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Effect of Partial Incision of the Zona Pellucida by Piezo-Micromanipulator for In Vitro Fertilization Using Frozen-Thawed Mouse Spermatozoa on the Developmental Rate of Embryos Transferred at the 2-Cell Stage

^c Chugai Pharmaceutical Co., Ltd. and ^d CSK Research Park, Inc., Gotemba, Shizuoka 412-8513, Japan

	ABSTRACT

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Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was r/6 µm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.

implantation, in vitro fertilization, sperm

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although cryopreservation of mammalian spermatozoa has been mostly applied to improve breeding of domestic animals and treatment of infertility in humans, there was no established method for cryopreservation of mouse spermatozoa until just 10 yr ago. Recently, cryopreservation of mouse spermatozoa has become available for use [1–9]. Cryopreservation of spermatozoa is simpler, less time consuming, and less costly than that of embryos in maintaining transgenic or gene-disrupted mouse strains [6, 8, 9]. Embryo freezing has been generally used for maintenance of established genetically modified mouse strains but requires obtaining a number of early pregnancy mice prior to the freezing procedures. A paradoxical operation is thus required such that a breeding colony must be expanded at considerable effort to reduce or abolish it.

In recent years, it has become possible to freeze a large number of spermatozoa immediately after collection from the epididymides of a small number of transgenic mice. Most transgenic mouse lines are established by DNA microinjection into a C57BL/6 zygote [10], but the fertilization rate using cryopreserved C57BL/6 spermatozoa is very low compared with the rate for other inbred strains [4]. We have recently solved this problem by in vitro fertilization (IVF) combined with partial zona pellucida dissection (PZD) [8], which was first developed as a method for improving fertilization rates in cases of severe male-factor infertility [11]. PZD significantly increases fertilization rates in vitro using cryopreserved C57BL/6J mouse spermatozoa with quite low incidence of polyspermy [8]. IVF using cryopreserved mouse spermatozoa combined with PZD is a promising technique for the maintenance of transgenic mouse colonies. However, one drawback of this technique is the requirement for the PZD zygotes to be cultured in vitro up to the morula or blastocyst stage prior to embryo transfer because blastomeres often escape from the slit of the zona pellucida during the early stages of embryonic development and attach to epithelial cells of the oviduct. In view of the advantages and disadvantages of these techniques, we used partial zona pellucida incision by piezo-micromanipulator (ZIP), which has been used for intracytoplasmic sperm injection (ICSI) in mice [12–15] and humans [16–18], for IVF with frozen-thawed spermatozoa from a C57BL/6J mouse.

	MATERIALS AND METHODS

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Experiment 1: ZIP of Fertilized Eggs

In the first series of experiments, we examined the effect of the ZIP technique on the pre- and postimplantation development of zygotes after embryo transfer into the oviduct of recipients. The holding pipette was prepared from a glass capillary tube (G1; Narishige, Tokyo, Japan). The capillary tube was heated and pulled using an automatic electromagnetic pipette puller (P-197; Sutter Instrument, Novato, CA). The tip of the pipette (about 80 µm in diameter) was polished using a microforge (De Fonbrune, Beaudouim, France). The micropipette for ICSI was used as a ZIP pipette, which was prepared from a glass capillary tube (Sutter) using a pipette puller (Sutter) and had a blunt end. The outer diameter of the tip of the ZIP pipette was approximately 7 µm. A small volume (about 0.5 µl) of mercury was introduced into the ZIP pipette from its proximal end. The pipette was connected to a Fluorinert (F77; Sumitomo 3M Co., Tokyo, Japan)-filled syringe system of the piezo electric actuator (Model PMM 150 FU; Prime Tech, Ibaraki, Japan) attached to a micromanipulator (Leica, Wetzlar, Germany). The blunt end of the micropipette was placed close to the surface of the zona pellucida of C57BL/6J (CLEA Japan, Tokyo, Japan) zygotes, which derived from natural mating following superovulation, at the pronucleus stage (Fig. 1). The zona pellucida was then incised by the micropipette through the application of piezo pulses (controller setting: speed 1, intensity 2) while the pipette was moved along the surface of zona pellucida. The arc length of the ZIP was r/6 µm. As a control, the zona pellucida of zygotes was partially dissected by a previously described method [8]. To create a larger perivitelline space, denuded zygotes were transferred onto 100-µl drops of a 0.3 M sucrose (no. 31365-0301; Junsei Chemical Co., Ltd., Tokyo, Japan) solution without BSA (no. 015-07491; Wako, Tokyo, Japan) in a culture dish (no. 351008; Falcon, Becton Dickinson, Franklin Lakes, NJ). When all the zygotes had sunk and become attached to the bottom of the culture dish, the 0.3 M sucrose solution was covered with liquid paraffin (no. 261-37; Nakalai Tesque, Kyoto, Japan). The zygotes carry a net negative charge, and the bottom of this culture dish carries a net positive charge because of the 0.3 M sucrose solution without BSA. After shaking the solution to confirm that all of the zygotes were bound to the bottom of the dish, the zona pellucida was dissected manually (dissection size: 20–40 µm) with the tip of an injection needle (30 gauge; Terumo, Tokyo, Japan) under a dissecting microscope (Fig. 2). After PZD, 20 µl of 0.3 M sucrose solution supplemented with 4% BSA was added to neutralize the electrostatic attraction of the surface of the zona. A micropipette was used to spray the PZD zygotes with 0.3 M sucrose solution containing BSA to remove them from the bottom of the dish; zygotes were then washed gently 3 times in Toyoda Yokoyama Hoshi (TYH) medium [19]. All zygotes were cultured in Whitten medium [20] containing 100 µM EDTA [21] up to the 2-cell stage in 5% CO₂ in air at 37°C. Zygotes were then transferred into the oviduct of ICR recipients (CLEA Japan, Tokyo, Japan) at 0.5 days postcoitum (dpc) of psuedopregnancy as described previously [22]. The recipients were killed at 19.5 dpc to determine the numbers of implantation sites and term fetuses.

View larger version (81K): [in this window] [in a new window]   FIG. 1. The ZIP procedure in mouse oocyte. The blunt end of the micropipette, tip diameter = 7 µm, touched the surface of the zona pellucida of the oocyte. The zona pellucida was incised by the micropipette by applying piezo pulses (controller setting: speed 1, intensity 2) while the pipette was moved along by the surface of zona pellucida (a). The orbit of the movement of the tip of pipette was not linear but round, like drawing an arc in a plane (b). The arc length of the ZIP was r/6 µm. To confirm complete breakage of the zona pellucida, the tip of the pipette was attached to the surface of the ooplasm through the slit in the zona pellucida (c). The pipette moved along the surface of the ooplasm without applying piezo pulses (d)

View larger version (39K): [in this window] [in a new window]   FIG. 2. Comparison of ZIP and PZD. The length of the incision made in the zona pellucida is approximately 26 µm with ZIP, whereas the arc length of the hole made with PZD is estimated as 92–136 µm; the dissection size of PZD was controlled manually between 20 and 40 µm

Experiment 2: IVF of Cryopreserved C57BL/6J Sperm Combined with ZIP and Embryo Transfer

In the second series of experiments, superovulated C57BL/6J oocytes with ZIP or PZD were inseminated with frozen-thawed spermatozoa from genetically modified C57BL/6J mice that have a human gene candidate for osteoblast differentiation factor in their genome (unpublished data). The procedures for spermatozoa cryopreservation were essentially the same as described previously [8]. Eighteen percent raffinose (no. R-7630; Sigma, St. Louis, MO) and 3% (w:v) skim milk (no. 0032-17–3; Difco, Detroit, MI) were dissolved in distilled water at 60°C. The solution was centrifuged at 10 000 x g for 15 min, and the supernatant was filtered and used as the cryopreservation solution. Two tails of the epididymides that were taken from 1 male mouse were minced in 100 µl of the cryopreservation solution in a 4-well multidish (no. 176740; Nunc, Roskilde, Denmark). The small volume of 100 µl was used to concentrate the sperm suspension as it was collected. The spermatozoa were dispersed from the organs at room temperature by gently shaking the dish for about 2 min. The sperm suspension at a concentration of 28–46 x 10⁷ cells/ml was then divided into 10 aliquots, 10 µl of each aliquot was put into a plastic straw (volume 0.25 ml; Fujihira Industry Co., Ltd,. Tokyo, Japan), and the straws were heat sealed. The straws were then cooled by placing them in the neck (gas layer) of a liquid nitrogen container (volume 2 L; Iuchi Seieido Co., Ltd., Osaka, Japan) for 10 min and then plunging them directly into liquid nitrogen, where they were stored for 3 mo before thawing. Each straw finally contained 28–46 x 10⁵ spermatozoa. The sperm were thawed by immersion of the frozen plastic straws in a water bath at 30°C. The ZIP and PZD methods were similar to those described above. In some oocytes, a r/6 µm long ZIP was repeated twice (a 2-ZIP). Those oocytes had 2 incisions in the zona pellucida approximately 20 µm apart. Superovulated oocytes were collected from the oviducts 15–16 h after injection of hCG. The oocytes were treated with 0.1% hyaluronidase (280 units/mg; H-3506, Sigma) in TYH medium to remove cumulus cells, then approximately 1 µl of thawed sperm suspension was added to 400 µl of the TYH medium for IVF at a sperm concentration of 1400–2300 cells/µl. Following incubation of the frozen-thawed spermatozoa for 0.5 h in 5% CO₂ in air at 37°C, the ZIP or PZD oocytes were introduced into TYH medium containing the frozen-thawed spermatozoa. At 6 h after insemination, the oocytes were washed 3 times in Whitten medium containing 100 µM EDTA and cultured up to the 2-cell or blastocyst stage in 5% CO₂ in air at 37°C. For untreated controls, cumulus-free oocytes were inseminated with frozen-thawed spermatozoa. Fertilization was defined by the number of embryos that had developed to the 2-cell stage 24 h after insemination. Two-cell embryos were transferred into the oviduct of the psuedopregnant recipients on 0.5 dpc [22], and blastocysts were transferred into the uterine horn of the recipients on 2.5 dpc of psuedopregnancy as described previously [23]. The recipients were killed on 19.5 dpc. To assess the influence of zona pellucida slit size on the preimplantation development of embryos in vivo, embryos with r/6 µm or r/3 µm long ZIP were transferred into the oviducts. The transferred embryos were subsequently flushed from both oviduct and uterus at 48 h after the transfer, and their morphology was examined.

All mice were housed in polycarbonate cages and maintained in a specific-pathogen-free environment in light-controlled (lights-on from 0500 to 1900 h), air-conditioned rooms (temperature: 24° ± 1°C, humidity: 50% ± 10%). Mice had free access to standard laboratory chow (CA-1; CLEA Japan). The animals used in this study were cared for and used under the Guiding Principles for the Care and Use of Research Animals promulgated by Chugai Pharmaceutical (Shizuoka, Japan).

Statistical Analysis

Data presented in this study were analyzed by the chi-square test and by the Tukey test for nonparametric multiple comparisons. For all tests, differences were considered significant at P < 0.05.

	RESULTS

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Experiment 1

When the zona pellucida of C57BL/6J zygotes was incised by r/6 µm long ZIP and these zygotes were then transferred at the 2-cell stage into the oviducts of recipient mice, 75% and 40% of the transferred embryos successfully implanted and developed to full-term fetuses, respectively (Table 1). These percentages were comparable to those of untreated control zygotes. However, only 5% of the embryos developed to newborns when PZD zygotes were transferred into the oviducts of recipient animals (Table 1). These results indicate that ZIP is less harmful than PZD with respect to embryonic development following transfer into the oviduct in mice.

Experiment 2

In the next series of experiments, cumulus-free oocytes prepared with ZIP, 2-ZIP, or PZD were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa (Table 2). The survival rate of oocytes prepared with PZD was significantly lower than that for the other experimental groups (P < 0.05 for both ZIP and 2-ZIP). Approximately half of the oocytes treated with ZIP, 2-ZIP, and PZD were fertilized by frozen-thawed spermatozoa. The fertilization rate was not significantly different between the experimental groups. None of cumulus-free oocytes were fertilized with frozen-thawed spermatozoa.

When fertilized eggs at the 2-cell stage were transferred into the oviducts, 18% and 2% of the transferred ZIP and PZD embryos developed to term, respectively (Table 3). This difference was significant (P < 0.05). The percentage development to term for embryos derived from 2-ZIP zygotes (12%) was also significantly higher than that for PZD embryos (2%) but was not different from that for ZIP embryos. When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, 17% and 21% of the transferred blastocysts developed to full term, respectively. This difference was not significant (P > 0.05). The percentage development to blastocyst stage of ZIP and PZD fertilized eggs were 85% and 89%, respectively.

To compare the duration required for ZIP and PZD procedures, over 200 oocytes were treated with either ZIP or PZD. ZIP required approximately 1.5 times less manipulation time than did PZD.

	DISCUSSION

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Cryopreservation of spermatozoa has become a commonly used tool for preserving transgenic mice and mice from saturation mutagenesis experiments. However, there are considerable differences among strains in the fertilization rate using frozen-thawed mouse spermatozoa, especially with regard to the inbred strain C57BL/6, which has exhibited a low fertilization rate compared with other strains [4]. We previously developed and reported a new system of IVF using cryopreserved spermatozoa and oocytes prepared with PZD [8]. The PZD technique has markedly increased the IVF rate of cryopreserved genetically modified C57BL/6J mouse spermatozoa [8]. However, transfer of PZD embryos into the oviduct is associated with a low success rate of development to term, as was shown in the present study (Table 3). This difficulty should be corrected for cryopreservation of mouse spermatozoa to become a basic technique used extensively in biomedical research.

The piezo electric actuator has been used as a tool for assisted reproductive techniques (ARTs) such as ICSI in the laboratory [12–15, 24] and in domestic animals [25–27] and humans [16–18], for production of chimeric embryos [28, 29] and assisted hatching [30, 31], and for transgenesis [14] and cloning [32–38]. The piezo electric actuator has been used mainly for injection of certain materials into the oocytes or embryos. We used this technique to develop a new ART for improvement of the fertility rate of cryopreserved mouse spermatozoa. The zona pellucida of oocytes was easily carved by ZIP without any additional treatment such as the use of hypertonic solution or subsequent successive washing procedures used in the PZD method [8]. The length of the incision made in the zona pellucida with the ZIP technique was 26 µm in the present study, representing about 1/12 of the perimeter of the zona pellucida of the mature mouse oocyte (approximately 314 µm). In comparison, the hole made in the zona pellucida with PZD is estimated to be 92–136 µm (Fig. 2) [8].

Almost all oocytes survived after the ZIP treatment (Table 2), and the survival rate of oocytes with ZIP was significantly higher than that with PZD (P < 0.05). Thus, PZD seems to be a less effective ART than ZIP because of the damage caused by the procedure; ZIP was associated with damage in less than 5% of the oocytes even when sucrose was used to shrink the cytoplasm [8]. The fertilization rate of ZIP oocytes was similar to that of PZD oocytes (P > 0.05) (Table 2). The 2-ZIP procedure (r/6 µm in length x 2) did not improve the rate of fertilization of oocytes. This result does not seem to be contradictory because the fertilization rate of PZD oocytes, which have an approximately 5 times larger slit than do ZIP oocytes, was equivalent to that of ZIP oocytes (Table 2). None of ZIP oocytes without insemination developed to 2-cell stage (data not shown), suggesting that parthenogenetic activation of oocytes was not induced by ZIP treatment, similar to the zona opening procedure [39]. The rate of development to term of 2-cell stage embryos with ZIP after transfer was significantly higher than that for PZD embryos (Table 3), indicating that ZIP could overcome the difficulty associated with PZD.

When the transferred embryos were flushed from both oviduct and uterus at 48 h after transfer into the oviduct, the embryos with r/6 µm long ZIP showed fully compacted morula, similar to the control embryos (Fig. 3). In comparison, the majority of r/3 µm long ZIP embryos contained 2 blastomeres or less with incomplete compaction. A couple of blastomeres in the r/3 µm long ZIP embryos probably escaped from the slit in the zona pellucida before the beginning of compaction, similar to PZD embryos. Because the size of blastomeres in r/3 µm long ZIP embryos was equivalent to that in the control and ZIP embryos, it seems that development within the zona pellucida was not affected in these embryos.

View larger version (67K): [in this window] [in a new window]   FIG. 3. The morphology of ZIP embryos at 48 h after transfer into the oviducts of recipient mice. ZIP embryos at the 2-cell stage were transferred and recovered from both the oviducts and uterine horns of the recipients. a) Control untreated embryos developed to morula. b) The embryos with r/6 µm long ZIP showed fully compacted morula similar to control embryos. c) The r/3 µm long ZIP embryos consisted of fewer blastomeres with incomplete compaction. Blastomere size is similar to that for control and ZIP embryos

Our results showed several advantages for ZIP with regard to IVF and subsequent embryo transfer. Zona incision was easily performed, and the length of incision could be voluntarily controlled. When large numbers of oocytes were treated, the time required for this procedure was short compared with that needed for PZD, yet high precision was obtained. The percentage of ZIP embryos that developed to term after transfer to the oviduct was comparable to that of intact control embryos, and ZIP was less traumatic to the oocyte than was PZD.

	FOOTNOTES

 
First decision: 13 August 2001.

¹ Correspondence. FAX: 81 155 49 5643; hisuzuki{at}obihiro.ac.jp

² Current address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13, Inada, Obihiro, Hokkaido 080-8555, Japan.

Accepted: September 17, 2001.

Received: July 13, 2001.

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