The improvement in fertilizing ability of cryopreserved mouse spermatozoa using laser‐microdissected oocytes

Abstract

Aim:  The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa.

Methods:  C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75 mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed.

Results:  When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments.

Conclusion:  The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. (Reprod Med Biol 2006; 5: 249–253)

INTRODUCTION

SUCCESSFUL REPORTS OF mouse sperm cryopreservation have been published since the 1990s. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ Subsequently, an improved cryopreservation method using a solution consisting of 18% (w/v) raffinose and 3% (w/v) skim milk was described by Nakagata and Takeshima. ⁷ This cryopreservation method was also fully applied to various strains, including wild mice and transgenic mice. ⁸ , ⁹ , ¹⁰ In recent years, large mouse sperm/embryo cryobanks have been established in several countries around the world. Sperm cryopreservation techniques have been contributing to the efficient maintenance of strains, and to transportation that is safer and cheaper than that with live mice. Furthermore, sperm preservation solved the serious problem of a lack in breeding space by the mass production of new mouse strains, such as transgenic, knockout and mutant. ¹¹

The C57BL/6 mouse strain is now commonly used for producing these transgenic and knockout strains. Unfortunately, several reports have been published that C57BL/6 mouse spermatozoa that had been cryopreserved using a raffinose/skim milk solution showed a decrease in fertilizing ability. ² , ⁸ , ¹² , ¹³ , ¹⁴ In order to increase fertilizing ability, micromanipulation techniques such as partial zona pellucida dissection (PZD), ¹⁵ partial zona pellucida incision by using a piezo‐micromanipulator (ZIP) ¹⁶ , ¹⁷ and intracytoplasmic sperm injection (ICSI) ¹⁸ , ¹⁹ have been established. However, these micromanipulation techniques require a considerable degree of technical skill.

It is known that partial zona pellucida dissection by laser beam has been used for assisted hatching to increase the implantation rate of human embryos. ²⁰ , ²¹ In the present report, the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved mouse spermatozoa was investigated.

MATERIALS AND METHODS

Animals

ALL ANIMALS WERE purchased from CLEA Japan (Tokyo, Japan). C57BL/6 J males and females for sperm and oocyte donors were aged over 12 weeks and 9–10 weeks, respectively. ICR females, recipients for the transfer of 2‐cell embryos, were aged 12–15 weeks. All animals were maintained in an air‐conditioned (temperature: 22 ± 2°C, humidity: 60 ± 10%) and light‐controlled room (light on 07.00–19.00 hours). The Animal Care and Use Committee of the Kumamoto University School of Medicine approved all procedures carried out in the present study.

Sperm cryopreservation

Spermatozoa were cryopreserved using the method described previously. ²² Briefly, two cauda epididymides were removed from males, and were then transferred to 100 µL of the cryopreservation solution consisting of 18% (w/v) raffinose and 3% (w/v) skim milk in a 4‐well multi dish (Nunc A/S). The cauda epididymides were minced well by micro spring scissors, and the spermatozoa were then dispersed by gently shaking the dish. Sperm suspension was loaded into 10 µL aliquots in 0.25 mL sampling straws (IMV Technologies, L’Aigle Cedex, France). Ten straws were heat‐sealed at both ends and cooled into liquid nitrogen vapor for 15 min. After this, these straws were plunged directly into the liquid nitrogen.

Oocytes collection

Oocytes were collected from females induced to superovulate by an intraperitoneal injection of 7.5 IU PMSG (Teikokuzoki Co., Tokyo, Japan), followed by an injection of 7.5 IU hCG (Teikokuzoki Co.) 48 h later. Cumulus‐oocyte complexes were collected from the oviducts at 15–16 h after hCG injection. Oocytes were freed from cumulus cells by treatment with HTF medium ²³ containing 0.1% hyaluronidase (H3506, Sigma‐Aldrich, St Louis, MO, USA). Cumulus‐freed oocytes were rinsed and kept in fresh HTF medium at 37°C in 5% CO₂ and 95% air until laser‐microdissection.

Laser‐microdissection of zona pellucida

The Saturn 3 laser system (Research Instruments Ltd, Cornwall, UK) was used for the microdissection of zona pellucida. The laser system was coupled with an inverted microscope (IX71, Olympus Co., Tokyo, Japan). An image of the oocytes was projected onto a PC/video monitor using a video camera. Each of the 50 oocytes were placed on the bottom of a 100 µL drop of PB1 medium ²⁴ containing 0, 0.25, 0.50 or 0.75 mol sucrose covered with paraffin oil in a culture dish. The zona pellucida were focused sharply and dissected individually by laser beam (output wavelength: 1480 nm, power output from patch lead: 350 mW) with different pulse lengths selected from 0.45 to 0.65 ms (Fig. 1). After this, oocytes were transferred to 90 µL of fresh HTF medium.

Figure 1.

Oocytes with their zona pellucida microdissected by laser. The arrow shows a hole microdissected by laser.

In vitro fertilization

A straw with spermatozoa was left at 37°C in a water bath for 15 min. The sperm suspension was pushed out in the 90 µL of HTF medium, after which it was kept for 30 min at 37°C in 5% CO₂ and 95% air. After this, the sperm suspension was introduced into 10 µL aliquots to 90 µL of HTF medium containing microdissected oocytes. Sperm and oocytes were then cultured at 37°C in 5% CO₂ and 95% air.

Embryo transfer

Oocytes were observed two pronuclei 5 h after insemination and embryos that developed to the 2‐cell stage were selected 24 h after insemination. Fertilization rates were calculated by the number of 2‐cell embryos from oocytes microdissected. These 2‐cell embryos were transferred into the oviducts of pseudopregnant females that had been mated with vasectomized males of the same strain on the day before embryo transfer. The number of offspring was counted on 19.5 days of gestation by cesarean section. The rates of offspring were calculated by number of offspring from oocytes transferred or microdissected.

Analysis of data

Each experiment was repeated over three times. The χ²‐test using Yates correction for continuity was used for the analysis of all data obtained from the present study.

RESULTS

IN EACH EXPERIMENT, over 200 oocytes were used for laser‐microdissection and the development of over one hundred embryos was assessed after transfer into pseudopregnant females. No oocytes damaged by laser beam and with polyspermy were observed in the present study. As shown in Figure 2, a high fertilization rate was obtained when oocytes microdissected in PB1 medium without sucrose were used. In contrast, no significant difference was obtained in the proportion of development to offspring of oocytes microdissected in PB1 medium containing a different concentration of sucrose used in the present study. All offspring obtained in the present experiment were apparently normal.

Figure 2.

The fertilization rate and the development to offspring of oocytes microdissected in PB1 medium containing 0, 0.25, 0.50 and 0.75 mol sucrose. Fertilization rate = number of 2‐cell embryos/number of oocytes microdissected. Percentages of offspring = number of offspring/number of 2‐cell embryos transferred. Significant difference, a vs b, d; b vs c, P < 0.05.

When oocytes were microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms in PB1 medium without sucrose, fertilization rates increased significantly as the pulse length was lengthened (Fig. 3) (P < 0.05). Although, the rate of offspring to the number of oocytes microdissected also increased significantly as the pulse length was lengthened, it decreased significantly when oocytes were microdissected by laser with pulse length of 0.65 ms (P < 0.05). All offspring obtained in the present experiment were also apparently normal.

Figure 3.

The fertilization rate and the development to offspring of oocytes microdissected by laser with a different pulse length selected from 0.45 to 0.65 ms in PB1 medium without sucrose. Fertilization rate = number of 2‐cell embryos/number of oocytes microdissected. Percentages of offspring = number of offspring/number of oocytes microdissected. Percentages with different lowercase letters are significantly different (P < 0.05). *Means that embryos were not transferred.

DISCUSSION

IN THE PRESENT study, it was shown that the fertilizing ability of cryopreserved mouse spermatozoa was improved when oocytes with their zona pellucida microdissected by laser were used. Furthermore, laser‐microdissected oocytes fertilized with spermatozoa finally developed into normal offspring.

Mouse spermatozoa, especially C57BL/6, are known to be sensitive to freezing, resulting in reduced motility and fertility. ² , ⁸ , ¹² , ¹³ , ¹⁴ Although, the reason for this phenomenon has been unknown, fertility could be increased by microdissection of the zona pellucida using a steel needle ¹⁵ and piezo‐micromanipulater ¹⁶ , ¹⁷ or injection of spermatozoa intracytoplasmically. ¹⁸ , ¹⁹ Presently, technical modification of the freezing protocol ²⁵ and freeze‐drying preservation that takes the place of cryopreservation ²⁶ , ²⁷ , ²⁸ , ²⁹ is being actively studied for further efficient sperm preservation. The laser‐microdissection showed in the present study also demonstrated an improvement in the fertilizing ability of cryopreserved mouse spermatozoa, as well as various micromanipulation techniques described previously (2, 3).

In the present study, the perivitelline space of oocytes was widened using a hypertonic solution containing a high concentration of sucrose to protect the oolemma from damage by the laser. However, a high proportion of oocytes were fertilized with cryopreserved spermatozoa and these embryos developed into normal offspring, even when oocytes were microdissected without the widening of perivitelline space in PB1 medium without sucrose (Fig. 2). This result suggested that microdissection by laser caused no damage to the oolemma near the hole and the subsequent development of embryos.

When oocytes were microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms in PB1 medium without sucrose, as shown in Figure 3, fertilization rates increased significantly as the pulse length was lengthened. Although the rate of offspring to the number of oocytes microdissected also increased as the pulse length was lengthened, it decreased significantly when oocytes were microdissected by laser with a pulse length of 0.65 ms. It is possible that blastomeres might escape from the hole of zona pellucida microdissected by laser in the oviduct of pseudopregnant females. ¹⁶ The results in the present study showed that the hole size of the zona pellucida microdissected by laser is important for the development and implantation of embryos after transfer into pseudopregnant females. Although a high fertilization rate was obtained by enlarging the hole size of the zona pellucida, optimal settings need to be defined. In the present study, we established a pulse length selected from 0.55 to 0.60 ms as the optimum setting of laser‐microdissection for the efficient production of embryos.

Intact zona pellucida works to prevent the transmission of pathogens to the oocytes. ³⁰ In order to examine the risk of pathogen infection to embryos, we fertilized laser‐microdissected oocytes with cryopreserved spermatozoa derived from mice infected with the mouse hepatitis virus (MHV), and the offspring obtained from these embryos were tested for MHV infection. All offspring had negative test results (data not shown). It is thought that the risk of pathogen transmission by the microdissection of zona pellucida is considerably low, ³¹ but further study is necessary for other pathogens.

The C57BL/6 mouse strain is now commonly used for producing transgenic and knockout strains. However, the fertilizing ability of these spermatozoa is still low after cryopreservation. The microdissection of zona pellucida by laser is a simple technique in which special skills are unnecessary. This technique will become a powerful tool for the mass production of embryos or offspring from cryopreserved mouse spermatozoa that show low fertilizing ability.