Intra-cytoplasmic sperm injection in a marsupial

REPRODUCTIONRESEARCH

Intra-cytoplasmic sperm injection in a marsupial

Nadine M Richings, Geoffrey Shaw, Peter D Temple-Smith and Marilyn B Renfree

Department of Zoology, The University of Melbourne, Victoria, 3010, Australia

Correspondence should be addressed to N M Richings; Email: [email protected]

Abstract

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus

eugenii ), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of

each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injec-

tion, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored

for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The

cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development

also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single

embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence

of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid

coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular

oocytes lacking a mucoid coat. Cell–zona and cell–cell adhesion occurred in embryos from follicular oocytes, suggesting that

the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal

oocytes and cell–cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the

integrity of the conceptus during cleavage.

Reproduction (2004) 128 595–605

Introduction

The only report of successful in vitro fertilization (IVF) in amarsupial is in a South American species, the grey short-tailed opossum (Monodelphis domestica), in whichmature follicular oocytes were placed with preincubatedepididymal spermatozoa in a complex culture mediumand 67% of fertilized oocytes cleaved (Moore & Taggart1993). Despite many efforts using standard inseminationmethods, fertilization has not been achieved in vitro inany Australian marsupial (Rodger 1994, Renfree & Lewis1996), although sperm binding and penetration ofthe zona pellucida (Mate et al. 2000, Sidhu et al. 2003),sperm–oocyte fusion (Sidhu 2003) and decondensationof the sperm head (Sidhu 2003) have been observedin vitro.

Intra-cytoplasmic sperm injection (ICSI) is a micro-manipulation technique in which a single spermatozoonis inserted into the cytoplasm of an oocyte. ICSI has beenused widely in eutherian mammals to achieve fertilizationand embryo development (Palermo et al. 1992, Catt &Rhodes 1995, Burruel et al. 1996, Hewitson et al. 1996,Pope et al. 1998, Martin 2000, Deng & Yang 2001,Yamauchi et al. 2002). In humans, 67% of injectedoocytes cleave and 82% of these embryos are deemed to

be diploid (two pronuclei) (Richings et al. 1999). Recently,ICSI was used to examine the timing and ultrastructure ofthe events of fertilization in a marsupial (Magarey & Mate2003b). Sperm collected from electro-ejaculated tammarwallabies (Macropus eugenii ) were injected into oocytescollected from hyperstimulated females. Oocyte acti-vation, sperm head decondensation and pronuclear for-mation were similar to that of some eutherian species,however syngamy did not occur. While tammar oocytesfrom hyperstimulated cycles can support most of theevents of fertilization, they may not be competent to com-plete fertilization. The ability of tammar oocytes fromnatural cycles to fertilize and cleave after ICSI has notbeen examined. There are no published reports of embryodevelopment after fertilization in vitro in any Australianmarsupial or after ICSI in any marsupial.

The reproductive biology of the tammar has been exten-sively studied (Tyndale-Biscoe & Renfree 1987). It is apolyoestrous, monovular, seasonal breeder and has a post-partum oestrus with mating occurring about 1 h after birth(Rudd 1994). In the wild, tammars normally only produceone young a year (Tyndale-Biscoe & Renfree 1987). Ovu-lation is spontaneous and occurs about 40 h after matingand about 24 h after the luteinizing hormone surge that is

q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00270

ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org

triggered by a rise in oestradiol produced by the Graafianfollicle (Harder et al. 1984, Shaw & Renfree 1984, Tyn-dale-Biscoe & Renfree 1987, Renfree & Lewis 1996). Asin all marsupials, fertilization occurs in the upper oviduct,the oocyte moves through the oviduct within 24 h andcleavage occurs in the uterus (Renfree & Lewis 1996).During passage through the oviduct and entrance to theuterus, the oocyte acquires two extra investments, amucoid coat and a shell coat (Tyndale-Biscoe & Renfree1987). Early cleavage in the tammar has been studied invivo and in vitro (Renfree & Lewis 1996). In most marsu-pials, cell–zona adhesion precedes cell–cell adhesion(Selwood 2001). In the tammar, from the pronuclear stagethe cells are attached to the zona pellucida with microvilliand the degree of attachment increases at the late 4-cellstage (Renfree & Lewis 1996). The degree of attachmentbetween individual cells appears to increase at the late4-cell or early 8-cell stage (Renfree & Lewis 1996). As inall marsupials, the cleavage divisions produce a unilami-nar blastocyst with no inner cell mass that expands anddevelops to form a bilaminar blastocyst (Tyndale-Biscoe &Renfree 1987).

In vivo fertilized tammar oocytes develop slower in cul-ture than in vivo (Renfree & Lewis 1996). Most embryoscollected from mated tammars at the 1-cell or 2-cell stageprogressed to the 4-cell stage in culture. While a few ofthese embryos progressed to the 8-cell stage, none devel-oped to blastocysts. In contrast, all embryos collectedfrom mated tammars at the 4-cell or 8-cell stage devel-oped to blastocysts (Renfree & Lewis 1996). This indicatesthat there may be a uterine signal to trigger cleavage pastthe third division in the tammar.

The aim of this study was to establish methods for ICSIand to examine embryo development in vitro after theintra-cytoplasmic injection of sperm, in a marsupial, thetammar wallaby.

Materials and Methods

Oocyte collection

Oocytes were collected from females undergoing naturalcycles at 2–3 days post-partum, and therefore only asingle oocyte could be collected from each animal.Female tammars (Macropus eugenii) (n ¼ 12) were killedby anesthetic overdose (100 mg/kg sodium pentobarbi-tone) and reproductive tracts were excised under sterileconditions. In females that had not yet ovulated (n ¼ 4),oocytes were dissected from Graafian follicles(4.83 ^ 0.24 mm) on the surface of the ovary into hand-ling medium (HEPES-buffered Dulbecco’s modified Eagles’medium (DMEM); L-glutamine, 2 mmol/ml; fetal bovineserum (FBS), 10%; penicillin G, 50 IU/ml; streptomycin,50mg/ml; all from Thermo Trace, Noble Park, Victoria,Australia). In females that had recently ovulated (n ¼ 8),oocytes were flushed from the oviduct with handlingmedium. When dissected from follicles, oocytes had few,

if any, cumulus cells attached to the zona pellucida. Thesecells were easily removed before injection by mechanicalmanipulation with needles. Oocytes flushed from theoviduct were surrounded by a mucoid coat that ranged inthickness from 9 to 28mm. All oocytes were examinedusing an inverted microscope with heated stage, differen-tial interference contrast (D.I.C.) Nomarski optics and anattached video camera and monitor. Each oocyte wasexamined on the highest magnification at which the entireoocyte could be viewed on the monitor. The thickness ofinvestments and the maximum diameter of the vitellusand the diameter perpendicular to it were recorded.The oocyte was then washed in fresh handling medium,transferred to equilibrated culture medium (DMEM;L-glutamine, 2 mmol/ml; FBS, 10%; penicillin G,50 IU/ml; streptomycin, 50mg/ml) and incubated at 37 8Cin a humidified gas environment of 5% CO2 in air untilthe time of micro-injection.

Sperm collection and preparation

Male tammars were anesthetized using 10 mg/kg Zoletil(Virbac, Peakhurst, NSW, Australia) and anesthesia wasmaintained using isoflurane (2% in O2; Abbot Australasia,Kurnell, NSW, Australia). Spermatozoa were collectedunder sterile conditions from the cauda epididymidis byeither hemicastration or epididymal aspiration. In hemi-castrated animals, the testis and epididymis wereremoved, the cauda epididymidis was dissected andwashed in phosphate-buffered saline (PBS; Thermo Trace)to remove any blood. Small pieces of epididymis wereplaced into handling medium for very brief periods oftime (2–10 s) to allow motile spermatozoa to be expelledfrom the tissue. Epididymal aspiration was performed bymicrosurgery or percutaneous aspiration (Bourne et al.1995b). For microsurgical aspiration, a small incision wasmade through the tunica to expose the cauda epididymidisand motile spermatozoa were aspirated from this regionusing a 26 gauge needle attached to a 1 ml syringe con-taining 0.1 ml handling medium. For percutaneous aspira-tion, the cauda epididymidis was palpated and a 26 gaugeneedle attached to a 1 ml syringe containing handlingmedium was inserted through the skin into the duct and asmall amount of fluid was collected. The epididymalsperm aspirate was resuspended in handling medium andkept at room temperature until injection. After sperm col-lection, incisions were sutured and males were allowed torecover and subsequently returned to the breeding colony.

ICSI

All oocytes were injected with spermatozoa within 6 h ofcollection. Oocytes were placed individually into drops(15ml) of handling medium surrounding a central dropof sperm suspension (0.5–1 £ 106/ml) on a Petri dish (Bec-ton-Dickinson-Falcon, Bedford, MA, USA) and coveredwith equilibrated mineral oil (Sigma, St Louis, MO, USA).

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The micro-injection procedure was based on the methoddescribed by Palermo et al. (1992) with modifications asdescribed by Bourne et al. (1995a). The sperm suspensiondid not contain polyvinylpyrrolidine (PVP) and motilespermatozoa were immobilized in the sperm suspensiondrop using the injection pipette (Cook Australia, Brisbane,Queensland, Australia) to crush the tail against the dish(Fig. 1a). A single immobilized spermatozoon was aspi-rated into the injection pipette and transferred to a dropcontaining an oocyte. The oocyte was held under suctionon a holding pipette (Cook Australia) with the polar body908 from the injection site (Fig. 1b). The spermatozoonwas then injected into the cytoplasm of the oocyte withthe injection pipette (Fig. 1c–h). The ease of injection wasinfluenced by the investments surrounding the oocyte. Theinjection of follicular oocytes was straightforward (Fig. 1).The thickness of the mucoid coat in tubal oocytes varied(9–28mm) and resisted penetration by the injectionpipette. Although the method used to inject these oocyteswas generally the same as that used to inject follicularoocytes, it required very precise and specific alignment ofthe holding and injection pipettes.

Oocyte and embryo culture

After injection, the oocytes were transferred to 50mldrops of equilibrated culture medium under mineral oil infour-well culture plates (Nunc, Roskilde, Denmark) andincubated at 37 8C in a humidified gas environment of5% CO2 in air. The medium was replenished everysecond day and culture was stopped when no furtherchanges were observed (up to 4 days). The injectedoocytes were examined using an inverted microscopewith D.I.C. Nomarski optics 16–20 h after injection forthe presence of pronuclei, extrusion of polar bodies andlysis or degeneration. Oocytes were evaluated twice dailyfor cleavage (cell number, retracting organelles, regularityof membranes), degree of fragmentation and signs ofdegeneration (retracting organelles, dark or granular cyto-plasm, cell lysis). All oocytes and embryos were carefullyexamined in a number of planes by gently shaking theculture dish, which caused the oocyte/embryo to roll andtherefore alter position. Four oocytes that did not cleaveafter injection were fixed in 4% glutaraldehyde/0.1 Mcacodylate buffer overnight at 4 8C, washed and stored infresh cacodylate buffer (0.1 M) at 4 8C until staining.Oocytes were rinsed thoroughly in PBS containing 0.3%bovine serum albumin (BSA; Sigma) and all debris wascarefully removed from the investments. The oocyteswere then placed in Hoechst 33258 (5mg/ml; Sigma; in0.3%BSA/PBS) for 30–45 min at 37 8C in the dark. Afterthis incubation, oocytes were rinsed thoroughly in threechanges of wash solution (0.1% Tween 20; Sigma; 0.3%BSA in PBS) and mounted on microscope slides in 10%glycerol (Sigma) under coverslips. The slides were exam-ined using a Leitz Dialux 20 microscope equipped witha u.v.-A filter block. After culture, four embryos were

processed by this method and the other embryos wereused in another study.

Results

Injection and fertilization

Once the holding and injection pipettes were preciselyaligned, there was no obvious difference in the ease ofinjection and compression of the vitellus during injectionof follicular oocytes (no mucoid coat) or tubal oocytes(with mucoid coat). No oocytes showed signs of mem-brane lysis or cytoplasmic degeneration after injection.Signs of fertilization (pronucleus-like structures or polarbody extrusion) were seen in all injected oocytes (Fig. 2).Two distinct polar bodies were seen in one-third ofoocytes (four out of twelve) and one distinct polar bodyand cytoplasmic fragments that were probably the firstpolar body were evident in seven oocytes. The day afterinjection, spherical refractile structures resembling pronu-clei were seen in most oocytes (ten out of twelve), but thestructural detail of these pronucleus-like structures wasdifficult to discern because of the character of the cyto-plasm (Figs 3c and d and 4a). Cleavage did not occur inoocytes which lacked these pronucleus-like structures.Hoechst staining was successful on three oocytes thatwere injected but did not cleave. Two oocytes had a non-decondensed sperm head, female chromosomes and apolar body and another oocyte had two pronuclei but noobvious DNA in the polar body.

Early cleavage in vitro

Pronucleus-like structures were seen in all oocytes thatcleaved. By 52 h post-injection (p.i.) half of the oocytes(six out of twelve) had completed at least the first cleavagedivision and 25% (three out of twelve) had completed thesecond cleavage division (Fig. 2). By 72 h p.i., 42% (fiveout of twelve) of the oocytes had not cleaved, 58% (sevenout of twelve) had completed at least the first cleavagedivision, 33% (four out of twelve) had completed at leastthe second cleavage division and 17% (two out of twelve)had begun or completed the third cleavage division. Nofurther cleavage occurred after 3 days of culture (Fig. 2).Hoechst staining was successful on three embryos. Anoocyte that appeared to be a 2-cell embryo had onlyreached the pronuclear stage, so although early stages offertilization had commenced, syngamy had not occurred.A 4-cell embryo had five nuclei and a cluster of densechromatin (Fig. 4k). Four of these nuclei were possiblyin two pairs which may each have been sister nucleiin blastomeres that had not yet undergone cytokinesis.The 8-cell embryo had seven nuclei and one dense clusterof chromatin (Fig. 4l). The cleavage rate varied betweenindividual embryos (Fig. 2).

Oocytes from the follicle and oocytes from the oviducthad many similarities in their subsequent development

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Figure 1 ICSI in the tammar wallaby. (a) Immobilization of sperm with the injection pipette. (b) Oocyte held on the holding pipette with thepolar body (PB1) positioned 908 to the injection site and sperm visible in the injection pipette (arrow); Z ¼ zona pellucida. (c) The injectionpipette inserted through the zona pellucida and oolemma and sperm visible in the injection pipette (arrow). (d) The injection pipette moved tothe centre of the ooplasm. (e) Ooplasm has been aspirated up the injection pipette in the direction of the arrow and is visible as granularmaterial in the lumen. (f) Ooplasm returned to the oocyte with sperm. (g) The injection pipette is then carefully and slowly withdrawn from theoocyte. (h) Sperm visible at the centre of oocyte (arrow). Scale bars ¼ 100mm.



in vitro after ICSI. A developmental sequence from collec-tion of the oocyte through early cleavage was constructedfrom observations of all oocytes that were injected andcleaved (Figs 3 and 4). After extrusion of the second polarbody and formation of the pronuclei (Figs 3c and d and4a), the vitellus shrank resulting in a larger perivitellinespace (Fig. 4b and c). The organelles retracted away fromthe oolemma in some regions of the cytoplasm (Fig. 4b)and the oolemma became irregular and less distinct inthese areas. One of these regions was near the polarbodies and another was opposite the polar bodies. Whenboth regions were obvious the oocyte had a peach-likeshape, with indentations on either side of the vitellus (Fig.4d). This was clearly the cleavage furrow of the first div-ision. At this time fragments were released near the clea-vage furrow (Fig. 3e) and cytoplasmic outgrowthsresembling ‘boxing-gloves’ or ‘palps’ were often present(Fig. 4e). These stages were also seen in oocytes that failedto complete the first cleavage division. At the 2-cell stageblastomeres were of similar size and a small cytoplasmicfragment, spherical to ovoid in shape and about33–50mm in dimension, was usually present near thecleavage furrow (Fig. 4f). There was only a small cleavagecavity (i.e. perivitelline space) and the cells appeared tobe pushed against the zona pellucida, though notattached. If the embryo failed to cleave further, by late onday 2 or day 3 the blastomeres had altered in shapebeing less ovoid, more irregular and attached to somedegree to the zona pellucida (cell–zona adhesion). Thecell–zona adhesion in these 2-cell embryos became more

pronounced during the culture period (Fig. 5a–c). At the4-cell stage the blastomeres were less regular in size butdid not follow an obvious pattern. Cell–zona adhesionwas evident throughout most of the 4-cell stage butbecame more pronounced over time (Fig. 5d–f). Contactbetween blastomeres (cell–cell adhesion) only occurred inlate 4-cell embryos. Only one embryo reached the 8-cellstage (Fig. 4i and j). The blastomeres differed in size andseemed to be in two groups, one small and one large. Thetwo cell types seemed to be at opposite sides or poles ofthe embryo. Attachment of cells to the zona pellucida wasapparent.

Despite these similarities, several differences were alsoseen in embryos from follicular oocytes and embryos fromtubal oocytes. At collection, oocytes from the ovary andoocytes from the oviduct differed in the number of invest-ments, in vivo environment to which they had beenexposed and post-ovulatory age. All follicular oocyteswere surrounded by a zona pellucida while all tubaloocytes had a mucoid coat and a zona pellucida (Fig. 2).All tubal oocytes had been ovulated in vivo, then exposedto the oviductal environment for an unknown period oftime. By 48 h p.i., cleavage had occurred in three out offour follicular oocytes and three out of eight tubal oocytes(Fig. 2). The proportion of blastomeres that cleaved ateach cell stage differed between embryos from tubal orfollicular oocytes (Figs 2 and 6). There was no obviousrelationship between the thickness of the mucoid coat andsubsequent development, since the oocyte with the thick-est mucoid coat (28mm) and the three oocytes with the

Figure 2 Dimensions of tammar oocytes at collection from either the follicle or the oviduct; timeline of observations after ICSI and treatment afterculture. Site: F ¼ follicle, O ¼ oviduct; Vitellus ¼ dimension of oocyte without investments (mm); ZP ¼ thickness of zona pellucida (mm); MC ¼thickness of mucoid coat (mm); n.a. ¼ not applicable; PBs ¼ number of polar bodies; 1 þ F ¼ 2nd PB and fragments of 1st PB; PN ¼ pronuclei,Y ¼one or two spherical to ovoid refractile regions seen in cytoplasm; N ¼ no pronucleus-like structures seen in cytoplasm. Time of observations:F ¼ fertilization check (results in Day1 column); X ¼ culture stopped; PCP ¼ post-culture processing; GH ¼ glutaraldehyde fixation and Hoechststaining; G* ¼ glutaraldehyde fixation, but lost during processing; S ¼ cell spreading.



Figure 3 A developmental sequence of a single oocyte, from collection to the 4-cell stage. (a) Tubal oocyte at collection with zona pellucida (Z),mucoid coat (M) and first polar body (PB1). (b) After ICSI with sperm visible near the centre of the ooplasm (arrow). (c) and (d) 16 h post-injec-tion (p.i.) with pronucleus-like structures (arrows) and polar bodies (PB1, PB2) obvious. (e) 40 h p.i. after completion of the first cleavage division(cells indicated by arrows) and cytoplasmic fragments obvious between blastomeres. (f) At 48 h p.i. the conceptus remained at the 2-cell stageand undulations were apparent in the zona pellucida (UZ). (g) At 63 h p.i. the second cleavage division was complete, the four cells wereobvious (arrows) and cell–zona adhesion (CZA) and zona undulations (UZ) were evident. (h) At 89 h p.i. the CZA was more prominent andcell–cell adhesion (CCA) was also evident. Scale bars ¼ 100mm.



Figure 4 Typical developmental features during cleavage in vitro after ICSI of tammar oocytes. (a) Day 1 p.i.: follicular oocyte with twopronucleus-like structures (arrow heads) and two polar bodies (PBs). (b) Day 1 p.i.: follicular oocyte with retracted organelles (RO) and shrinkage(S) of the vitellus resulting in a large perivitelline space. (c) Day 1 p.i.: tubal oocyte with shrinkage (S) of the vitellus and cleavage furrow(arrow). (d) Day 1 p.i.: tubal oocyte with cleavage furrow (arrows) evident on opposite sides of the vitellus giving a ‘peach-like’ shape. (e) Day 1p.i.: tubal oocyte with ‘palp-like’ structures that form in the cleavage furrow. (f ) Day 2 p.i.: tubal oocyte that has cleaved to a 2-cell embryo,with the cytoplasmic fragment (CF) often evident after this cleavage division. (g) Day 2 p.i.: follicular oocyte that has cleaved to a 4-cell embryo.Early cell–zona adhesion (CZA) is evident and a large cleavage cavity typical of embryos from follicular oocytes. (h) Day 2 p.i.: tubal oocytethat has cleaved to a 4-cell embryo (arrows indicate cells). CZA is evident. Minimal cleavage cavity and zona pellucida undulations (UZ) typicalof embryos from tubal oocytes. (i) and (j) Day 3 p.i.: tubal oocyte that cleaved to an 8-cell embryo. Blastomeres are numbered (1–8) and thelines from each digit indicate the centre of the blastomere. Blastomeres 1–4 are larger, blastomeres 5–8 are smaller. * ¼ above focal plane. (k)A 4-cell embryo had two polar bodies (arrow heads), a cluster of dense chromatin (1), and five nuclei, four appeared to be in two pairs (2, 3)and the fifth structure was on its own (not visible in this plane of focus). (l) An 8-cell embryo had seven nuclei (1–6, and one not visible in thisplane of focus), one dense cluster of chromatin (7) and two polar bodies (arrow head, only one shown in this plane of focus). Z ¼ zona pellu-cida, M ¼ mucoid coat. Scale bars ¼ 100mm.



Figure 5 Adhesion of blastomeres to the zona pellucida in 2-cell and 4-cell embryos became more prominent over time. (a–c) A single 2-cellembryo from a tubal oocyte: (a) 43 h p.i., (b) 63 h p.i. and (c) 87 h p.i. (d–f ) A single embryo from a follicular oocyte from the 2-cell to 4-cellstage: (d) 41 h p.i., (e) 54 h p.i. and (f ) 67 h p.i. CZA ¼ cell–zona adhesion, CCA ¼ cell–cell adhesion, CF ¼ cytoplasmic fragment, UZ ¼undulating zona pellucida. Scale bars ¼ 100mm.



thinnest mucoid coats (9, 16, 20mm) all failed to cleave(Fig. 2).

The cleavage cavity and intercellular spaces betweenblastomeres seemed to be more expansive in embryosfrom follicular oocytes (2-cell, see Fig. 5a compared withFig. 5d; 4-cell, see Fig. 4g compared with 4h). Undula-tions developed in the zona pellucida of embryos fromtubal oocytes after 2 or more days in culture (Figs 3 and5a–c), but were never evident in embryos from follicularoocytes (Figs 4g and 5d–f).

Discussion

This is the first published report of embryo developmentafter ICSI in a marsupial. Mature oocytes collected frompreovulatory follicles or flushed from oviducts andinjected with epididymal spermatozoa could undergocleavage as demonstrated by morphology and DNA(Hoechst) staining. At best, the injected oocytes developedto the same stage in vitro as oocytes fertilized in vivobefore collection and culture (Renfree & Lewis 1996). Wewere unable to demonstrate any difference in develop-mental potential between embryos from tubal oocytespossessing a mucoid coat and follicular oocytes lacking amucoid coat, although morphological differences wereevident in culture.

The number of pronuclei and extrusion of the secondpolar body are used to ascertain the ploidy of fertilizedoocytes in vitro (Braude 1987, Vanderhyden & Armstrong1989, Donoghue et al. 1992). The refractile structuresobserved in all oocytes that cleaved were assumed to bepronuclei. The presence of the pronuclei and extrusion ofextra polar body material in the injected tammar oocyteswere both good indicators of fertilization or oocyte acti-vation. It was not possible to determine the number of

pronuclei and therefore the ploidy of the resultantembryos. Extrusion of the second polar body in theinjected oocytes was a clear demonstration that theembryos were not triploid as a result of a retained polarbody (digyny). Although the injection procedure can acti-vate the oocyte of tammars, the injected spermatozoon isusually involved in fertilization (Magarey & Mate 2003a).This is also the case in eutherians (Tesarik et al. 1994, Catt& Rhodes 1995). It is therefore unlikely that more than asmall proportion of cleaved oocytes were parthenogenotesas a result of oocyte activation by the injection procedure.

Tubal oocytes are older than follicular oocytes in termsof post-ovulatory age and they have an extra investment,the mucoid coat (Tyndale-Biscoe & Renfree 1987).Possible functions of the mucoid coat are that it may be abarrier to polyspermy, provide nutrients to the embryo andact as an osmotic stabilizer during development (reviewedin Selwood 2000). Post-ovulatory aging is a continualprocess beginning within hours of ovulation (Mintz 1967,Kaufman 1983, Williams 2002) and, in eutherians,includes changes in organelles, cytoskeleton, corticalgranule release, protein synthesis, spindle structure,plasma membrane and chromosomes (fragmentation,scattering, decondensation) (Tarin 1996). Tubal oocytesmay have undergone post-ovulatory aging which is associ-ated with an increased incidence of parthenogenesisin eutherian oocytes (Kaufman 1983). However, if thick-ness of the mucoid coat reflects time in the oviductand therefore post-ovulatory age, the oldest oocyte clearlydid not undergo parthenogenetic activation since it didnot cleave.

In tammars, fertilization occurs before the mucoid coatis deposited (Tyndale-Biscoe & Renfree 1987), but theoocytes retain the potential for fertilization and cleavageafter this time since half of the tubal oocytes cleaved after

Figure 6 The number and proportion of blastomeres at each cell stage that cleaved from follicular and tubal oocytes. Light grey, blastomeres didnot cleave; dark grey, blastomeres cleaved.



ICSI. Embryos from tubal oocytes had a small cleavagecavity and closely apposed blastomeres and their mor-phology was similar to embryos from in vivo fertilization(Renfree & Lewis 1996). Embryos from follicular oocyteshad a more obvious cleavage cavity and greater separationbetween blastomeres. Although cell–zona adhesionseemed to be unaffected in these embryos, the blastomereswere more spherical and there was reduced cell–celladhesion. It is likely that these differences would affectfurther development, particularly blastocyst formation.Embryos of Monodelphis domestica generated from IVF offollicular oocytes were able to develop to the 16- to 32-cell stage in vitro, but halted development at blastocystformation (Moore & Taggart 1993). The observations inthis study support previous suggestions that the mucoidcoat is involved in providing an appropriate environmentfor the conceptus (Selwood 1989, 2000) by modulatingthe osmotic pressure in a manner that minimizes thevolume of the cleavage cavity and promotes close contactbetween blastomeres.

The morphology of tammar embryos generated fromtubal oocytes after ICSI was similar to that of in vivofertilized tammar embryos (Renfree & Lewis 1996). Thecell–zona and cell–cell adhesions that are established inearly cleavage of marsupial embryos (reviewed inSelwood 2001) were evident in embryos generated fromboth follicular and tubal oocytes indicating that themucoid coat is not essential for the development of thesecontacts. Both cell–zona and cell–cell adhesions becamemore pronounced during the culture period, even inembryos that had ceased cleavage, suggesting that theseprocesses are time dependent rather than cell numberdependent.

In vitro fertilized (injected) oocytes developed moreslowly than was reported for in vivo fertilized oocytes(Renfree & Lewis 1996). Only 43% of the injected tammaroocytes in this study that underwent cleavage had reachedthe 4-cell stage within 24 h of fertilization, compared with86% of comparable in vivo fertilized oocytes collectedfrom mated tammars and placed in culture (Renfree &Lewis 1996). There was a broad range of cleavage timesfor each cell stage, reflecting the different cleavage ratesof individual embryos. In humans, faster growing embryoshave a significantly higher implantation rate than slowergrowing embryos (Edgar et al. 2000). In this study, thefaster cleaving embryos developed further, possibly reflect-ing the better potential of these embryos.

In this study, oocytes from natural cycles cleaved to the8-cell stage after the injection of a single epididymalspermatozoon. However, oocytes collected from hyper-stimulated tammars only develop to the pronuclear stageafter injection of ejaculated spermatozoa (Magarey &Mate 2003a,b). The stimulation regimen used in thosestudies was probably not optimal since oocytes wereunable to complete fertilization. Although spermatozoawere prepared by swim-up from the semen after electro-ejaculation (Magarey & Mate 2003a,b), they were

exposed to seminal and prostatic components. Tammarepididymal spermatozoa have a relatively high rate ofendogenous metabolism that can support motility and res-piration for prolonged periods of time (Murdoch & Jones1998). However, preparation of ejaculated spermatozoaby swim-up reduces or impairs their motility, ultrastruc-ture, metabolism and rate of intracellular accumulation ofsugars (Murdoch et al. 1999). PVP was used in that tam-mer ICSI study for sperm immobilization and injection(Magarey & Mate 2003b) and, since no cleavage wasreported, it is possible that tammar oocytes or spermato-zoa are sensitive to this polymer. PVP is not required toachieve fertilization with ICSI (Bourne et al. 1995a,b) andembryonic development, implantation and live birth ratesare similar in embryos generated from ICSI or fromstandard IVF insemination (Richings et al. 1999). In thepresent study, cleavage was achieved after ICSI withoutthe use of PVP in the injection procedure, so it is clearlynot required.

The embryos generated from ICSI were morphologicallysimilar to those previously described from in vivo fertilizedembryos (Renfree & Lewis 1996), but further research isneeded to confirm the viability of embryos created fromsperm injection in marsupials. This study demonstrated thepotential for IVF in marsupial oocytes using ICSI, and haslaid the foundation for developing techniques such astransgenesis, as well as assisted reproductive techniquesfor conservation of endangered species.

Acknowledgements

We thank everyone in the Wallaby Research Group for helpwith animal handling, with special thanks to Sue Osborn andMichael Leihy for help with animals in surgery. We thank DrDebra Gook for valuable advice and technical help with theHoechst staining technique and for the use of her laboratory,materials and equipment. We thank David Paul for prep-aration of photographic plates. This work was supported bythe Australian Research Council (DP0344941) and an Austra-lian Postgraduate Award to N M R.

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Received 2 April 2004First decision 9 July 2004Revised manuscript received 30 July 2004Accepted 9 August 2004



Intra-cytoplasmic sperm injection in a marsupial

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