50 ,tg of antide(Sigma) in 20% (vol/vol) propylene glycol, injected subcuta-neously 2 h prior to hormone injections to prevent release ofendogenous LH during the course of the experiment. Samples(1 ,ug) of CHO LH, CHO LH-CTP, and purified hCG(Calbiochem) were dissolved in 1 ml of 0.9% NaCl. Allinjections and blood sampling were performed by accessing thejugular veins under ether anesthesia. LH (14) and hCG (15)levels were determined by RIA.

Abbreviations: LH, luteinizing hormone; FSH, follicle stimulatinghormone; hCG, human chorionic gonadotropin; CTP, C-terminalpeptide; PCOS, polycystic ovarian syndrome; b, bovine.*To whom reprint requests should be addressed.

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Immunohistochemistry. Male bLH,3-CTP pituitaries wereperfusion-fixed in 2% (vol/vol) paraformaldehyde, mountedin paraffin, and sectioned. After deparaffinization, sectionswere incubated with the following antibodies: (i) guinea piganti-rat LH,3 (AFP22238790GPOLHB, National Institute ofDiabetes and Digestive and Kidney Diseases, Bethesda) for 1h at 37°C, diluted 1:200; (ii) fluorescein isothiocyanate-conjugated anti-guinea pig IgG for 30 min at 37°C, diluted1:300; (iii) rabbit anti-CTP (P79R) for 1 h at 37°C, diluted1:100; (iv) rhodamine-conjugated anti-rabbit IgG for 30 min at37°C, diluted 1:500. Sections were washed at room tempera-ture with phosphate-buffered saline for 20 min between eachincubation. Secondary antibodies were from Organon Tekni-ka-Cappel.

RESULTS AND DISCUSSIONCTP Increases the til2 ofLH. To test directly the effect of the

CTP on t112, we generated recombinant bLH and bLH-CTPheterodimers and measured their elimination from the serumof rats (Fig. 1). The majority of each hormone was eliminatedby first-order kinetics. Therefore, t,l2 values were estimated forthis phase by the amount of time it took to clear from 40 to 20%of hormone. The ti,2 ofbLH obtained by this analysis was 20-25min. The addition of CTP to bLH,3 dramatically affectedclearance of the heterodimer, causing its tyl2 to increase 2- to3-fold. In fact, the elimination curve of bLH-CTP closelyapproximated the elimination of hCG. These experimentsdemonstrated that addition of CTP markedly decreased theclearance of bLH-CTP heterodimers in serum and providedimpetus for introducing the bLHf3-CTP construct into trans-genic mice.

Generation of bLHj-CTP Transgenic Mice. The bovinea-subunit promoter was used to direct expression of the trans-gene to gonadotropes (8,9). Addition ofCTP to bLHB3 allowedimmunologic identification of cells expressing the chimericsubunit. Double immunohistochemical labeling of pituitarieswith CTP- and LHj3-specific antibodies demonstrated that allLH6 cells contained the CTP signal (Fig. 2), confirming thatexpression of the transgene occurred in all gonadotropes.Significantly, no CTP signal was detected in pituitaries fromnontransgenic mice (data not shown).Transgenic Mice Are Infertile. Attempts were made to

breed all founder animals. Male mice, however, were subfer-tile. Although one animal never bred, another male bred after

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FIG. 1. Analysis of serum ti/2 of recombinant bLH-CTP in rats.Recombinant bLH and bLH-CTP heterodimers were purified fromCHO cells and injected intravenously into rats to measure eliminationin vivo. Samples containing 1 ,tg of purified hCG (-), CHO LH-CTP(*), or CHO LH (-) were injected into the jugular vein of female rats(n = 3 or 4 per group) pretreated with antide, a gonadotropin releasinghormone antagonist, and blood was collected as indicated for 540 min.The ti1/2 was estimated by measuring the time it took to clear from 40to 20% of hormone from the serum. The addition of CTP to bLHcaused its t,'2 to increase 2- to 3-fold.

FIG. 2. Immunohistochemical detection of bLH,B-CTP in mousepituitaries. (x180.) Sections were incubated with primary and sec-ondary antibodies as follows. (A) With guinea pig anti-LH,B andfluorescein isothiocyanate-conjugated anti-guinea pig IgG. (B) Withrabbit anti-CTP and rhodamine-conjugated anti-rabbit IgG. All cellsexpressing LH,B contained CTP.

a prolonged (2 month) delay. This permitted establishment ofa bLHI-CTP line. Initial analysis of bLH,B-CTP males indi-cated that neither LH nor testosterone levels were different intransgenic vs. control males, although testes in transgenicmales were significantly smaller than in controls (data notshown).

Analysis of transgenic females established that expression ofbLH,B-CTP had a profound impact upon female reproduction.Two founder females never bred, whereas a third had one litterwith one pup that did not survive. Analysis of ovarian cyclesby vaginal smear in 12- to 16-week-old F2 transgenics revealedchronic anovulation. In a subsequent experiment utilizing8-week-old bLHf3-CTP females from the F3 generation, 9 of10 transgenic females mated multiple times, but only 1 gen-erated a litter compared to 5 of 6 littermate controls. SerumLH levels were elevated in 13 transgenic (39.7 ± 8.7 ng/ml; P< 0.01) vs. 8 control (2.7 ± 0.48 ng/ml) females (14), dem-onstrating that hypersecretion of LH had been achieved.Ovarian Pathology. Morphologic analysis of ovaries from

bLH,3-CTP founder and F1-F3 generations (age, 4-8 months)revealed three classes of pathologic changes unique to trans-genic females: enlarged ovaries packed with corpora lutea,ovaries containing multiple cysts, and ovaries with granulosaand theca-interstitial cell tumors (Fig. 3). The incidence ofphenotypic ovarian changes in bLH-f-CTP females is summa-rized in Table 1. Breeding of four F1 animals revealed that theinfertility and ovarian pathology were transmitted onlythrough two males and not observed in offspring from two F1females with normal ovaries. It is not known whether this is dueto segregation of multiple integration sites or due to geneticimprinting.

Fig. 3B demonstrates a markedly enlarged ovary containingnumerous corpora lutea. Mice with this ovarian pattern havevastly elevated progesterone levels (data not shown) and ap-pear to exhibit a prolonged luteal life span. To test whetherchronically elevated LH could lengthen the function of cor-pora lutea, we experimentally induced new corpora lutea andanalyzed progesterone production. In rodents, the life span ofthe corpus luteum during pseudopregnancy (12-14 days) canbe measured by mating females with a vasectomized male anddetermining the length of time to a new estrous cycle. Unfor-tunately, transgenic females were anovulatory, making thisapproach problematic. Therefore, we initiated ovulation byhemiovariectomy-a method utilized in rats with pharmaco-logically induced cystic ovaries (16). Surgeries were performedon both transgenic and nontransgenic littermates and the micewere placed immediately with vasectomized males. Mating wasdocumented by the presence of a semen plug in the vagina.Progesterone levels were analyzed every 3 days and the

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FIG. 3. Ovaries from transgenic females fall intothree pathologic phenotypes: enlarged, cystic, andtumorigenic. (X9.) (A) A normal ovary is showncontaining multiple corpora lutea and follicles. (B)Ovaries from multiple bLHI-ECTP females weregrossly enlarged and contained multiple corporalutea (CL), typical of a pseudopregnant ovary. Thefollicles are pressed to the outside of the ovary andare atretic. (C) Other ovaries obtained from bLHI--CTP females contained numerous cysts-hemor-rhagic (h) or fluid-filled (f) and derived from bothfollicular and luteal tissue. (D) Granulosa cell tumors(*) were observed in ovaries from Fl to F3 gener-ation bLH,B-CTP females. Tumors were noted inmice from age 4 to 9 months and contained variousamounts of other stromal cells.

termination of the pseudopregnancy was documented by theobservation of a new mating or demonstration of a cornifiedvaginal epithelium by vaginal smear. Both were indicative ofovulation.

Control animals were unaffected by hemiovariectomy andexhibited a normal pseudopregnancy with progesterone levelspeaking at day 3 and returning to baseline by day 12, followedby another ovulation (Fig. 4). Two out of four hemiovariec-tomized transgenics mated and became pseudopregnant.These mice secreted large amounts of progesterone with peaklevels occurring at day 12. Remarkably, progesterone re-mained elevated at least until day 18 and no ovulation wasobserved. Thus, the progesterone and ovulation data demon-strate that transgene expression prolonged the luteal life spanbeyond a normal pseudopregnancy.An increase in luteal life span suggests that chronically

elevated LH overcomes the physiologic signals that ordinarilycontrol luteal cell death. The mating stimulus induces pseu-dopregnancy in rodents by causing twice daily surges ofprolactin (17). In turn, prolactin supports progesterone pro-duction by maintaining LH receptors on luteal cells (18). Thus,an increase in luteal life span in transgenic mice could reflecta direct action of hypersecreted LH through binding to LHreceptors on luteal cells or an indirect effect that sustains theaction of prolactin. Although distinguishing between these twomechanisms requires additional experiments, hypersecretionof LH clearly alters the life span of the corpora lutea.Development of multiple cysts represents another class of

phenotypic changes noted in bLHf-CTP ovaries (Fig. 3C).Testosterone in transgenic females was elevated -5-fold overcontrol females [transgenic, 1.95 + 0.61 ng/ml (n = 5);control, 0.38 ± 0.02 ng/ml (n = 5), P < 0.05]. Estradiol wasalso elevated [transgenic, 65.2 ± 12 pg/ml (n = 5); control,24.8 ± 9 pg/ml (n = 5); P < 0.05], although only 2-fold. Thus,

Table 1. Summary of ovarian phenotypes in bLHf3CTP femalesfrom founder animals and subsequent generations derived frommale founder 94

No.

Mice Normal Enlarged Cystic Tumor Other*

Founders 2 1F1 2 1 2 1F2t 1 4 2F3t 2 2 2 2

*Normal architecture is lacking; remaining stroma is luteinized.tIncludes female offspring from F1 males only (see text for discussion).

these changes result in an elevated androgen/estrogen ratio,analogous to that observed in women with PCOS (19). Al-though FSH was not measured in these mice, the elevations inestradiol and LH suggest that the LH/FSH ratio may bealtered as well. Consequently, these mice represent a modelthat may be relevant to the study of PCOS, a disease that mayaffect 20-30% of women (20).

In humans, the etiology of PCOS is multifactorial. Presen-tation often includes hyperandrogenemia and insulin resis-tance (2, 21, 22). Hypersecretion of LH is associated withPCOS, but it is unknown whether increased LH alone triggersalteration of the other hormones. Our data indicate thatchronically elevated LH is sufficient to cause the PCOS-likesyndrome observed in bLH3-CTP mice. It should be noted,however, that cysts are often more numerous in ovaries fromPCOS women than in ovaries from bLHj-CTP mice. Inaddition, the thickened fibrous capsule of the ovary typicallyseen in PCOS (23) is not present in transgenic mouse ovaries,suggesting that other genetic or environmental factors are

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FIG. 4. Demonstration of a prolonged luteal life span in femalesexpressing the bLHl3-CTP transgene. Transgenic females were hemi-ovariectomized to induce ovulation and then placed immediately withvasectomized males to establish a new pseudopregnancy. Functionalluteolysis was analyzed by measuring serum progesterone levels.Progesterone secretion returned to minimal levels by day 12 in controlmice (512, 0; 463, t), typical of a normal rodent pseudopregnancy. Incontrast, progesterone levels in bLH3-CTP females were markedlyelevated (508, *; 509, *), reaching a peak by day 12 and remainingelevated even until day 18.

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necessary to achieve the full spectrum of ovarian pathology.This further underscores the significance of the bLH(-CTPtransgenic mouse model, since additional transgenic allelesencoding PCOS candidate proteins can be bred into thebLHS-CTP genetic background.

Interestingly, cysts in transgenic ovaries appear to be derivedfrom follicular or luteal tissue and may be present in ovariesthat contain corpora lutea (Fig. 3C). This suggests that cysticchanges may occur during any stage of the estrous cycle, ifelevated LH is present. One treatment of PCOS that has beenrelatively successful in achieving ovulation is wedge resectionof the ovary (2), analogous to the hemiovariectomy describedabove. The two transgenic mice that ovulated after hemio-variectomy had been anovulatory for at least 2 months prior tothe surgery, and their ovaries, when inspected upon removal,contained cysts (transgenic mouse 508) or granulosa celltumors (transgenic mouse 509). Ovaries surgically removedfrom the two transgenic mice that did not ovulate containedabundant luteal tissue but no cysts or tumors. Thus, it istempting to speculate that cysts and tumors secrete factors thatinhibit ovulation. If so, then a fall in concentration of thesefactors, which could occur by removal of one ovary, may allowa subsequent ovulation.

Cystic ovaries contained both hemorrhagic and fluid-filledcysts. In addition, hyperemia was found in both cystic andtumor-bearing ovaries. The combination of hyperemia andhemorrhagic cyst formation is reminiscent of ovaries fromtransgenic mice generated by estrogen receptor gene knockout(24). We suggest that this phenotype is probably due to chronicLH hypersecretion from disruption of the estrogen feedbackloop in estrogen receptor knockout mice or from expression ofthe additional bLH( allele in our transgenic model.A subset of bLH3-CTP females developed ovarian granu-

losa and theca-interstitial cell tumors by 4-8 months of age(Fig. 3D), indicating that stimulation of granulosa and otherstromal cells by LH-CTP was tumorigenic. Gonadotropinhyperstimulation has been suggested as a mechanism for tumorformation in ovaries transplanted to the spleen in ovariecto-mized rats (25), in women undergoing ovarian stimulation fortreatment of infertility (26), and in ovaries of inhibin-deficientmice (27). The tumors observed in each of these examples

include granulosa cell and other stromal cell tumors. Despitethe association of elevated gonadotropins and tumor forma-tion in each of these examples, a direct link between gonado-tropin hyperstimulation and tumorigenesis has never beenestablished. Therefore, the finding of granulosa and stromalcell tumors in transgenic mice whose only genetic alteration isthe addition of a gene encoding a chimeric gonadotropinstrongly suggests that abnormal gonadotropin stimulation istumorigenic. Interestingly, the recently derived crystal struc-ture of hCG has been used to suggest that glycoproteinhormones can be classified as members of a superfamily ofcystine-knot growth factors that includes nerve growth factor,transforming growth factor 13, and platelet-derived growthfactor (3 (28). It is feasible that excessive levels of LH intransgenic mice reveal growth factor-like properties ofLH notpreviously realized, resulting in angiogenesis and growth ab-errations. Thus, these possibilities illustrate the potential util-ity of the transgenic model for further study of the molecularmechanisms involved in LH-mediated tumor formation.

Transgenic Females Have Renal Abnormalities. Approxi-mately 25% of infertile females expressing the bLHI-CTPtransgene demonstrated a unique nongonadal phenotype (Fig.5). These females had enlarged bladders, dilated ureters, andhydronephrosis. In some animals, the hydronephropathy wascomplicated by acute pyelonephritis, whereas others appearedto have chronic interstitial nephritis. The development of renalpathology was unexpected but may be related to chronicallyelevated steroids since hydronephrosis has been observed inrats chronically administered estradiol (29). In addition, hy-dronephrosis is well documented during pregnancy in womenand nonhuman primates, although it is disputed whether thisresults from ureteral obstruction or from physiologic dilata-tion due to elevated progesterone (30).CTP Exaggerates the Effect of Hypersecreted LH. We have

shown that the presence of CTP on bLHP greatly reduces theelimination rate of heterodimers from serum. It is probablethat the peptide extension contributes to both elevated serumLH levels and the severe gonadal and nongonadal phenotypes.To test this, we generated several additional lines of miceoverexpressing the wild-type bLH(3 gene by using the samebovine a promoter. All three female founders expressing the

FIG. 5. bLHf-CTP females developed hy-dronephrosis and subsequent pyelonephritis. (A)Gross morphology of a normal kidney (Left) andan infected dilated kidney (Right). (B) Normalkidney histology with collapsed pelvis and intactcortex and medulla. (C) Kidney from femaletransgenic, with markedly dilated pelvis and col-lecting ducts, typical of hydronephrosis. (D) His-tologic section from the infected kidney picturedin A. The pelvis and collecting ducts containbacteria and polynuclear cell infiltrates. There isabscess formation in the cortex and extensioninto the surrounding capsule (B-D, x5).

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bLH,B transgene demonstrated a delay in breeding but, incontrast to bLHj-CTP females, eventually had litters ofnormal size. Males expressing bLHf3 were fertile, althoughanimals from one line had slightly smaller testes. Females fromthis particular line were analyzed extensively. Serum LH levelsin transgenic females were compared to controls. LH waselevated in 13 transgenic (46.5 ± 13.8 ng/ml; P < 0.01) vs. 12control (3.76 ± 0.62 ng/ml) females (14), demonstrating thathypersecretion of LH led to hormone elevations that weresimilar to those seen in bLH3-CTP females. Like bLHf3-CTPmice, many females from the bLHf3 line ovulated infrequentlyand developed cysts. Hemiovariectomy led to ovulation in 3 of3 mice, and mice induced to pseudopregnancy showed aprolonged luteal life span. Neither tumors nor kidney pathol-ogy has been noted in these mice, although fewer animals havebeen analyzed. Collectively, these data suggest that hyperse-cretion of wild-type LH can lead to aberrant gonadal pheno-types and that addition of the CTP potentiates the effect ofLH.There are several possibilities for the increased impact of the

bLH3-CTP transgene: (i) increased average levels of serumLH, (ii) altered pulse pattern of serum LH, or (iii) increasedactivity of bLHf3-CTP heterodimers at the LH receptor. Byassuming similar levels of transgene expression, the slowerelimination of bLHf-CTP, compared to bLH, would lead toan overall increase in average LH levels in bLH,BC-TP vs.bLHO3 founder animals. In addition, the decreased rate ofclearance of bLHf3-CTP may alter the shape of the LH pulsein the serum by decreasing the slope of the elimination curveand elevating trough levels of LH. Finally, we have not yetdetermined whether addition of the C1TP to bLH affects thebinding characteristics of the chimera. If the CTP slowed therate of dissociation of the chimera from the receptor, forexample, it may increase the bioactivity of the hormone in theserum. Distinguishing between these three possibilities re-quires further experimentation.One additional feature of both bLHI-CTP and bLHf3

transgenes is the bovine a-subunit promoter. We chose thispromoter to target transgene expression to gonadotropesbecause tissue-specific regulatory elements of LH,3 promotersare undefined. Nevertheless, our data suggest that the apromoter may be less tightly regulated in females than the LH,3promoter since expression of the transgene led to hypersecre-tion of LH despite elevated steroid levels. Additionally, thispromoter demonstrates sexual dimorphism in our transgeniclines since hypersecretion is not achieved in males. Thissuggests that the promoter is less efficiently regulated in thefemales. Finally, in the rat the a-subunit gene is expressedearlier in development than the 03 gene (31). Although we havenot determined the exact day of transgene expression, it ispossible that LH may be secreted earlier than usual in bLHf-CTP and bLH,B mice and contribute to the pathologic phe-notype observed. Interestingly, another study (32) showed thatexpression of a transgenic human FSH,B gene, driven by its ownpromoter, had little impact upon female or male fertility.Consequently, it should be instructive to test whether linkageof the LH,B promoter to wild-type and chimeric bLH3 subunitsleads to the same level of LH overexpression achieved by thea-subunit promoter in our transgenic model.Summary. We have generated transgenic mice with an allele

encoding a chimeric gonadotropin that markedly impairsfertility. In males, expression of the transgene led to reducedfertility and smaller testes, despite normal hormone levels. Infemales, expression of the transgene led to elevated levels ofLH, increased testosterone and estradiol secretion, and exten-

sive pathologic changes in the ovaries. This phenotype wasexaggerated by but not solely due to the CTP, since bLH,Bfemales were also affected. Our data also suggests that bLHf3-C1TP females have a severely reduced rate of fertility, despiteevidence of mating. This may be due to inappropriate lutealsupport and/or subnormal fertilization. Since excess LH hasbeen implicated in infertility and increased miscarriage rate inwomen (3, 4), the impact of elevated LH levels on thefertilization of the oocyte and subsequent pregnancy should bestudied. The bLH3-CTP transgenic mouse model will beinvaluable for these and other studies as it provides a uniqueopportunity to study the mechanisms whereby elevated LHleads to prolonged luteal life span, cyst formation, tumorigen-esis, and perhaps abnormal fertilization and implantation.

We thank Vernon Stevens for the gift of the CTP antibody andDavid Schomberg for valuable discussions concerning reproductivephysiology.

1. Catt, K. J. & Dufau, M. L. (1991) in Reproductive Endocrinology, eds. Yen,S. S. C. & Jaffe, R. B. (Saunders, Philadelphia), 3rd Ed., pp. 105-155.

2. Franks, S. (1989) Clin. Endocrinol. 31, 87-120.3. Shoham, Z., Jacobs, H. S. & Insler, V. (1993) Fertil. Steril. 58, 1153-1161.4. Regan, L., Owen, E. J. & Jacobs, H. S. (1990) Lancet 336, 1141-1144.5. Gibson, M. J., Miller, G. M. & Silverman, A.-J. (1991) Endocrinology 128,

965-971.6. Karsch, F. J. (1987) Annu. Rev. Physiol. 49, 365-382.7. Niswender, G. D., Nett, T. M. & Akbar, A. M. (1974) in Reproduction in

Farm Animals, ed. Hafez, E. S. E. (Lea & Febiger, Philadelphia), 3rd Ed.,pp. 57-81.

8. Kendall, S. K, Saunders, T. L., Jin, L., Lloyd, R. V., Glode, L. M., Nett,T. M., Keri, R. A., Nilson, J. H. & Camper, S. A. (1991) Mol. EndocrinoL5, 2025-2036.

9. Hamernik, D. L., Keri, R. A., Clay, C. M., Clay, J. N., Sherman, G. B.,Sawyer, H. R., Nett, T. M. & Nilson, J. H. (1992) Mol. Endocrinol. 6,1745-1755.

10. Matzuk, M. M., Hsueh, A. J. W., Lapolt, P., Tsafriri, A., Keene, J. L. &Boime, I. (1990) Endocrinology 126, 376-383.

11. Fares, F. A., Suganuma, N., Nishimori, K., LaPolt, P. S., Hsueh, A. J. W. &Boime, I. (1992) Proc. NatI. Acad. Sci. USA 89, 4304-4308.

12. Wagner, T. E., Hoppe, P. C. M., Jollick, J. D., Scholl, D. R., Hodinka, R.& Gault, J. G. (1981) Proc. Natl. Acad. Sci. USA 78, 6376-6380.

13. Kaetzel, D. M., Browne, J. K, Wondisford, F., Nett, T. M., Thomason,A. R. & Nilson, J. H. (1985) Proc. Natl. Acad. Sci. USA 82, 7280-7283.

14. Niswender, G. D., Midgley, A. R., Monroe, S. E. & Reichert, L. E. (1968)Proc. Soc. Exp. Biol. Med. 128, 807-811.

15. Vaitukaitis, J., Braunstein, G. D. & Ross, G. T. (1972) Am. J. Obstet.Gynecol. 113, 751-758.

16. Farookhi, R., Hemmings, R. & Brawer, J. R. (1985) Biol. Reprod. 32,530-540.

17. Terkel, J. (1986) Ann. N.Y Acad. Sci. 474, 76-93.18. Niswender, G. D. & Nett, T. M. (1994) in The Physiology ofReproduction,

eds. Knobil, E. & Neill, J. D. (Raven, New York), 2nd Ed., pp. 781-816.19. Coney, P. J. (1984) FertiL Steril. 42, 667-682.20. Polson, D., Wadsworth, W. J., Adams, J. & Franks, S. (1988) Lancet ii,

870-872.21. Dewailly, D. & Cortet-Rudelli, C. (1992) Horm. Res. 38, 41-45.22. Poretsky, L. (1991) Endocrinol. Rev. 12, 3-13.23. Stein, I. F. & Leventhal, M. L. (1935) Am. J. Obstet. Gynecol. 29, 181-191.24. Lubahn, D. B., Moyer, J. S., Golding, T. S., Couse, J. F., Korach, K. S. &

Smithies, 0. (1993) Proc. Natl. Acad. Sci. USA 90, 11162-11166.25. Biskind, G. R., Bernstein, D. E. & Gospe, S. M. (1952) Cancer Res. 13,

216-220.26. Matzuk, M. M., Finegold, M. J., Su, J-G. J., Hsueh, A. J. W. & Bradley, A.

(1992) Nature (London) 360, 313-319.27. Willemsen, W., Kruitwagen, R., Bastiaans, B., Hanselaar, T. & Rolland, R.

(1993) Lancet 341, 986-988.28. Lapthom, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield,

R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W. (1994) Nature (London)369, 455-461.

29. Andriole, V. T. & Cohn, G. L. (1964) J. Clin. Invest. 43, 1136-1145.30. Roberts, J. A. (1976) Urology 8, 1-4.31. Simmons, D. M., Voss, J. W., Ingraham, H. A., Holloway, J. M., Broide,

R. S., Rosenfeld, M. G. & Swanson, L. W. (1990) Genes Dev. 4, 695-711.32. Kumar, T. R., Fairchild-Huntress, V. & Low, M. J. (1992) Mol. Endocrinol.

6, 81-90.

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