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Development of In Vivo-Matured Porcine Oocytes Following Intracytoplasmic Sperm Injection

^a Nextran, Albany, Ohio 45710

ABSTRACT

The objective of this study was to assess the development of porcine ova fertilized by intracytoplasmic sperm injection (ICSI). Allyl trenbolone (Regumate) was used to synchronize estrus in 13 postpuberal gilts. Gilts were superovulated with pregnant mare serum gonadotropin and hCG. Ova were aspirated from 5- to 8-mm follicles at 36 h after hCG. Cumulus cells were removed by blunt dissection and pipetting in Beltsville embryo culture medium (BECM) supplemented with 0.1% hyaluronidase. Sperm were washed and resuspended in BECM + 8% polyvinylpyrrolidone. Ova (n = 237) that exhibited a polar body were centrifuged at 15 000 x g for 6 min and injected with a single spermatozoon. One hundred fifty-four ova were cultured in NCSU-23 medium in a 5% CO₂ in air environment for 168 h. Ova were fixed in acetic acid/ethanol and stained with 1% orcein. Sixty-nine ICSI ova were cultured for 24 h and transferred (mean = 23) to three recipients. Eighty-one ova (69%) that survived ICSI cleaved within 48 h. Thirty-eight percent (31/81) of these ova became blastocysts (mean ± SEM = 24.7 ± 1.1 cells). One recipient gave birth to three pigs. These results demonstrate that porcine embryos derived from ICSI can develop into live pigs.

fertilization, oocyte development, ovum, sperm

INTRODUCTION

Intracytoplasmic sperm injection (ICSI) has been used to generate live human [1], rabbit [2], sheep [3], bovine [4], equine [5], and murine [6] offspring. In swine, Catt and Rhodes [7] found that injection of in vitro-matured oocytes with spermatozoa resulted in a high (60%) fertilization rate. In a more recent study, ICSI of porcine oocytes using immature, round spermatids yielded embryos that were capable of developing into blastocysts in vitro [8]. The birth of live pigs as a result of ICSI has not been reported.

The objectives of this study were to characterize in vitro development of porcine embryos produced by ICSI and to determine if ICSI-derived porcine embryos can generate live offspring.

MATERIALS AND METHODS

Estrous Synchronization

All experimental procedures involving the use of animals were conducted in accordance with the Guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching [9]. All protocols were approved by an internal Institutional Animal Care and Use Committee. Postpuberal (>6 mo of age) gilts were fed allyl trenbolone (AT) (Regumate; Hoechst-Roussel Vet, Warren, NJ; 10 mg/gilt/day) for 16–19 days. Twenty-four hours after the last day of AT consumption, gilts received 10 mg of prostaglandin F₂ (PGF₂) (Lutalyse; Pharmacia-Upjohn, Kalamazoo, MI). Pregnant mare serum gonadotropin (PMSG: 1000 IU) and hCG (750 IU) were administered at 48 and 128 h, respectively, after the last day of AT consumption.

Ova Recovery and Preparation

At 36 h following hCG administration, gilts were laparotomized and follicles 5–8 mm in diameter were aspirated using an 18-gauge needle and 3-ml syringe that contained 1 ml of Beltsville embryo culture medium (BECM) [10]. Cumulus (expanded) enclosed ova were washed twice in BECM and placed in an organ well tissue culture dish. Cumulus cells were initially removed by blunt dissection in BECM. The remaining cumulus cells were removed by repeated pipetting in BECM supplemented with 0.1% hyaluronidase. Denuded ova were washed twice and stored in NCSU-23 [11] medium supplemented with 1x basal medium Eagle's (BME) amino acids, 1x minimal essential medium nonessential amino acids, and 100 µg/ml cysteine. Ova were maintained in a 5% CO₂ in air environment at 39°C prior to ICSI.

Sperm Preparation

Semen was collected 24 h prior to the day of ICSI and initially extended (1:4 v/v) in Beltsville thawing solution (BTS). Extended semen was slowly cooled down to room temperature (23°C) and stored overnight at 17°C. Immediately prior to ICSI, 2 ml of extended semen was washed, and the pellet was resuspended in 8 ml of BECM. Sperm were stored at 38°C until ICSI.

ICSI Procedure

Oocytes that exhibited the first polar body were centrifuged in BECM at 15 000 x g for 6 min. Oocytes were centrifuged to permit visualization of sperm following injection [12]. ICSI was performed in 4-µl drops of NCSU-23 medium under oil (M8410; Sigma, St. Louis, MO). Movement of live sperm was slowed by adding 2 µl of the sperm suspension to a 4-µl drop of BECM supplemented with 8% polyvinylpyrrolidone (PVP). Five 4-µl drops were arranged in a column on the lid of a 50- x 9-mm Petri dish. All drops were covered by mineral oil that had been equilibrated with NCSU-23 medium overnight. The top drop contained sperm in PVP; the four remaining drops contained one oocyte each.

ICSI was performed on a heated microscope at 200x magnification using a Nikon TE3000 inverted microscope with Hoffman optics. Injection and holding pipettes were supplied by Humagen, Inc. (Charlottesville, VA) and regulated by two Narishige micromanipulators. Sperm were individually immobilized by scoring the tail. A single sperm was aspirated from the sperm droplet and moved to a droplet containing an oocyte. The oocyte was captured by the holding pipette and immobilized with its polar body at either the 6 or the 12 o'clock position (Fig. 1). The spermatozoon was injected using the type B (oolemma was pricked once with no suction) or type C (oolemma was pricked once followed by small suction) method described by Nagy et al. [13].

View larger version (116K): [in this window] [in a new window]   FIG. 1. Intracytoplasmic injection of a spermatozoon into an in vivo-matured porcine oocyte that has undergone centrifugation. A spermatozoon is visible in the injection pipette

Ova Culture and Staining

One hundred fifty-four oocytes injected with a single spermatozoon were cultured for 168 h in vitro. Ova were cultured in 50-µl microdrops of NCSU-23 medium under oil in a 5% CO₂ atmosphere at 39°C. At the end of the culture period, ova were fixed in 1:3 acetic acid:ethanol and stained with 1% orcein. Ova that failed to divide by 48 h post-ICSI were fixed and stained immediately. Oocytes in which the metaphase II plate was absent and/or two pronuclei were visible were considered to be activated.

Short-Term Culture and Embryo Transfer

Eighty-three oocytes that had been injected with live sperm were cultured for 24 h as described previously. Sixty-nine ova that survived ICSI were transferred (mean = 23) to three recipients that had exhibited estrus 24 h later than the donors. Immediately prior to transfer, the number of ova that had cleaved was noted and any degenerate ova were discarded. Pregnancy was determined on Day 25 of gestation by ultrasound (EI Medical, Loveland, CO).

RESULTS

One hundred eighteen of 154 (77%) ova survived ICSI. Activation did not occur in 23 (19%) of the ova that survived ICSI. Of the ova that cleaved (81 of 118, 69%), 46% and 54% did so within 24 h and between 24 and 48 h, respectively, following ICSI. Thirty-eight percent (31 of 81) of the ova that cleaved formed blastocysts that contained an average (±SEM) of 24.7 ± 1.1 nuclei (Table 1 and Fig. 2).

View larger version (131K): [in this window] [in a new window]   FIG. 2. ICSI-derived porcine embryos after 168 h in vitro. Five blastocysts are visible.

Pregnancy was confirmed in one of three recipients. This recipient received 22 ova and delivered three live (two females, one male) pigs on Day 113 (Day 0 = date of transfer). The mean (±SEM) birthweight was 1.8 ± 0.1 kg.

DISCUSSION

Early studies on ICSI of mouse ova led to the conclusion that "the complex set of interactions normally required for sperm to penetrate the egg investments and to fertilize the egg [were] biologically unnecessary" in mammals [14, 15]. This conclusion has since been confirmed by the birth of ICSI-derived live offspring in other mammalian species and now in swine.

In the current study, the fertilization rate, i.e., the proportion (69%) of porcine ova that cleaved following ICSI, and the proportion of embryos that developed into blastocysts were similar to values reported by Catt and Rhodes [7] and Kim et al. [16], respectively. The proportion of ICSI embryos that developed into blastocysts (38%) and the mean number of nuclei present were also similar to those results observed following culture of in vitro-matured/in vitro-fertilized porcine oocytes [17, 18]. These findings suggest that the in vitro developmental rates of embryos derived from ICSI and from in vitro fertilization are similar.

The chronology of events leading up to the initial cleavage of ICSI ova appears to be prolonged compared with the chronology for ova naturally fertilized in vivo. Hunter [19] found that the interval from activation to the appearance of the two-cell stage for naturally fertilized ova was 14–16 h. In the present study, 46% of the oocytes injected with sperm failed to cleave until at least 24 h after ICSI. The delay in cleavage may have been due to differences in morphology between injected sperm and those that fertilize the ovum naturally. During natural fertilization, the plasma membrane over the acrosomal cap is lost before the sperm enters the ooplasm. This membrane remains intact when sperm are introduced into the ovum by ICSI. Because disintegration of the plasma membrane must occur before the sperm's nucleus can enter the ooplasm [20], the presence of this "extra" membrane may delay release of the nucleus and formation of the pronucleus [21].

The protocol developed for ICSI of in vivo-matured porcine oocytes was similar to that currently being used for ICSI of human oocytes. Some differences, however, were noted between human and porcine ICSI with regard to the spermatozoon's response to washing and culture in PVP. Prior to human ICSI, semen is typically centrifuged through a percoll [13] gradient or a commercially prepared biphasic gradient medium (ISOLATE; Irvine Scientific, Santa Ana, CA) to remove dead sperm and cellular debris. ISOLATE is a colloidal suspension of silica particles stabilized with covalently bound hydrophilic silane in Hepes-buffered human tubal fluid. Attempts to "clean" porcine semen by centrifugation through ISOLATE, however, consistently yielded sperm preparations with greatly reduced motility. We observed this result even when porcine sperm were diluted to a concentration that was similar to that found in human semen, e.g., 40 to 90 x 10⁶ sperm/ml, prior to the wash with ISOLATE. Because of this finding, porcine sperm in the current study were washed with BTS and BECM only.

In contrast to human sperm, porcine sperm also exhibited agglutination (head to head) in doublets, triplets, etc., within 15 min of their addition to PVP. This phenomenon made it difficult to isolate and aspirate individual sperm for ICSI after a relatively short (<30 min) time. Addition of a fresh aliquot (1 µl) of sperm to the PVP drop temporarily alleviated this problem and usually provided a sufficient number of singlet sperm to allow completion of an ICSI session. This agglutination phenomenon was also observed when sperm were washed with ISOLATE.

The low proportion (3 of 69, 4%) of transferred ICSI embryos that developed to term may have been caused by one or more of several factors. Long-term (168 h) culture of ICSI embryos in the present study yielded blastocysts with nucleus numbers that were significantly lower than those reported for blastocysts generated by injection of a spermatid or spermatid nucleus and cultured in a similar medium [16]. Nucleus numbers in ICSI blastocysts were also lower than those observed in normally fertilized embryos that had been cultured for 120 h in utero [22]. These differences may reflect an inadequacy in the current culture system. This concern was addressed by performing ICSI as soon as possible after recovery of in vivo-matured oocytes and limiting the post-ICSI in vitro culture period to 24 h.

Factors previously identified in bovine ICSI studies may also have contributed to the low proportion of ICSI embryos that developed into pigs. These factors include a low rate of oocyte activation and/or male pronucleus formation. Both concerns were addressed by Rho et al. [11] while attempting to generate calves by ICSI of in vitro-matured oocytes. Pretreatment of sperm with dithiothreitol (DTT) and activation of injected oocytes with ionomycin and 6-dimethylaminopurine increased the proportion of injected sperm that formed pronuclei, the rate of blastocyst development, and the overall efficiency of bovine ICSI. However, transfer of ICSI embryos to recipients after 8 days in vitro resulted in no pregnancies that were carried to term. In the current study, 19% of the porcine oocytes injected with sperm failed to undergo activation. The majority of these oocytes contained sperm heads that had not undergone decondensation. Pretreatment of porcine sperm with DTT may promote nuclear decondensation and increase the proportion of oocytes that are activated.

In the absence of sperm decondensation or deposition of the sperm in the vitellus, some oocytes in the current study may have undergone parthenogenetic development. Kim et al. [16] found that 9% of oocytes subjected to sham ICSI developed into blastocysts after 8 days in vitro. The mean (±SEM) number (29 ± 6) of nuclei observed in these parthenogenetic blastocysts was similar to the number of nuclei observed in ICSI-derived blastocysts produced in the current study. Because blastocysts produced in the present experiments were not subjected to chromosomal analysis, a proportion of these embryos may have undergone parthenogenetic development.

To our knowledge this is the first report of pigs derived from in vivo-matured oocytes fertilized by ICSI. The birth of ICSI pigs assumes added importance with the finding that exogenous DNA can be delivered into the oocytes of other species by ICSI [23]. The use of ICSI for the production of transgenic animals appears to have at least two advantages over pronuclear microinjection: the integration frequency is high, and the diameter of the ICSI pipette is at least 10 times greater than that of pronuclear DNA microinjection pipettes. This increase in diameter may allow introduction of large DNA molecules such as yeast or mammalian artificial chromosomes (250 kilobases in size) into an oocyte without the threat of "shearing" of the DNA molecule. The birth of live pigs in the present study suggests that sperm-mediated transfer of DNA via ICSI may be possible in swine.

ACKNOWLEDGMENTS

I thank Dr. Jana Copelin, McFarland Clinic, P.C., Ames, IA, for my training in the art of human ICSI.

FOOTNOTES

First decision: 20 January 2000.

¹ Correspondence: Michael J. Martin, Nextran, 901 Carpenter Rd., Albany, OH 45710. FAX: 740 698 2518; nexmartin{at}earthlink.net

Accepted: February 14, 2000.

Received: December 20, 1999.

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