Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids

FERTILITY AND STERILITY VOL. 77, NO. 4, APRIL 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids Laura Hewitson, Ph.D., a,b,c,d Crista Martinovich, B.S., a,c Calvin Simerly, Ph.D., a,b,c,d Diana Takahashi, M.Sc., a,c and Gerald Schatten, Ph.D. a,b,c,d Received June 18, 2001; revised and accepted November 1, 2001. Supported by the National Institute of Child Health and Human Development/ National Institutes of Health through a cooperative agreement (U54 HD18185) as part of the Specialized Cooperative Centers Program in Reproduction Research and by National Institutes of Health Grant RR13632. Presented at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 21 26, 2000. Reprint requests: Gerald Shatten, Ph.D., PDC, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213 (FAX: 412-641-2410; E-mail: pdcgs@mail.magee.edu). a Division of Reproductive Sciences, Oregon Regional Primate Research Center, Oregon Health and Science University. b Department of Obstetrics and Gynecology, Oregon Regional Primate Research Center, Oregon Health and Science University. c Present address: Pittsburgh Development Center (PDC) of the Magee-Womens Research Institute, Pittsburgh, Pennsylvania. d Present address: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania. 0015-0282/02/$22.00 PII S0015-0282(01)03281-2 Oregon Regional Primate Research Center, Oregon Health and Science University, Beaverton, Oregon, and Pittsburgh Development Center, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania Objective: To establish pregnancies in rhesus monkeys using testicular sperm and elongated spermatids injected into oocytes. Design: Comparative animal study. Setting: Regional Primate Research Center. Animal(s): Prime, fertile rhesus monkeys. Intervention(s): Oocytes collected by laparoscopy from gonadotropin-stimulated female rhesus monkeys were injected with testicular sperm or elongated spermatids obtained from the testis of males. Cleavage stage embryos were transferred to surrogate females. Main Outcome Measure(s): Fertilization, embryo cleavage, and the establishment of pregnancies. Fertilization failures were fixed and processed for the detection of microtubules and chromatin configurations. Result(s): Fertilization, assessed by the presence of two pronuclei within 15 hours after injection, was 60% for intracytoplasmic sperm injection with testicular sperm and 47% for elongated spermatid injection. Fertilized zygotes co-cultured in Connaughts Medical Research Labs (CMRL) medium on a Buffalo Rat Liver cell monolayer resulted in hatched blastocysts after testicular sperm extraction intracytoplasmic sperm injection and elongated spermatids. Embryos transferred at the 4- to 8-cell stage gave rise to three pregnancies: 2/3 from testicular sperm and 1/1 from an elongated spermatid. Three healthy infants were delivered by cesarean. Oocytes that failed to fertilize typically remained arrested in metaphase of meiosis. Conclusion(s): Testicular sperm and elongated spermatids can be used for fertilization in the rhesus monkey resulting in live births. (Fertil Steril 2002;77:794 801. 2002 by American Society for Reproductive Medicine.) Key Words: Assisted reproduction, non-human primates, animal models, male infertility, spermatids, fertilization Intracytoplasmic sperm injection (ICSI) was first introduced to enable oligospermic men to father children (1, 2). Its unrivaled success has since led to the development of sperm retrieval techniques so that even men with obstructive azoospermia can be offered the chance of parenthood. Testicular sperm can be retrieved by a number of ways, reviewed by Tournaye (3), including fine needle aspiration, testicular sperm aspiration (TESA) and testicular sperm extraction (TESE), and when used for ICSI, result in successful pregnancies (4, 5). However, in men with nonobstructive azoospermia, where defects or blocks in spermatogenesis prevent the formation of sperm, the injection of immature spermatids has been proposed (6). This was quickly followed by the introduction of several novel assisted reproductive technologies (ART) using round (7) or elongated (8, 9) spermatids, reviewed by Al-Hasani et al. (10), and even secondary spermatocytes (11). However, there have been considerable concerns raised regarding the unambiguous proof that these immature germ cells retain reproductive potency, as well as ongoing concerns regarding the normalcy of these, and other ART, offspring (12 16). Furthermore, without a clinically relevant animal model to look at gener- 794

ational effects of ART, the long-term effects of assisted reproduction on ICSI children may not necessarily be addressed for several decades (17). Numerous animal models have been proposed to study ART. The mouse has typically enjoyed the most success (18 20), but it is difficult to extrapolate these data to humans as the mouse does not rely on a paternally inherited centrosome during fertilization, as is typical of humans (21, 22). Spermatid injections in rabbits (23) and domestic species (24, 25) have been successful but oocytes from these species require an additional chemical or electrical stimulus to achieve these results. Assisted reproduction in Old World monkeys, including rhesus, cynomolgus, and pig-tailed macaques, has recently been proposed as being relevant animal models for studying human ART (26). The ICSI has been well established in these species (26 31), resulting in several live births (26), although preliminary experiments with round spermatids were not successful (27). Interestingly, the cytoskeletal events of fertilization in rhesus monkeys (28, 32) closely mimic those described for humans (33) and both species follow a paternal method of centrosome inheritance (32 35). These similarities, coupled with the relatively short generational time in rhesus monkeys, highlight their use as a model for understanding human assisted reproduction. The present study was therefore undertaken to investigate whether rhesus oocytes fertilized using testicular sperm and elongated spermatids would support embryonic development in vitro, and after transfer to surrogate females, give rise to normal offspring. MATERIALS AND METHODS Hormonal Stimulation and Oocyte Collection Female rhesus monkeys (Macacca mulatta) exhibiting normal menstrual cycles were superovulated with a regimen of exogenous gonadotropic hormones as previously described (29). Follicles were aspirated by laparoscopy at 27 hours after hcg injection and the oocytes immediately assessed for nuclear maturity and cytoplasmic quality. Immature oocytes that had undergone germinal vesicle breakdown were cultured at 37 C in preequilibrated microdrops of modified CMRL-1066 medium (36) supplemented with 10 g/ml porcine FSH (Vetrepharm Research Inc., Athens, GA), 5 IU/mL hcg (Calbiochem, La Jolla, CA), and 3 mg/ml bovine serum albumin (BSA) to support maturation to metaphase II-arrest. Mature oocytes with a distinct polar body and even cytoplasm were cultured for up to 6 hours in a preequilibrated, modified Tyrode s medium (TALP, 37) containing 0.2 mmol/l pyruvate and 3 mg/ml BSA before fertilization. Preparation and Selection of Testicular Sperm and Elongated Spermatids A small tissue sample was removed from the testis of two sedated adult, fertile rhesus males by fine needle aspiration. Briefly, a 21- to 23-gauge butterfly needle was inserted through the scrotal sac and the testicular contents aspirated into 0.5 ml of HEPES-buffered TALP medium (TALP- HEPES) by applying a strong negative pressure with a 10-mL syringe. The testicular tissue was washed twice in TALP-HEPES and then subjected to repeated pipetting to produce a roughly homogeneous cell suspension of spermatogenic cells. Rhesus testicular sperm (Fig. 1A) and elongated spermatids with no visible flagellum (Fig. 1B) were identified using a Nikon inverted microscope equipped with 20 and 40 Hoffman modulation contrast objectives. The testicular sperm generally exhibited slightly twitching tails. Elongated spermatids were characterized by an oval-shaped nucleus signifying nuclear condensation. A flagellum was not visible in the spermatids selected for this study suggesting that either the tail was still forming and therefore embedded within the caudal cytoplasm or that the tail had been lost during preparation. Elongated spermatids with a visible tail of varying lengths could also be identified, but these were not included in this study. Intracytoplasmic Injection of Sperm and Elongated Spermatids Microinjection pipettes (Humagen, Charlottesville, VA) were beveled to 50 degrees with an outer diameter of 6 or 9 m for sperm or elongated spermatids, respectively. Holding pipettes had an outer diameter of 120 m and an inner diameter of 20 m. All manipulation procedures were performed at 37 C in100 L of TALP-HEPES under mineral oil (Sigma Chemical Co., St. Louis, MO). Spermatogenic cells were suspended in TALP-HEPES before selection, whereas ejaculated sperm were suspended 1:100 in 10% polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA) to aid in cell handling. Oocytes were immobilized with the polar body in the 11 o clock position and injected with testicular sperm or elongated spermatids, as previously described (28, 29). Gentle aspiration of the oocyte cytoplasm assured that the oolemma had been breached during injection. Injected oocytes were returned to culture in TALP at 37 C in5%co 2. Assessment of Fertilization and Embryo Culture Oocytes were examined between 12 and 15 hours after injection with a Nikon TE300 inverted microscope equipped with Hoffman modulation contrast optics. Oocytes containing two pronuclei and two polar bodies at this time were considered normally fertilized and selected for culture, whereas unfertilized or arrested oocytes were prepared for immunocytochemistry (discussed later). Fertilized oocytes FERTILITY & STERILITY 795

FIGURE 1 Selection of testicular sperm and elongated spermatids (A, B) and after injection into metaphase arrested rhesus oocytes (C F). Testicular sperm (A) were characterized by a condensed nucleus and an enlarging flagellum with slight motility. Elongated spermatids (B) were characterized by an elongating nucleus with the migrated cytoplasm at its basal region, containing the axial filament. No visible flagellum was apparent. Pronuclear formation 15 hours after the injection of testicular sperm (C) and elongated spermatids (D) into rhesus oocytes. Cleavage stage ICSI (E) and ELSI (F) embryos used for transfer. All images were captured with a Nikon 35-mm camera attached to a Nikon TE300 inverted microscope, equipped with Hoffman modulation contrast optics. Bar 10 m A, B; 20 m C, D; 30 m E, F. were co-cultured from the two-cell stage on Buffalo Rat Liver cells (American Type Culture Collection, Rockville, MD) in CMRL medium containing 10% FCS (Hyclone Laboratories, Inc., Logan, UT) as previously described (29) and scored for development daily. ET and Pregnancy Monitoring Rhesus females with normal menstrual cycles synchronous with the oocyte donor were selected as embryo recipients based on serum progesterone (P) and estrogen (E) levels. Surgical ET were performed on day 2 or 3 after ovulation by transferring two 4- to 8-cell embryos into the oviduct using a mid-ventral laparotomy. Implantation was assessed by hormonal analyses of daily blood samples and pregnancy was confirmed by transabdominal ultrasound on day 30 after transfer. Infants were delivered by a scheduled cesarean section between 155 and 160 days gestation. Cytoskeletal Imaging Fertilization failures were processed for the immunocytochemical detection of microtubules and DNA as previously described (26, 28, 38). Briefly, zonae were first re- 796 Hewitson et al. TESA-ICSI and ELSI in non-human primates Vol. 77, No. 4, April 2002

TABLE 1 Number of oocytes produced and fertilization, cleavage, and ET data in the rhesus monkey. n b No. (%) of fertilized oocytes No. (%) of cleaved embryos e No. of embryos transferred Pregnancy rate (%) No. (%) of offspring ICSI a 20 12 (60) c 10 (83) d 6 2/3 (66) 2 (66) ELSI 19 9 (47) c 5 (55) d 2 1/1 (100) 1 (100) Total 39 21 (54) 15 (71) 8 3/4 (75) 3/4 (75) a ICSI with testicular sperm. b No. of oocytes surviving injection. c,d Not statistically significant (ANOVA). e No. of embryos reaching the 4- to 8-cell stage. moved by incubation in 0.1% pronase (CalBiochem) for 5 10 minutes. After recovery, zona-free oocytes were attached to polylysine-coated coverslips and then fixed in 2% formaldehyde (Polysciences, Warrington, PA). Coverslips with attached oocytes were permeabilized in PBS containing 2% Triton X-100 detergent (Sigma) for 40 minutes and then transferred to a blocking solution (PBS with 150 mmol/l glycine and 3 mg/ml BSA) for 30 minutes. To visualize microtubules, oocytes were incubated in a mouse monoclonal antibody to -tubulin (E7) for 1 hour. Oocytes were rinsed in PBS and 0.1% Triton for 30 minutes. E7 was detected using an FITC-conjugated goat anti-mouse IgG secondary antibody (1:40; Zymed, San Francisco, CA). DNA was labeled with 5 g/ml DAPI (Sigma) for 10 minutes. Coverslips were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA) to retard photobleaching. Conventional epifluorescent microscopy was performed using a Zeiss Axiophot microscope. Images were digitally captured with a chilled CCD camera (Princeton Instruments Inc., Trenton, NJ) using Metamorph Imaging software (Universal Imaging, West Chester, PA). Images were archived on CDs and processed as described above. RESULTS A total of 92 oocytes were retrieved by laparoscopy from two stimulated females, 26 28 hours after hcg injection. Oocyte maturity at the time of collection was as follows: 12/92 (13%) oocytes were arrested in metaphase II, 67/92 (73%) oocytes had undergone germinal vesicle breakdown, and 13/92 (14%) oocytes had an intact germinal vesicle. After 8 hours of culture in CMRL supplemented with hormones, 64/67 (95%) germinal vesicle breakdown oocytes completed maturation yielding a total of 76 metaphase II oocytes, 49 of which, were allocated to this study. The 49 in vivo or in vitro matured oocytes were randomly divided so that 27 were allocated for ICSI using testicular sperm (TESA-ICSI; Fig. 1A, arrow) and 22 were used for the injection of elongated spermatids (Fig. 1B, arrows). Of 27 metaphase II oocytes undergoing TESA-ICSI, 20 (74%) survived the injection process and, by 15 hours after sperm injection, 12/20 (60%) had extruded the second polar body and developed two apposed pronuclei indicative of normal fertilization (Fig. 1C, arrow). For ELSI, 19/22 metaphase II oocytes survived, of which 9/19 (47%) were fertilized (Fig. 1D, arrow; Table 1). By 60 hours after injection, 10 (83%) TESA-ICSI and 5 (55%) ELSI embryos had cleaved to the 4- to 8-cell stage. The rates of both fertilization and embryo cleavage after ICSI with testicular sperm and ELSI were not statistically different (ANOVA; Table 1). Embryos resulting from TESA-ICSI and ELSI appeared morphologically similar (Fig. 1E, F) and did not appear to differ in the timing of cell divisions. Further embryo development is not reported as a significant number of embryos were either selected for transfer or allocated to other projects, although blastocysts were attained from both fertilization methods. Three TESA-ICSI ETs were performed (two embryos transferred per female), which resulted in two (66%) singleton pregnancies confirmed by ultrasound. Two ELSI embryos, transferred to a surrogate female, also gave rise to a singleton pregnancy (Table 1). All three pregnancies were delivered by cesarean section at 157 days of gestation (with the normal gestation length for rhesus monkeys being 165 days) to ensure survival of the offspring. Tess and Tickler (weighing 420 and 550 g, respectively; Fig. 2A and 2B) were produced by TESA-ICSI and Elsie (weighing 430 g; Fig. 2C) was produced by ELSI. Tess and Elsie are biological sisters (sibling oocytes were injected with sperm or spermatids from the same male on the same day). Tickler was produced using gametes from different animals. Oocytes injected with a testicular sperm that did not develop pronuclei (8/20) by 16 hours after injection were fixed for immunocytochemical analyses to determine the cause of fertilization failure (Table 2). The majority of these (5/8) remained arrested at metaphase of meiosis characterized by an anastral, barrel-shaped spindle radially oriented at FERTILITY & STERILITY 797

FIGURE 2 Rhesus offspring produced by the intracytoplasmic injection of testicular sperm and elongated spermatids. Tess (A) and Tickler (B) were conceived using testicular sperm and Elsie (C) was conceived using an elongated spermatid. the oocyte cortex (MII, red) with chromosomes aligned across the spindle equator (MII, blue; Fig. 3A). The sperm (S) appeared unchanged within the oocyte cytoplasm. One additional oocyte contained both a maternal (MII) and paternal (P) meiotic spindle (Fig. 3B) and two oocytes remained arrested in metaphase but did not contain a sperm. The majority of fixed oocytes injected with an ELSI remained arrested in metaphase of meiosis (5/10; Table 2 and Fig. 3C). The injected spermatid typically remained unchanged, identified by its association with manchette microtubules (Fig. 3C, arrow). An additional two ELSI oocytes underwent activation but arrested at an early stage of female pronuclear formation (Fig. 3D). The second polar body (PB) remained anchored to the oocyte by the mid-body (red), whereas the decondensing female pronucleus (F) resided close to the mid-body. The spermatid did not form a male pronucleus or organize a microtubule aster (spermatid, arrow). One oocyte underwent premature chromosome decondensation (PCC) containing both a female and male meiotic spindle. DISCUSSION We report the first successful application of ICSI using testicular sperm and elongated spermatids to non-human primates. The implanted rhesus embryos, derived from either testicular sperm or elongated spermatids, gave rise to three healthy babies. The ability to produce rhesus monkey offspring using novel methods of ART not only provides a model for studying human assisted fertilization, but also suggests the possibility of using surgically retrieved sperm TABLE 2 Causes of fertilization failure after the injection of testicular sperm or elongated spermatids into rhesus oocytes. No. (percentage) of oocytes arresting at each stage n b Met II with sperm/spermatid Met II without sperm/spermatid Small female PN with sperm/spermatid Large female PN with sperm/spermatid Met II and PCC c ICSI a 8 5 (62.5) 2 (25) 0 0 1 (12.5) ELSI 10 5 (50) 0 2 (20) 2 (20) 1 (10) a ICSI with testicular sperm. b No. of oocytes failing to form pronuclei by 15 hours after injection. c Premature chromosome decondensation (oocyte contains both a maternal and paternal meiotic spindle). 798 Hewitson et al. TESA-ICSI and ELSI in non-human primates Vol. 77, No. 4, April 2002

FIGURE 3 Microtubule and chromatin configurations of rhesus oocytes fixed 24 hours after TESA-ICSI (A, B) or ELSI (C, D). After TESA-ICSI (A) or ELSI (C), the oocyte typically remained arrested in metaphase of meiosis, characterized by an anastral, barrel-shaped spindle radially oriented at the oocyte cortex (MII, red) with chromosomes aligned across the spindle equator (MII, blue). The injected sperm (S, blue) or spermatid (S tid, blue) did not form a male pronucleus or assemble a microtubule aster within the oocyte cytoplasm. In B, the oocyte was injected with a testicular sperm that has undergone premature chromosome decondensation resulting in both a maternal (MII) and paternal (P) meiotic spindle. The sperm tail, separated from the sperm, may still be associated with the sperm centrosome because a small aster of microtubules has formed by the tail (arrow). In D, an activated ELSI oocyte has extruded the second polar body (PB), which remains anchored to the oocyte by the mid-body (red). The decondensing female pronucleus (F) resides close to the mid-body. The spermatid is characterized by a slightly swollen nucleus associated with manchette microtubules (green, arrows) surrounding the basal region of the spermatid. Bar 10 m. or spermatids for the propagation of rare or endangered species, which may suffer some degree of spermatogenic impairment. Although several techniques have been developed to aid in the identification of spermatogenic cells for ART (39 43), the selection criteria can be confusing and have met with considerable skepticism (44). The identification criteria for ELSI used by several investigators include spermatids with varying degrees of nuclear condensation and tail formation suggesting that different stages of spermatids were used for each study (45 48). This is only confounded by the exclusion of specific images demonstrating the stages of spermatids that were used (9, 49, 50). Furthermore, there are several reports that were able to identify sperm in samples from men with nonobstructive azoospermia as long as elongated spermatids were present, suggesting that spermatid arrest may not be the correct terminology to describe these cases (51, 52). This also creates the possibility that maturing sperm, not elongated spermatids, may have been used for some elongated sper- FERTILITY & STERILITY 799

matids cases. Clearly, the criteria for spermatid selection are complicated and, more importantly, can be interpreted differently by the various clinics. In this study, elongated spermatids were characterized using 20 or 40 Hoffman modulation contrast optics according to the Clermont classification (53) as illustrated by de Kretser and Kerr (54), by an oval nucleus (suggesting nuclear condensation) already demonstrating a reorganization of cytoplasm to the caudal region. Fertilization rates using rhesus testicular sperm (60%) and elongated spermatids (47%) from fertile adult males were lower than with ejaculated sperm (77%) (29), although similar to clinical trials (10, 55, 56). It appears that elongated spermatids, like testicular and ejaculated sperm, contain an oocyte-activating factor, although oocyte activation was the most common cause of fertilization failure after both TESA- ICSI (Fig. 3A) and ELSI (Fig. 3C). In one TESA-ICSI oocyte, the sperm underwent PCC forming a paternal meiotic spindle (Fig. 3B). Furthermore, the sperm head and tail had separated, most likely leaving the sperm centrosome with the tail, as a small area of microtubule assembly was associated with the sperm tail. Interestingly, in two elongated spermatid injection cases, the oocyte activated whereas the spermatid appeared arrested (Fig. 3D). This suggests that the oocyte either underwent parthenogenetic activation or that the oocyte was activated by the spermatid, which then failed to begin decondensation or microtubule nucleation. Because the gametes used in this study were obtained from fertile animals, the fairly high rates of fertilization failure suggest that the technique would be less effective using gametes from animals with impaired fertility, although this would provide a more suitable preclinical model. The timing of embryo development after both rhesus testicular sperm and spermatid injection was comparable to that documented for conventional rhesus ICSI (29). Furthermore, despite lower fertilization and cleavage rates after ELSI, the morphology of spermatid embryos was similar to that of testicular ICSI embryos (Fig. 1C F). The establishment of pregnancies from TESA-ICSI and ELSI embryos was 2/3 (66%) and 1/1, respectively, and similar to conventional rhesus ICSI (66%) (26). However, these figures cannot be easily extrapolated to the clinical data as the rhesus sperm and spermatids used for this study were obtained from fertile males as opposed to the infertile men typically seen in the clinics. Cognitive testing is currently being performed on rhesus offspring conceived by both natural and assisted reproduction at the Infant Primate Research Lab at the Washington Regional Primate Research Center and includes analyses using a mental development index derived for non-human primates, modified from the Bayley Scales of Infant Development (57). The Bayley mental development index, used to evaluate ICSI children (12, 14), has suggested that there may be an increased risk of mild delays in the development of ICSI children at 1 year when compared with children conceived by routine IVF or conceived naturally (14). Once the rhesus offspring reach sexual maturity, their reproductive potential will be investigated. Interestingly, since Elsie (produced by elongated spermatid injection) is a sibling of Tess (produced by testicular ICSI), this enables us to compare two different methods of ART on related animals. The ability to use testicular sperm and spermatids for animal production in rhesus monkeys not only benefits research into human reproduction but also contributes to captive breeding programs to aid in the survival of endangered species. An integrated approach for cryobanking both ejaculated and epididymal sperm along with testicular biopsies from non-human primates would benefit future species conservation. Finally, the application of spermatid injection to the rhesus monkey underscores its value as a preclinical model for examining and understanding many aspects of assisted human fertilization. Acknowledgments: The authors thank Dr. John Fanton and Darla Jacob for surgical expertise, the DAR staff at ORPRC for animal husbandry; Michelle Miller and Tonya Swanson for technical assistance and Kevin Grund, Kevin Mueller, and Ethan Jacoby for semen collection and preparation. Hormones for oocyte stimulations were gratefully provided by Ares Serono, Inc., and Organon, Inc. 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