Introduction
Intracytoplasmic sperm injection (ICSI) has been widely applied in human medicine and has greatly enhanced our ability to overcome some forms of infertility (Palermo et al., Reference Palermo, Cohn and Rosenwarks1996a). However, the overall ICSI efficiency in animals, including rabbits, is lower than that found in humans. The rabbit is an ideal laboratory animal due to its body size, strong productivity and short pregnancy cycle. It is well known that the ICSI fertilization technique has several advantages for its application. Therefore, a good ICSI protocol for the rabbit will improve its use and the development of some research work. However, the current efficiency of ICSI in rabbits is not sufficient for our present needs. Establishing a highly efficient ICSI method is therefore necessary.
Several factors are related to the fertilization and developmental efficiency of sperm-injected oocytes, such as the state of the spermatozoa (Ogonuki et al., Reference Ogonuki, Inoue, Miki, Mochida, Hattoti, Okada, Takeiri, Shimozawa, Sankai and Ogura2005), the quality of the oocytes (Zheng et al., Reference Zheng, Jiang, Zhang, Sun and Chen2004), activation of the oocytes (Gomez et al., Reference Gomez, Catt, Evens and Maxwell1998; Kolbe & Holtz, Reference Kolbe and Holtz1999; Chung et al., Reference Chung, Keefer and Downey2000; Li et al., Reference Li, Morris and Allen2000; Liu et al., Reference Liu, Kusakabe, Chang, Suzuki, Schmidt, Julian, Pfeffer, Charles, Bormann, Tian, Yanagimachi and Yang2004), concentration of PVP and the injection technique (Suttner et al., Reference Suttner, Zakhartchenko, Stojkovic, Mtillner, Alberio, Medjugorac, Brem, Wolf and Stojkovic2000). The choice of sperm injection protocol should be the most important factor compared with other considerations in the production of an ICSI animal. The traditional mechanical injection method was the only option before the invention of the Piezo driven system. The Piezo has contributed largely to the success of mouse ICSI, but it is not necessary for rabbits. Compared with the Piezo system, the traditional mechanical injection method has the benefits of easy operation and less cost. There are a few reports on rabbit ICSI research using the traditional mechanical injection technique after 1988 (Hoshi et al., Reference Hosoi, Miyake, Utsumi and Iritani1988, Reference Kasai, Oshi and Yanagimachi1992, Reference Hoshi, Yazawa and Yanagida1994; Keefer, Reference Keefer1989; Yang et al., 1990; Hosoi & Iritani, Reference Hosoi and Iritani1993; Sofikitis et al., Reference Sofikitis, Miyagawa, Agapitos, Pasyianos, Toda, Hellstrom and Kawamura1994). Only two of these papers reported full-term development of offspring with fresh semen (Hoshi et al., Reference Hosoi, Miyake, Utsumi and Iritani1988; Deng & Yang, Reference Deng and Yang2001). The survival rate of injected oocytes was only about 70%, and was far lower than using the Piezo method in mice. After transfer, the development rate in vivo was not more than 10%. Overall, the ICSI protocol for rabbits needs to be improved.
In this present study, we used very sharp injection pipettes in order to make the pipettes enter the cytoplasm of oocytes directly. In operation process, it is not necessary to breakdown the cytoplasm membrane by an aspiration step. This is not only a simplified injection procedure, but also has increased the survival rate of sperm-injected oocytes. After being transferred, those ICSI-derived embryos fertilized by an infertile male rabbit were surgically transferred to the oviducts of the recipients. Some of the embryos completed full-term development. The efficiency was higher than that of previous studies; this is first report that resolves this infertility problem using the ICSI technique in rabbits.
Materials and Methods
Experimental animals
Donors for oocytes used in the experiment were sexually mature New Zealand white female rabbits aged from 6 to 12 months. Two sperm donors were used. One had good spermatozoa quality and could fertilize naturally; the other was an infertile male. Two adult females of synchronous estrous were used as recipients and they were treated with hCG just before being caged together with the vasoligated bucks. Each animal was caged individually and housed at a temperature of 23 ± 2 °C, with 30–60% relative humidity, and fed commercial feed and water ad libitum.
Preparation of pipettes
Embryo-holding pipettes were pulled by hand in an alcohol flame and were cut to an outer diameter of 150–200 μm and the tips of the pipettes were processed on a microforge to an inner diameter of 20–30 μm. An angle of 30–40° was made 4 mm from the tip. Sperm-injection pipettes were pulled on a micropipette puller (Narishige PD–5). The pipettes were cut to an inner diameter of 6–7 μm and the tips were beveled to 60° on a grinding wheel. The pipettes were rinsed in 20% hydrofluoric acid for 10 s, then several times in distilled water, and a sharp ‘thorn’ about 5 μm in length was pulled out from the tip with a microforge.
Preparation of oocytes and zygotes
Donor New Zealand white rabbits were superovulated by intramuscular injection with pregnant mare stimulating gonadotropin (PMSG, Tianjin Hormone Factory) at a 130 IU/head dose, and injected with human chorionic gonadotropin (hCG, Ningbo Hormone Factory) at 130 IU/head 72 h later. At 14–15 h after hCG injection, oocytes were collected by flushing each oviduct with 5 ml HEPES-buffered TCM199 containing 10% FCS. Collected oocyte masses were digested with 0.1% hyaluronidase for 5 min in a 5% CO2 incubator and cumulus cells were removed by repeated pipetting. Oocytes were firstly washed three to five times with HEPES-buffered TCM199 and then twice with TCM199 (Gibco BRL) medium supplemented with 10% fetal calf serum (FCS, HyClone), 1.25 mM sodium pyruvate and 0.1 mM EDTA at 37 °C in 5% CO2. Mature oocytes were placed in culture droplets under oil until injection.
At 18–20 h post coitus, the superovulated rabbits were killed with sodium pentobarbital and zygotes were harvested by flushing the oviducts.
Preparation of semen
Semen was collected from both fertile and infertile mature males by an artificial vaginal method. After collection, the semen was centrifuged at 2000 rpm in a 1.5 ml tube for 5 min. The supernatant was discarded and the pellet of sperm cells was transferred to the bottom of a 3 ml centrifuge tube containing pre-warmed HEPES-buffered TCM199 solution with 10% FBS. After incubation for 30 min, the upper 100 μl that contained swim-up sperm was transferred to another tube and used as the donor spermatozoa.
Sperm injection
Droplets in several rows were made in the lid of a 90 cm cell culture plate and were covered with 15–20 ml of light mineral oil. There were four drops in each row. The droplet on the extreme left containing 5 μl of 12% PVP solution was for the store of sperm cells, the one on the extreme right containing 15 μl of HEPES-buffered TCM199 plus 10% FCS was for store of oocytes, and the remaining two droplets in the middle were 15 μl HEPES-buffered TCM199 for washing the holding pipettes and injection pipettes. A group of 10–15 eggs were transferred to the oocyte droplet and was allowed to equilibrate for 1–2 min before injection. A single spermatozoon was picked up tail first into the injection pipette. Afterwards the pipette was washed in the adjacent droplet to ensure no extra spermatozoa were carried over, the pipette was then moved into the injection droplet containing the oocytes. Prior to injection, oocytes were held by the holding pipette at the 9 o'clock position with the first polar body positioned at 6 or 12 o'clock. The zona pellucida (ZP) and cytoplasm membrane were pieced at the 3 o'clock position by pushing the injection needle steadily forwards to the opposite side until it entered the cytoplasm. Without the aspiration step, the spermatozoon was directly placed deep into the cytoplasm with as little injection medium as possible. Then the injection needle was gently withdrawn. As the needle tip was removed, negative pressure forced some cytoplasm and cytomembrane into the pipette to about 7 μm in length. Finally, the injected oocyte was released from the holding pipette. All the manipulations were done at room temperature.
Embryo culture
The oocytes were allowed to equilibrate for 3–5min after injection, and were washed two to three times in TCM199 following transfer into the fresh equilibrated media droplets. Then they were incubated in culture medium with 5% CO2 in air at 38.5 °C. After 2 h of culture, embryo viability was observed and the collapsed oocytes were discarded. The surviving ones were examined for the formation of pronuclei after 5–7 h following microinjection. The cultured embryos were observed for their developmental stages once every 24 h under a dissecting microscope. Half of the culture medium was replaced every 2 days. The embryo developmental state and numbers were recorded.
Embryo transfer
In order to evaluate the developmental ability of oocytes injected with fresh spermatozoa derived from an infertile male rabbit, embryos at the 2–4-cell stage were surgically transferred into oviducts of synchronized recipient rabbits. For any individual recipient only one side of its oviduct (ovulating side) was used to accept embryos. The pregnant recipients were allowed to deliver kits naturally.
Data analysis
The rates of survival, pronuclear, cleavage, and blastocyst formation were subjected to the chi-squared test. Differences were considered to be significant at p < 0.05.
Results
Development of ICSI embryos in vitro
Without any additional treatment for activation, sperm-injected oocytes possess the normal developmental competence from fertilization to blastocyst stage in vitro culture. They formed pronuclei and extruded a second polar body after ICSI 5–7 h. Most zygotes developed to the 2–4-cell stage at 18 h of culture. On the third day, embryos at the 8–16-cell stage and morulae were present. The early blastocysts were observed after about 70 h of development, expanded and hatched blastocysts were found on day 4 and day 5 (the fertilization day being day 0).
Survival rates and development in vitro of oocytes injected with fresh living spermatozoa are shown (Table 1). A total of 317 oocytes were injected with a single spermatozoon and 50 with sham treatment. The survival rate of ICSI oocytes was 86.1% and for sham injection was 90.0%, and there was no significant difference between groups of ICSI and sham (p < 0.05). 59.0% of the survival oocytes were fertilized after culture. The cleavage rate of ICSI oocytes was not significantly lower than that of natural fertilization (88.2% versus 91.7%, p < 0.05). However, their blastocyst developmental competence was lower than that of embryos derived from naturally fertilized embryos (45.3% versus 80.6%, p < 0.05).
In vitro development of embryos derived from ICSI, sham injection and in vivo
a, b, cValues with different superscripts within the same column differ significantly (p < 0.05).
Development of ICSI oocytes in vivo
After ICSI with fresh rabbit spermatozoa, 57 out of 60 oocytes survived 1 h following injection. There were 27 oocytes that formed pronuclei. On day 2 after ICSI, 21 had developed to the 2-cell and 4-cell stages (Table 2) and were transferred into oviducts of two recipients. One of the recipients was pregnant and naturally gave birth to two (one male and one female) healthy offspring on day 31 after transfer. The survival rate of ICSI embryos in vivo was 18.2%.
In vivo development of embryos following microinsemination with infertile rabbit's spermatozoa
Discussion
The injection pipette used in DNA microinjection for transgenics is about 1 μm diameter and was able to piece the membrane of the embryo easily without making serious injury to the structure of the embryos. The pipette used for sperm injection is usually 8–10 μm in external diameter because it must accommodate the sperm head, which is about 5.5–6.5 μm in width. It is inevitable that there will be damage to the membrane and the subcellular structures of the oocytes by the action of pressuring and squeezing, even when a bevel is fabricated on the tip of the pipette. In the present study, the pipette's tip was sharp, like that of the needle used in pronuclear microinjection. This made the egg plasma membrane break easily without an aspiration procedure. We think that this fact is the first reason that the injected oocytes had such a high survival rate (86.1%), which was much better than found in previous studies (Hosoi et al., Reference Hosoi, Miyake, Utsumi and Iritani1988; Sofikitis et al., Reference Sofikitis, Miyagawa, Agapitos, Pasyianos, Toda, Hellstrom and Kawamura1994; Deng & Yang, Reference Deng and Yang2001; Li et al., Reference Li, Chen, Lian, Sun, Wang, Liu, Li and Han2001; Zheng et al., Reference Zheng, Jiang, Zhang, Sun and Chen2004). On the other hand, we added an aspiration step action when withdrawing the pipette. This procedure might allow the broken plasma membrane to reform more quickly. It was beneficial in decreasing the death rate of the ICSI eggs, as failure to heal the opening and an outflow of cytoplasm from the injection wound could induce death of the oocytes (Deng & Yang, Reference Deng and Yang2001). Our results indicated that a pipette drawn to a sharp thorn could piece through the membrane of rabbit oocytes directly and only make limited damage to the treated eggs, as with the Piezo driven system (Kimura & Yanagimachi, Reference Kimura and Yanagimachi1995).
Pretreatment of spermatozoa is usually needed in some species of animals in order to induce complete release of oocyte-activation factor (OAF) from the spermatozoa. The use methods that have DTT or Triton X-10-treated spermatozoa, and breaking of the sperm membrane, can aid efficient activation of oocytes after ICSI (Catt & O'Nell, Reference Catt and O'Nell1995; Kuretake et al., Reference Kuretake, Kimura, Hoshi and Yanagimachi1996; Kasai et al., Reference Kasai, Oshi and Yanagimachi1999). In our ICSI experiment, fresh spermatozoa were managed without any treatment prior to injection into the ooplasm. Our results were consistent with those of Deng & Yang's. Therefore, our study proved that the use of pretreatments for rabbit spermatozoa before ICSI, such as sperm capacitation, immobilization or demembranation (Hosoi et al, Reference Hosoi, Miyake, Utsumi and Iritani1988; Keefer, Reference Keefer1989; Hosoi & Iritani, Reference Hosoi and Iritani1993; Catt & O'Nell, Reference Catt and O'Nell1995; Gerris et al., Reference Gerris, Mangelschots, Van Royen, Joostens, Eestermans and Ryckaert1995; Palermo et al., Reference Palermo, Schlegel, Colombero, Zaninovic, Moy and Rosenwarks1996b; Rho et al., Reference Rho, Kawarsky, Johnson, Kochhar and Betteridge1998a, Reference Rho, Wu, Kawarsky, Leibo and Betteridgeb; Kasai et al., Reference Kasai, Oshi and Yanagimachi1999) is not necessary for the production of offspring by ICSI.
The choice of sperm injection method is only one of the factors that affects the overall efficiency of ICSI (Chen & Seidel, Reference Chen and Seidel1997; Suttner et al., Reference Suttner, Zakhartchenko, Stojkovic, Mtillner, Alberio, Medjugorac, Brem, Wolf and Stojkovic2000). Other factors, such as quality of oocytes, sperm state (Ogonuki et al., Reference Ogonuki, Inoue, Miki, Mochida, Hattoti, Okada, Takeiri, Shimozawa, Sankai and Ogura2005), or activation treatment (Gomez et al., Reference Gomez, Catt, Evens and Maxwell1998; Kolbe & Holtz, Reference Kolbe and Holtz1999; Li et al., Reference Li, Morris and Allen2000; Liu et al., Reference Liu, Kusakabe, Chang, Suzuki, Schmidt, Julian, Pfeffer, Charles, Bormann, Tian, Yanagimachi and Yang2004), may affect ICSI oocyte fertilization and developmental ability. As indicated in this study, the rate of fertilization was 59.1%, and this value is neither the lowest (Hosoi et al., Reference Hosoi, Miyake, Utsumi and Iritani1988) nor the highest (Ogonuki et al., Reference Ogonuki, Inoue, Miki, Mochida, Hattoti, Okada, Takeiri, Shimozawa, Sankai and Ogura2005) reported in rabbit ICSI research. However, once successfully fertilized by ICSI, cleavage rate can reach 88.2%. There is no significant difference compared with universal IVF; however, the blastocyst rate of ICSI embryos was far lower than that of embryos in vivo under the same culture conditions. This may be due to insufficient oocyte activation (Liu et al., Reference Liu, Kusakabe, Chang, Suzuki, Schmidt, Julian, Pfeffer, Charles, Bormann, Tian, Yanagimachi and Yang2004). Other factors, such as mechanical damage during sperm injection (Yavas et al., Reference Yavas, Roberge, Khamsi, Shirazi, Endman and Wong2001), residual PVP in the cytoplasm (Tsai et al., Reference Tsai, Huang, Kung, Lin, Chang, Wu and Chang2000), effects of operation in vitro, etc., might also cause a decrease in developmental competence.
ICSI technique has become a routine practice for male factor infertility such as obstructive azoospermia (Tesarik & Mendoza, Reference Tesarik and Mendoza2003) and non-obstructive azoospermia (Dalit et al., Reference Dalit, Yoger, Hauser, Haim, Faud and Israel1999). Microinsemination also allows oocytes to be fertilized with spermatozoa with low or no motility (Szczygiel et al., Reference Szczygiel, Kusakabe, Yanagimachi and Whittingham2002; Sakamoto et al., Reference Sakamoto, Kaneko and Nakagata2005) in mice. In the present study, we found that spermatozoa from an infertile male rabbit can successfully fertilize oocytes by ICSI. These embryos could develop to term in vivo, and produce live offspring. This result demonstrates that the ICSI technique might have more applications for reproduction in the rabbit. This is the first report of ICSI using infertile male spermatozoa in rabbits.
In summary, the present study demonstrates that the injection protocols we established have the characteristics of easy manipulation, time saving, slight damage to oocytes and result in a high rate of development for ICSI embryos. To date, the efficiency of full-term development in this experiment is higher than that of previous reports, in which spermatozoa were injected by mechanical methods. Moreover, embryos fertilized by this ICSI method using spermatozoa from infertile rabbits resulted in normal pups after a short period in vitro culture and transfer.
