There are mutant and transgenic mouse strains which lack the ability to breed, but where the females have functional ovaries. Ovary transplantation is an important tool for maintaining and producing crosses with these non-breeding strains.We have evaluated an ovary transplantation method by transplanting ovaries from females belonging to a non-reproduetive BALB/cByJ mutant mouse strain, All transplanted mice, 10 BALB/c.C57BL/6By, produced offspring and 94% of the progeny originated from the transplanted ovaries. The mean litter size and the mating period needed for productive mating to occur were similar to what is observed for corresponding control mice

Inzunza, José

Iwarsson, Krister

Lavebratt, Catharina

Petersson, Susanna

Schalling, Martin

Ährlund-Richter, Lars

English

Journals from University of Tartu

Scand. J. Lab.Anim Scii No 2, 1998 Vol. 25Ovary transplantation method resulting inhigh reproductive performance in miceby Catharina Lavebmrt ‘) , Jusé [nzunza 2). Susanna Peterssan ”, Krister [warsmn ,3)Martin Schalling ” & Lars A'hrhmd-Richter 1}.” Neurogenetics Unit, Center for Molecular Medicine, Karolinska Hospital L800, 8-171 76 Stockholm.2) Clinical Research Center, Karolinska Institute, Huddinge University Hospital, 3-141 86 Huddinge.3’ Laboratory Animal Unit, Karolinska Institute. S—171 77 Stockholm,Correspondence: Catharina Lavebratt, Center for Molecular Medicine,Karolinska Hospital L8200, S-171 76 Stockholm, SwedenIntroductionA number of mutant and transgenic mouse strainsdo not breed, The infertility can be due to non-functional germ cells, prepubertal lethality, inabi-lity to mate or incapacity to complete a successfulfertilization. gestation or parturition. When thereason for infertility is other than non-funetionalgerm cells ovary transplantation and / or artificialinsemination can be used. Such techniques arenecessary for maintaining the genotype of strainswith dominant inheritance of any of these breedingdysfunctions The techniques are very useful alsofor maintaining the genotype of strains that reces-sively inherit the breeding dysfunction. They allowbreeding with homozygous rather than hete-rozygous mice resulting in twice the ratio of ab-normal versus normal offspring in the litter. Inaddition, the use of heterozygous genotype forboth parentals would necessitate progeny testingto assure presence of the mutant genotype in theoffspring with which the strain maintenance is tobe continued Often, the only way of testing this isto breed the offspring to find out whether thesecond generation litter include any phenotypi-cally mutant mouse. Depending on when thephenotype can be ascertained, the progeny testingcan be very consuming regarding time, labour andnumber of animals needed. In outcrosses betweena normally functional strain and a breeding dys—functional strain where an affected sex is to beused, the profits of ovary transplantation andartificial insemination are the same as in incrossesiOvary transplantation methods with moderatesuccess were described in several publications inthe 19605 (Stevens, I95 7). Applications of thesemethods. 0r of modifications of them. showingvarying success have been reported until recently(Parkent'ng et a1, [985; Brent (’1 al, 1990; Cox 2!al, 1996; Gunasena et al, 1997a; Gunasena et al,1997b). The ovary transplantation method pre-sented here is similar to what has previously beendescribed by Stevens (1957) and Cunliffc-Beamer(1983). This method is used at the Jackson Labo-ratory, Maine, and has recently been establishedalso at the Karolinska Institute, Sweden, In thispublication we evaluate the performance of thismethod using an example with the non—breedingmouse mutant megenecphaly, mceph, with aBALB/cByJ background.Materials and MethodsAnimals:Three females of inbred BALB/eByJ mice h0-mozygous for the Spontaneous mutation megen-cephaly, mceph, (Jackson Laboratory, Bar Harbor,Maine) were used as donors of ovaries (Donahueet a1, 1996). These homozygous, phenotypieallymutant mice do not breed but they have fullyfunctional ovaries, Ovaiy recipients were 10 fe-males of the congenie strain 0864”" 1-1121) "(Jackson Laboratory, Bar Harbor, Maine). Thegenome of this strain is a background of BALB/C-+T’” Tyrp] " with the HM) locus originating fromthe C57BL/6By strain. The alleles of this locusdiffer between the BALB/eByJ and theC57BL/6By strain allowing them to be used asmarkers for determination ofmaternul germ-line inthe offspring. The animals were housed in a barrieranimal facility and kept at 12 h lightzl2 h dark.ness. a temperature of 21 - 22" C. and relativehumidity of 41) - 50%. They were caged in indivi-dually ventilated microisolalors in Vent—A—CageScand. J. Lab.Anim.Sei, N0. 2. 1998 Vol. 25racks (Iwarsson & Norén, 1992) with BeekayBedding (B&K Universal AB, So1lentuna, Swe-den), Rodent breeding diet R36 (protein 18.5 %,fat 4.0 “/o, ash 6.3 %, Vitamin and mineral fortified;Lactamin AB, Stockholm, Sweden) and waterwere provided ad [ibitumOvary transplantation:Both ovary recipient and ovary donor mice weredivided into age groups: recipients 42-45 and 95days old. donors 45 and 70 days old. Ovary trans-plantation was performed for three age combina-tions. The ovary transplantation technique wascarried out essentially as previously described(Cunlife»Beamer, 1983). The donor mice wereeuthanized by cervical dislocation. The two ovari—es from each mouse were removed under asepticconditions. They were placed in a petri dish con—taining 2 ml of M2 medium (Sigma, St. Louis,M0) at room temperature. The ovaries were clea-ned from the attached fat pad and the coveringbursa under microscope at 2 X magnification. Thecleaned ovaries were cut in halves. The recipientmice were anaesthetized with 0.2 m1 / 10 g bodyweight of 2.5 % tribromo-ethanol and tert-amyialcohol (“Avertin”) solution. After preparing foraseptic surgery. a single incision of the skin wasmade at dorsal, transversally across the lumbarregion, making both ovaries accessible. The ovary-attaehed fat pad was grasped and pulled out withthe ovary following. Under microscope at 2 Xmagnification a small incision was made in thebursa which then was peeled back over the surfaceof the ovary. allowing removal of the whole ovary.Both native recipient ovaries were removed andone halfot'a donor-ovary was placed into only theright bursa of the recipient mouse. Thus. ovariesfrom 1 donor was enough for transplanting 4recipients.Breeding:The transplanted mice were observed at standardconditions for 32 days. They were then mated witha reproductive male of the strain CAST/Ei(Jackson Laboratory, Bar Harbor, Maine). Theywere mated in 2 » 3 periods to allow for moresignificant evaluation of reproducibility, since it iscommon that the birth rate per mating is lower at54the first mating period than at subsequent periods(Gunasena el al. 1997a). The offspring werecounted 12 - 72 h after delivery and at weaning.The numbers of offspring given in results are fromthe counting at weaning. The pups were matedwith eachother and the size of the F2 litters wasdetermined at weaning.Typing of the hemoglobin tb-ehain (be):Whole blood (50 pl) was taken from the tip of thetail of each offspring mouse at 3 weeks ofage. Theblood was diluted with water (0.5 m1), and samplebuffer (10 ul of 0.5 M eystamine (Sigma, St.Louis MO), 0.01 M DTT. 0.74 M NlhOH) wasadded. The Ebb pattern was analyzed with poly-acrylamidc gel electrophoresis (T = 5 %, C = 3 0/u)at 50 V in electrophoresis buffer (0.025 MTriS(hydroxymethyl)aminomethane, 0.19 M glyeinpH 8.6) using the Mini-Protean II ElectrophoresisSystem (BlO-RAD Laboratories, Richmond, VA).Under these conditions the relative migrationdistance of the be from the donor (BALB/cByJ)as compared to the ilbb from the recipient(C57BL/GBy) is approximately 80%, allowingunambiguous typing, Having also be fromCAST/Ei in the blood sample does not interferewith the distinguishing between BALB/CBy] beand C57BL/6By be (Figure 1).ABCbebeB13Figure 1. Electrophoresis pattern of the hemoglo-bin B-ehain (be). A = CAST/Ei, 13 = donor(BALB/eByJ), C : recipient (C57BL/6By) Bb =Bromphenol blue.ResultsTable 1 shows the outcome from the first two, mating periods for each transplanted mouse.All transplanted mice produced at least onelitter. The mean litter size for all the transplan-ted mice from the first two mating periods was6.0 i 2.7 (mean 1: SD) offspring per litter.For untransplanted BALB/eJ the mean littersize is reported to be 5.2 (Green & Witham.I991). The mean litter size for the transplantedmice when grouped by age of recipient and ageof donor mice as 42—45 / 70 days, 95 / 70 daysand 42 / 45 days (recipient age / donor age) was6.0 I 3.7, 6.4 i 2.2 and 5.0 i 2.3 (mean i SD)offspring per litter, respectively. There was nosignificant difference in mean litter size betwe-en the age combinations (p > 0.05, t—test). TheScand. J. Lab. Anim. Sci. No 2. 1998. V01. 25time from start of the mating period until deli-very for the transplanted mice ranged between17 and 54 days, with a mean of 28.9 i 10.4(mean .t SD) days. Laboratory female mice arereceptive at intervals of 4 - 6 days with gestati-on periods of 19 - 20 days. For BALB/e] only47% of matings result in a delivery (Green &Witham, 199]). Thus, for the transplanted miceproductive matings occurred within a normaltime range of the breeding pair being together.Eight of the 10 transplanted mice producedoffspring that survived beyond 3 weeks. Todetermine the maternal origin ofthese offspringthey were typed for hemoglobin B—chain vari-ant.Table 1. Reproductive performance of the transplanted mice from the first two mating periods.  1-‘ irst mating period Second matinu eriod1D of Age of ID of Age of ID of Nglive No_dead ID of Ngiive Ngdeadrecipient recipient donor donor male offspring offspring male offspring otispring(days) (davs)1A* 42 1425 70 V 0 0 V 0 ND2A 42 1425 70 V 0 0 Y 0 ND3A 42 1425 70 Z 0 0 Z l 14A 42 1425 70 Z 2 O Y 9 01C 45 1426 70 X 8 0 X 8 118 95 1426 70 V 6 0 V 0 ND28 95 1426 70 V 7 0 Z 3 03 B 95 1426 70 V 6 1 V 9 02C 42 1431 45 X 7 0 V 0 03C 42 143 1 45 Y 6 0 Y 2 0          * Euthanized by cerviteal dislocation at the first delivery due to delivery complications.ND Whole litter dead, Number of dead offspring not determined.Scand. J.Lab Anim. Sci No 2. 1998. V01. 25Table 2 shows the maternal origin of" the offspringfrom the 2 first mating periods, except for female18 where the data are from the 3 first matingperiods. 18, but no one else, had 3 mating periods.For 6 of the 8 transplanted tnice all their offspring,50 mice, were derived from the transplanted ovari-es. The 2 remaining transplanted mice produced14 out of 15 and 12 out of 16 offspring mice ori-ginating trom the donor ovary. A total of 5 off—spring consequently represented the recipientsnatural progeny. In total 76 of the 81 survivingoffspring mice, 94%, were derived from the trans-planted ovaries. The offspring mice did not appearto have any defects and the mean size of the 1’2litters from the Iirst mating period of the offspringwas 9.4 i 2.2 (mean i SD).Table 2. Distribution ofdonor versus recipientmaternal germ-line in the offspring. Number of1D Offspring Offspring Deadreeipie from from offspringnt donor recipientovary ovary1A* 0 0 ND2A 0 0 ND3A 1 0 14A 1 l 0 01C 12 4 118 13 0 ND28 10 O 03B 14 1 12C 7 0 03C 8 0 0Total 76 5 ND* l-luthanized by cervical dislocation at the firstdelivery due to delivery complications.ND Number ofdead offspring not determined.56DiscussionThis evaluation showed a very high performanceof the ovary transplantation method using theprotocol presented here, also compared to previ-ously published techniques (Stevens. 1957; Par—kening er a1, 1985; Brem er a1. 1990, Cox e1 a],1996; Gunasena et al, 1997a; Gunasena et ul,1997b). All transplanted mice were reproductiveand 94% of the offspring were derived from the-transplanted ovaries. In addition, the mean littersize for the females was normal, despite the factthat the two native ovaries were replaced by onlyone half 01' an ovary. Previously reported percen—tages of ovary transplanted mice becoming preg—nant or producing offspring are 33% (Cox et al,1996), 48% (Parkem‘ng er a1, 1985), 60% (Brem eta1, 1990), 66% (Gunasena et al, 19971:), 80%(Stevens, 1957) and 100% (Gunasena 61 L11,l997a). These data were obtained after bilateralovariectomy of the recipients and replacementwith one half or two whole ovaries per female. Themean litter size for transplanted mice has previ-ously been reported to be similar (Cox et a1, l996)or reduced (Gunaserta et a1, 1997a) compared tocontrol mice.One 01 the CAST/Ei males, Z, produced onlysmall litters of2 — 3 offspring. Al] the other malesgave litters of 6 offspring or more, with the samefemales as the male Z had produced litters with.There was no apparent delay by the transplantationin the production of litter. Four of the transplantedmice had productive matings at the age 019 weeks.The average age at first mating for femaleBALB/e] is 8 weeks (Green& Witham, 1991). Allthe transplanted mice became pregnant within anormal time range after being put together with amale. From the first mating period 70% of thefemales delivered, whereas 90% delivered fromthe second mating period. These values are nor-mal, especially since 2 - 3 females simultaneouslywere together with 1 male. The birth rate permating result is similar to what is reported byGunasena et al (1997a).There is a risk of not completely removing thenative ovaries during transplantation and thus ofthe transplanted mouse producing her naturalprogeny. Delivery of natural progeny occurred in6% of the offspring. Four of these 5 endogenousoffspring were delivered from one and the samefemale, suggesting that incomplete ovary removalis infrequent. However, to be sure of the genotypeof the offspring it is important to type the progenyfor the maternal germ-line. This can often beperformed easily with coat colour markers. Withthe protocol used, only one donor mouse is neededto provide ovary transplants to four recipient mice.Surplus ovaries can be frozen for transplantation ata later time. In summary, the ovary transplantationtechnique used proved to be an efficient toolregarding time, labour, cost and number of ani—mals needed for the maintenance of mouse strainsthat do not breed but have functional ovaries.SummaryThere are mutant and transgenic mouse strainswhich lack the ability to breed, but where thefemales have functional ovaries. Ovary transplan-tation is an important tool for maintaining andproducing crosses with these non-breeding strains.We have evaluated an ovary transplantation met—bed by transplanting ovaries from females be-longing to a non-reproduetive BALB/CByJ mutantmouse strain, A11 transplanted mice, 10BALB/e.C57BL/6By, produced offspring and 94%of the progeny originated from the transplantedovaries. The mean litter size and the mating periodneeded for productive mating to occur were simi-lar to what is observed for corresponding controlmice.Sammanfattm'ngDet flnns mutanta och transgena musstammar somsaknar formagan att reproducera sig, men darhonoma har fungerande aggstoekar. Aggstoeks-transplantation ar ett viktigt instrument for attbehalla och skapa korsningar med dessa ieke—reproduktiva staminar. Vi har utvarderat enaggstoekstransplantationsmetod genom at‘t trans-plantera aggstoekar Tran honor tillhorande en icke-reproduktiv mutant musstam uppkommen iBALB/cByJ. Alla transplanterade moss, 10BALB/c.C57BL/6By, gav avkomma och 94% avden harstammadc fran de transplanteradeaggstoekarna. Medelva‘rdet pa kullstorlek ochtidsatgz‘mg av hane och hona tillsammans Fer pro-duktiv parning var snarlika med varden [Iir mots-varandc kontroll moss.Seand. J.1,ab.Anim.Sei. No.2. 1998.V01, 25AcknowledgmentsWe are grateful to Dr L. Mobraatcn. Dr L. R.Donahue and J. M. Sztein at the Jackson Laborato-ry, Bar Harbor, Maine. We also thank the person-nel at the Animal Facility, Center for MolecularMedicine, Karolinska Institute, Stockholm. Swe-den, for excellent technical assistance. The estab-lishment of the ovary transplantation technique atthe Karolinska Institute was financially supportedby a technology transfer grant from the SwedishMedical Research Council (MFR). Also, MFR issupporting Catharina Lavebratt with a post docto-ral fellowship and Martin Sehalling with a projectgrant.ReferencesBrem G, E Baunack, M Muller rt? EL Winnacker.‘Transgenic offspring by transcaryotic implanta-tion of transgenic ovaries into normal mice. MoiReprod Dev. 1990, 25, 42-44.Cox SL, J Shaw & (1' Jenkin' Transplantation oferyopreserved fetal ovarian tissue to adult reci-pients in mice. J Reprod Fertil. 1996, 10.7, 315-322.Cunlifi’e-Beamer TL: Biomethodology and surgi-cal techniques, In: Foster HL, JD Small & JGFox (eds): The mouse in biomedical research.Academic Press, New York. 1983. 3, 402-430.Donahue LR, SA Cook, KR. Johnson, RTBronson& MT Davisstm: Megeneephaly: a new mousemutation on chromosome 6 that causes hyper-trophy of the brain. Mamm Genome. 1996. 7.871-876.Green MC & BA Witham (eds): Handbook ongenetically standardized JAX mice 4th edition.The Jackson Laboratory, Bar Harbor. 1991.Gunasena KT, I’M Vt'llz'nes, ES Crz'lser & Jk'Critser: Live births afier autologous transplantof eryopreserved mouse ovaries. Hum Reprod.1997a, 12. 101-106.Gunasena KT. JR Lakey, PM 11/111123: [CS Critser& .lK Critscr: Allegeneie and xenogeneic trans-plantation of eryopreserved ovarian tissue toathymie mice. Biol Reprod. 1997b. 57, 226-231.Scand. .1. Lab.Anim. Sci. No. 2. 1998. Vol. 25lwarsson K & L Nore'rt: Comparison of mieroen-vironmental conditions in standard versus for-ced-air ventilated rodent filter-top cages. Scand JLab Anim Sci. 1992, 19, 167-173.Parkcm'ng TA, TJ Collins & FF Elder: Orthotopicovarian transplantation in young and aged58C57BL/6J mice: Biol. Reprod. 1985, 32, 989-997.Stevens LC: A modification of Robertson’stechnique of homoiotopic ovarian transplanta-tion in mice. Transplant Bull. 1957. 4 .106—107.

Ovary transplantation method resulting in high reproductive performance in mice

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Ovary transplantation method resulting in high reproductive performance in mice

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