N OCIETY' - the Society for Reproductive Biology

THE EFFECTS OF TARGETED DISRUPTION OF THE CYP 19 (AROMATASE) GENE ONFOLLICULOGENESIS IN MICEKara L Britt, Ann E Drummond, Margaret E E Jones, Mitzilee Dyson, Nigel G Wreford 1 ,Evan R Simpson and Joc.k K FindlayPrince Henry's Institute ofMedical Research, PO Box 5152, Clayton, Victoria 3168 andIDepartment of Anatomy, Monash University, Clayton, Victoria 3168, Australia.IntroductionA primary function of estrogen is to maintain the femalereproductive tract, notably ovarian folliculogenesis.Aromatase, a cytochrome P450 (P450 arom ) encoded by thecyp19 gene, catalyses the conversion of C l9 androgens toestrogens. Estrogen exerts its actions via the estrogenreceptors (ER), ERa and ERp, which are prevalent ongranulosa cells [1]. An aromatase knockout mouse(ArKO), generated via disruption of exon 9 of the cyp 19gene [2], allowed us to examine the local role of estrogenin ovarian folliculogenesis.Materials and MethodsBody, ovarian, gonadal fat and uterine weights fromwildtype, heterozygous and ArKO mice at 10-12, 16-18,21-26, weeks and one year of age were measured. Serumand ovaries were utilised for further analyses. Ovarieswere either fixed in formalin and paraffin embedded, orfrozen in OCT compound. Paraffin sections of wildtype,heterozygote and ArKO ovaries were histologically· andstereologically assessed. The numbers of primary,secondary and antral follicles were calculated using avariation of the fractionator method [3].Results and DiscussionThe ovarian weights of ArKO mice were reducedcompared to wildtype littermates up to 21-26 weeks ofage, at which time the weight of the ArKO ovaries were< 50% of wildtype. By one year of age ovarian weighthad increased, possibly due to the increase in connectivetissue. Uterine mass was decreased in the ArKO, to 50%of wildtype at 10-12 weeks and< 25% by one year. Gonadal fat pad weights of ArKOmice were increased by 2-fold compared to wildtype at 21­26 weeks. ArKO ovaries at 10-12 weeks contained anincreased number of primary follicles compared toheterozygote littermates, and less secondary follicles (Fig1). At 21-23 weeks of age the number of primary follicleswas still greater than that observed in wildtype andheterozygote mice. By one year of age there were nosecondary or antral fo,llicles present in the ArKO ovary.At 21-23 weeks the oocytes of heterozygote primaryfollicles had greater nuclear diameters than wildtype (Het111lm vs WT 9Ilm). Secondary and antral follicles showeda similar difference when compared to ArKO (Het 151lmvs ArKO llllm; Het l61lm vs ArKO 121lm respectively).A block in follicle development and a lack of corpora lutea(CL) within the ovaries was reflected in infertility at allages studied. Histological examination demonstrated thatthe ArKO ovary degenerated with age. Haemorrhagiccystic follicles appeared in the ovary at 21 weeks of ageand atresia, as measured by TUNEL assay, was observedto increase as a function of age. Coincident with the lossof follicles, extensive tissue remodelling, exemplified byan influx of macrophages and collagen deposition, wasobserved in the ArKO ovaries.8620015010050o~~ 250oi. 200j § 150c~ 100;:f 50o20015010050oo WT• HET1 0 -1 2 wee ks• KOFig 1: Effect ofage (a,b)and genotype (X,y) onfollicle number.Different letters denoting significance (* no follicles at thisstage ofdevelopment observed).Serum gonadotrophin (FSH and LH) levels measured bvradioimmunoassay were found to be elevated in the ArKOmice, and continued to increase with age. Interestingly at 10­12 weeks heterozygote animals had increased FSH and LHlevels compared to wildtype, whilst remaining lower thanArKO levels. Earlier studies showed an increase intestosterone and undetectable estradiol levels in the ArKOmice [2]. The impact of the 10% soy contained within themouse chow on the ovarian phenotype was investigated usingmice raised from birth on soy-free mouse chow. ArKO micemaintained on a soy-free diet displayed an earlier onset of thephenotype, with reduced ovarian weight, increased gonadal fatpads, and the appearance of haemorrhagic cysts at an earlierage (16 weeks). These results imply that phytoestrogenswithin the diet were acting as estrogens within the ovary andfat deposits of the mice. Thus mice on a soy-free diet providea more comprehensive model of estrogen deprivation.In summary, age-dependent disruption of folliculogenesis andovulation was observed in ArKO female mice, accounting fortheir infertility. It is not clear whether the ovarian phenotypeobserved in the ArKO mouse is a direct result of the loss ofestrogen, or an indirect result of the elevated gonadotrophinsand testosterone or both.[1] Drummond AE et al., Mol. Cell. Endo. 1999 149:153[2] Fisher CR et al., PNAS. (USA). 1998; 95: 6965-6970.[3] Gundersen HJG et al., APMIS 1988; 96:857-881.Supported by NH&MRC of Australia (Regkey 983212)Polyovular Follicles in the Pouch Young and Adult KangarooSusan N. Reinke l , Ian M. Gunn!, Marilyn B. Renfree 2 , Alan O. Trounson l1Institute ofReproduction and Development, Monash University, Clayton, Victoria, 3168, Australia; 2 Department ofZoology, University ofMelbourne, Parkville, Victoria, 3052, Australia.Introduction. Polyovular follicles (follicles containingmore than one oocyte)-have been observed in a numberof mammalian species including the human (1), dog (2),mouse (3), wild Norway rat (4) and goat (5). Generally,polyovular follicles occur more frequently in immatureovaries although they are also found in adults. Inmarsupials, the occurrence of polyovular follicles hasbeen observed in the American opossum, Didelphismarsupialis (6) and the Australian native cat, Dasyurusviverrinus (7). Similarly, it is noted that polyovularfollicles were first, and only, observed in the kangarooby van Beneden during the 1880's as previously cited(6). The aim of this study was to investigate whetherpolyovular follicles occurred in two species of sexuallyimmature and mature kangaroos.Materials and Methods. Both eastern grey kangaroos(Macropus giganteus) and red kangaroos (Macropusrufus) were shot at Dubbo and Gunbar in NSW. Redkangaroos were also collected at Nymagee (NSW) andeastern grey kangaroos at Dunkeld (Vic) and Narromineand Warren in NSW. One ovary was scavenged fromeach female pouch young (PY) (n=6, ages 120 to 261days) and adult (n=28) eastern grey kangaroos, and fromfemale PY (n=3, ages 67 to 135 days) and adult (n=14)red kangaroos. The age of adult kangaroos was notdetermined. Whole, uncut ovarian tissue was fixed forlight microscopy and the top ten sections were examinedfor polyovular follicles. Polyovular follicles wereclassified according to the system used for opossumovaries (6): Germ cell nests: oocytes in mutual contactand surrounded by a layer of flattened granulosa cells(Fig. 1); Polyovular follicles type I: oocytes separated bygranulosa cells but still within a single follicle (Fig. 2);Polyovular follicles type II: oocytes in mutual contact(Fig. 3); Polyovular follicles type III: oocytes withinfollicle found in a linear arrangement.Results and Discussion. The follicle populationconsisted of monovular follicles although polyovularfollicles were also found in PY and adult material. Alltypes of polyovular follicles with the exception of typeIII were observed. In the adult red kangaroo polyovularfollicles never contained more than two oocytes whilepolyovular follicles in the adult eastern grey kangaroofrequently had more than two oocytes. The mostcommonly observed polyovular follicle type was that ofgerm cell nests which were found in both species of PYand only in eastern grey adults. As the ages of adultswas not recorded, it is possible that some of theseindividuals were juveniles that had not yet completedovarian development and that the germ cell nests presentwere in the process of separation. Another explanationfor the presence of polyovular follicles may beenvironmental conditions which, as previously reported,?lays .a significant part in reproductive developmentmcludmg the delay of sexual maturity (8). Therefore,the incidence of polyovular follicles observed in thisstudy could be a result of environmental factors, such aschemical sprays and toxins, which have been known toeffect reproductive organs.Conclusions. Polyovular follicles occur in both red andeastern grey kangaroos. Adult eastern grey kangarooshad three types of polyovular follicles but they appearedless common than in the eastern grey pouch young.Fig. 1. Germ cell nest from adult female eastern greykangaroo. H & E, x 250. Bar =33J.l.m.Fig. 2. An atretic Graafian follicle containing two ova fromadult female eastern grey kangaroo. H & E, x 63. Bar =135J.l.m.Fig. 3. Biovular follicle from adult female eastern greykangaroo. H & E, x 160. Bar=53 J.l.m.References1. Arnold L (1912) Anat Rec. 6: 413-422.2. McDougall K, Hay MA, Goodrowe KL, Gartley CJ, King WA(1997) J Reprod Fert. 51: 25-31.3. Fekete E (1950) Anat Rec. 108: 699-707.4. Davis DE, Hall 0 (1950) Anat Rec. 107: 187-192.5. Joshi C, Nanda BS, Saigal RP (1976) Anat Anz. 139: 299-304.6. Hartman CO (1926) Am]Anat. 37: 1-51.7. O'Donoghue CH (1912) AnatAnz. 41: 353-368.8. Newsome A E (1965) Aust J 'hJo. 13: 735-759.87