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Regular Article |
a Laboratoire d'Histologie et de Biologie de la Reproduction, Equipe d'Accueil 2138, UFR Science Médecine Biologie Humaine, Université Paris 13, 93017 Bobigny, France
b U 332-INSERM, Institut Cochin de Génétique Moléculaire, 75014 Paris, France
ABSTRACT
The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56°C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.
conceptus, implantation, in vitro fertilization
INTRODUCTION
The mammalian spermatozoon has a unique chromatin structure that renders its genome very resistant to physical disruption. Sperm DNA is highly condensed and stabilized by an extensive cross-linking of protamines by disulfide bonds. In previous studies, mammalian spermatozoa were extremely resistant to temperature variation. Normal mouse offspring were obtained after microinjection of freeze-dried spermatozoa [1] and frozen spermatozoa without cryoprotection [2], demonstrating sperm nucleus resistance to low temperature. Mammalian sperm genome resistance to heat has also been reported. Isolated sperm nuclei of hamster, mouse, and human exposed to high temperature (90°C for 30 min) were able to form pronuclei when microinjected into hamster oocytes [3]. Rabbit zygotes derived from spermatozoa heated at 60°C for 30 min could cleave and reach the six- to eight-cell stage [4]. However, the formation of apparently normal pronuclei and the occurrence of the first embryonic cell divisions do not guarantee sperm nucleus integrity. Sperm nuclei damaged by DNA-specific dye coupled with ultraviolet light excitation during flow cytometric sorting have been shown to permit morphologically normal pronucleus development after microinjection into hamster oocytes, although they exhibited profound abnormalities in chromosome structure at the first mitotic metaphase [5]. However, the ability of heat-treated spermatozoa to support full embryonic development has not yet been demonstrated. In the present study, we used intracytoplasmic sperm injection (ICSI) in the mouse to assess the ability of spermatozoa exposed to heat to support fertilization and embryonic development.
MATERIALS AND METHODS
Reagents
All inorganic and organic compounds were purchased from Sigma Chemical Company (St. Louis, MO) unless stated otherwise.
Media
CZB medium [6] supplemented with 5.55 mM D-glucose and 5 mg/ml BSA (fraction V) was used for oocytes and embryos culture. Modified CZB medium (CZB-Hepes) with 20 mM Hepes and reduced NaHCO3 (5 mM) and 0.1 mg/ml polyvinyl alcohol (30–70 kDa, cold water soluble) was used for spermatozoa, oocyte, and embryo collection, subsequent treatments, and ICSI. A Ca2+-free CZB medium containing 5 mM SrCl2 (Sr2+-CZB) was used for activation of microinjected eggs. All cultures were performed in CZB at 37.5°C under 5% CO2 in air. Isotonic NaCl solution supplemented with polyvinylpyrrolidone (PVP; Mr 360 x 103) was used to suspend spermatozoa prior to ICSI.
Collection of Oocytes and Zygotes
Oocytes to be microinjected were recovered from superovulated B6CBA female mice (3–12 wk old). Females were injected twice 48 h apart with 5 IU eCG (Folligon; Intervet, France) and 5 IU hCG (Chorulon; Intervet). Oocytes were collected from oviducts 13–14 h after hCG injection and freed from the cumulus by a 3-min treatment with 0.1% hyaluronidase in CZB-Hepes. Cumulus-free oocytes were rinsed several times in CZB-Hepes and then incubated in CZB at 37.5°C under 5% CO2 in air for up to 2 h before microinjection.
Zygotes at the two-pronucleus stage were collected from superovulated CD1 (Swiss Webster, albino) females that had been mated with males of the same strain during the previous night. Zygotes were cultured in CZB medium for up to 24 h before embryo transfer at the two-cell stage.
Preparation of Spermatozoa
Spermatozoa were collected from one or two excised caudae epididymides after squeezing them in a 500-µl drop of CZB-Hepes under mineral oil. Spermatozoa were allowed to disperse at 37.5°C for 30 min. The sperm suspension was transferred to a 500-µl Eppendorf tube for centrifugation at 600 x g for 5 min. Motile spermatozoa were allowed to swim up in the supernatant for 30 min. The upper part of the supernatant (about 200 µl) was recovered in a 500-µl Eppendorf tube and kept at room temperature (RT). This sperm suspension contained nearly 100% motile sperm cells. For heat treatment, the suspension of motile spermatozoa in a 500-µl tube was heated in a water bath for 30 min at 56°C and then kept on ice until ICSI was performed. Before and after heating, sperm viability and integrity were assessed. Vitality staining was performed by adding two drops of eosin Y to one drop of the sperm suspension and mixing for 30 sec. Two drops of nigrosine were added to this suspension. After mixing for 30 sec, smears were prepared using one drop of the final suspension. Spermatozoa integrity was investigated using transmission electronic microscopy (TEM). For TEM, spermatozoa were fixed for 1 h at RT with 2% glutaraldehyde, 0.1 M cacodylate buffer (pH 7.2) and processed routinely.
ICSI Procedure and In Vitro Culture of Zygotes and Embryos
Injection of sperm heads into mouse oocytes was performed according to the method of Kimura et al. [7] using a piezo-driven micropipette. Immediately before injection, 0.25 µl of heated or nonheated sperm suspensions was mixed with 5 µl of 12% PVP-saline medium. A single spermatozoon was drawn head first, and a few piezo-pulses (high intensity, low speed) were applied in the neck region to separate the head from the flagella. The injection of the sperm head into the oocyte was performed immediately. Microinjection of about 10 oocytes at RT was performed within 30 min. ICSI was completed within 2 h after oocyte collection.
Oocytes Activation after ICSI and Zygote/Embryo Culture
Heated spermatozoa were unable to activate oocytes after ICSI. To induce activation, microinjected eggs were incubated in Sr2+-CZB for 45 min and then transferred to CZB [8].
Embryo Transfer into Foster Mothers
CD1 females (Swiss Webster, albino) 8–13 wk old were used as recipients of the embryos. These mice were mated with vasectomized males of the same strain the night before the transfer. Two-cell embryos were transferred to one oviduct of the recipient female on the day on which a vaginal plug was found (Day 1 of pregnancy). In some experiments, embryos derived from microinjection of heated spermatozoa were cotransferred with embryos collected from superovulated mated CD1 females. In this case, pups derived from ICSI and those derived from mating were distinguished according to their coat and eye color [9].
Examination of Oocytes/Zygotes/Embryos
Fertilization and embryonic development were assessed through microscopic observation. Five to 7 h after ICSI, oocytes with two distinct pronuclei and the second polar body were considered normally fertilized. Normally fertilized oocytes were cultured for up to 120 h to evaluate embryo development to the blastocyst stage.
Data Analysis
Data were compared using the Student t-test from the StatView package. Differences were significant at P < 0.05.
RESULTS
Effect of Heat Treatment on Spermatozoa Vitality and Structures
Ninety-five percent of spermatozoa after swim-up and before heat treatment were viable, as assessed by vital staining. Heat treatment resulted in motility impairment and alteration of sperm membrane integrity in 98% of spermatozoa. TEM showed that heated spermatozoa had a disrupted plasma membrane and acrosome (Fig. 1). However, the appearance of the chromatin in the nucleus was comparable to that of controls.
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Fertilization and In Vitro Development of Oocytes after ICSI
Fertilization did not occur after microinjection of heat-treated spermatozoa unless activation was artificially induced. When activation was performed by incubation of microinjected oocytes in Sr2+-CZB, the fertilization and cleavage rates were comparable to those obtained after microinjection of nonheated spermatozoa (Table 1). However, the rate of development of two-cell embryos in vitro to the blastocyst stage was dramatically lower, 15.6% vs. 61.6% (Table 2).
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Development of Embryos after Transfer to Foster Mothers
Fifty-eight two-cell-stage embryos derived from heated spermatozoa were transferred to four pseudopregnant females (Table 3). The first two female recipients received embryos derived from heated sperm. The second female received 10 embryos and gave birth, 1 day after the expected term, to two stillborn pups of normal phenotype but of dramatically increased body size. In the mouse, when too few embryos implant they may grow too big to be born without damage [9]. Therefore, in the following transfer experiments, embryos derived from heated spermatozoa were cotransferred with embryos collected from mated superovulated CD1 females to ensure sufficient litter size. Of 16 embryos derived from heated spermatozoa and transferred to two females (females 3 and 4), two embryos (12.5%, one male and one female) developed into viable offspring. Both grew into normal adults and produced normal litters when mated with B6CBA partners. In the control group, the transfer of 25 embryos to three recipient females (females 5, 6, and 7) led to the birth of 11 (44%) offspring.
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DISCUSSION
Our data show that the mouse spermatozoon heated at 58°C for 30 min and microinjected into the oocyte can support full embryonic development leading to the production of a normal, healthy pup. TEM revealed severe alteration of the plasma membrane and the acrosome in all mouse spermatozoa after heat treatment. This observation confirmed previous reports that spermatozoa of different species do not need to be motile and structurally intact to support full embryonic development and to produce normal offspring as long as the sperm genome is preserved [1, 10–12]. In the mouse, normal embryonic development may require only an intact sperm nucleus and nuclear matrix [13]. Thermostability of mammalian sperm genome is probably linked to the extensive disulfide bonds cross-linking nuclear protamines. In mammals, during postmeiotic maturation of the spermatid, elongation and condensation of the nucleus occur with the replacement of the somatic cell DNA-binding proteins, the histones, by the small and more basic sperm-specific protamines [14]. In the resulting mature spermatozoon, the DNA is condensed and tightly packaged into a nucleoprotamine complex. The mouse sperm DNA stability probably reflects its high concentration in protamines; more than 98% of nuclear proteins are protamines [15].
The embryos derived from heated spermatozoa were less competent to develop to term than were control embryos, suggesting that the heating procedure was detrimental to embryo developmental ability. In vitro, the initial cleavage rate was unaffected but subsequent development stages were impaired. However, the implantation capacity of blastocysts derived from heated sperm was comparable to that of the controls. Of the 58 embryos derived from heated spermatozoa that were transferred to surrogate mothers, 10 (16.6%) should have developed to the blastocyst stage according to in vitro data. Because at least nine embryos implanted, the implantation rate was in the same range as that of controls similarly estimated (90% vs. 69%). These data suggest that embryonic development failure preferentially occurred during the very early preimplantation stage and may have resulted from alteration of sperm chromatin and subsequent impairment of embryonic genome activation that occurs at the two-cell stage in the mouse [16].
The ultrastructural TEM study of heated spermatozoa did not reveal any modification of the chromatin appearance, suggesting that extensive sperm chromatin alteration was not the cause of embryo development failure. However, heating may have induced subtle alterations of DNA or DNA-protein complexes, resulting in perturbation of sperm DNA remodeling at fertilization and thus in paternal genome expression in the early embryo. The male pronucleus plays an essential role in the early postfertilization transcriptional activity of the zygote because it supports a higher level of transcription than does the female pronucleus [17].
The impairment of embryonic development may also be related to sperm chromatin alteration in the storage cell culture medium after heat treatment. Mouse sperm chromosomes deteriorate rapidly (in less than 2 h) in conventional media after spermatozoa immobilization [18]. Sperm chromosome damage may be caused or mediated by sodium cations. The ability of membrane-damaged mouse spermatozoa and spermatid nuclei to participate in normal development is maintained significantly longer in a potassium-rich than in a sodium-rich cell culture medium [12, 18, 19]. In our study, nonheated spermatozoa were microinjected immediately after the head was separated from the flagella, whereas heated spermatozoa were kept up to 2 h in the CZB-Hepes medium before microinjection. Modified media to handle and store heated spermatozoa may improve the embryo development rate.
The artificial activation procedure used here, although stimulating pronucleus formation at a high rate, may have been inefficient in promoting normal embryonic development. Nuclear transfer experiments have indicated clearly that the type of activation procedure greatly influences the developmental ability of zygotes [20, 21]. The inability of heated sperm to activate oocytes was not surprising because the sperm oocyte activating factor was previously reported to be heat sensitive [22].
This study provides important data on mammalian sperm genome resistance to in vitro physical stress. This knowledge may be of importance for the development of future in vitro sperm processing procedures in assisted reproductive technologies.
FOOTNOTES
First decision: 24 January 2001.
1 Correspondence: Jean Philippe Wolf, Laboratoire d'Histologie et de Biologie de la Reproduction, UFR Science Médecine Biologie Humaine, 74 Rue Marcel Cachin, 93017 Bobigny, France. FAX: 33 1 48 38 77 77; jean-philippe.wolf{at}jvr.ap-hop-paris.fr 
Accepted: June 20, 2001.
Received: January 16, 2001.
REFERENCES
This article has been cited by other articles:
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  C. Li, E. Mizutani, T. Ono, and T. Wakayama Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI Reproduction, May 1, 2009; 137(5): 779 - 792. [Abstract] [Full Text] [PDF]
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 K.-B. Lee and K. Niwa Fertilization and Development In Vitro of Bovine Oocytes Following Intracytoplasmic Injection of Heat-Dried Sperm Heads Biol Reprod, January 1, 2006; 74(1): 146 - 152. [Abstract] [Full Text] [PDF]
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 B.M. Acton, A. Jurisicova, I. Jurisica, and R.F. Casper Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development Mol. Hum. Reprod., January 1, 2004; 10(1): 23 - 32. [Abstract] [Full Text] [PDF]
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O. Lacham-Kaplan, J. Shaw, L. G. Sanchez-Partida, and A. Trounson Oocyte Activation after Intracytoplasmic Injection with Sperm Frozen Without Cryoprotectants Results in Live Offspring from Inbred and Hybrid Mouse Strains Biol Reprod, November 1, 2003; 69(5): 1683 - 1689. [Abstract] [Full Text] [PDF]
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