Eppendorf Piezo-actuated Mouse ICSI User Manual

Userguide

No 037 | September 2011 - revised August 2012

Piezo-actuated Mouse ICSI (intracytoplasmic
sperm injection) using the Eppendorf PiezoXpert®

Hadi Hajarian, Laboratory of In Vitro Fertilization, Agro-Biotechnology Institute (ABI), Malaysia
Ko Kwan Mor, Eppendorf Asia Pacific Headquarters, Malaysia

Abstract

ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted reproductive technology in
both, humans and animals. The Mouse is one of the most common model organisms of choice to study mammalian
fertilization. However, the ability to fertilize mouse eggs successfully by sperm injection has been hard to achieve due
to the fact that the metaphase II mouse oocytes are extremely sensitive and convential ICSI gives low survival rates
[1]. This problem can be solved with piezo-actuated micromanipulation where the capillary advances a very short
distance at a very high speed. This enables the capillary to penetrate the cell membrane with minimum distortion of
the cell and yields to high survival rates. The microinjection workstation required for this technique is very similar to
standard ICSI, but with the addition of a piezo-assisted unit attached to the capillary holder. In this Userguide, the

®

use of the Eppendorf PiezoXpert in combination with the Eppendorf TransferMan
parameter settings as well as optimization of the piezo-actuated microinjection procedure itself are discussed.

NK 2 workstation is shown and

Introduction

Fig. 1: Actuator of PiezoXpert mounted onto the right arm of

the manipulator (Eppendorf TransferMan NK 2).

Intracytoplasmic sperm injection (ICSI) is a technique that
involves the direct transfer of a single sperm into the oocyte
cytoplam via a glass capillary with a spike.

While this conventional ICSI technique has been very suc­cessful in humans, it has proven unsuccessful in mice [2].
This is due to a lower viscosity of the ooplasm. Thus wound
healing capacity of mouse oocytes is inferior to that of hu­man oocytes. Furthermore, the oolemma of mouse oocytes
is much more elastic than that of human oocytes. Suc­cessful ICSI in mice was first demonstrated by Kimura and
Yanagimachi [3] using piezo-actuated micromanipulation,
which is far less traumatic than the conventional method.
This method proved to increase survival as well as fertiliza­tion rates of oocytes after sperm injection [3].

Eppendorf has a long tradition in the area of conventional
ICSI. In particular, the Eppendorf TransferMan NK 2 system
is an electronic micromanipulation system that offers a num­ber of useful features for ICSI. In combination with the piezo
impact unit PiezoXpert, Eppendorf is offering a complete
system for both, conventional ICSI and piezo-actuated ICSI
(Figure 1, 2).

Userguide No 037 | Page 2

Fig. 2: Eppendorf piezo-actuated mouse ICSI system. Worksta-

tion with 2 TransferMan NK 2, PiezoXpert, mounted onto a Ni­kon Eclipse TE 2000 microscope and incubator GALAXY 14 S.

head separation methods (sonication method and piezo­assisted method) are shown as below.

ICSI dish with sonicated sperm

An example with sperm heads that are separated using
sonication is shown in Figure 3 [4]. Here, a 5 μL flat drop of
sperm suspension is positioned in the center of a flat Petri
dish. 3 x 5 μL drops of CZB-HEPES with 12 % PVP are
positioned at one end of the dish along the midline. In addi­tion, 2 x 5 μL injection drops of CZB-HEPES are placed at
the other end of the dish along the midline. Add approx. five
eggs in one of the injection droplets. Cover with mineral oil.

Materials and Equipment

Animals, media, consumables and devices were used as
described previously [4] with the following modifications:
Media:
CZB-HEPES (CZB-H)
CZB
CZB-HEPES with 12 % polyvinylpyrrolidone (PVP)
Density gradient (e.g. Percoll)
Fluorinert C-77 (FC-77), Fluorinert C-770 (FC-770)

Consumables:

100 μL pipette tips
Piezo impact unit:
Eppendorf PiezoXpert

Methods

1 Preparation of spermatozoa

Mouse sperms (fresh or frozen-thawed) are prepared based
on the method of sperm head isolation.
If the sonication method is used, sperms are prepared by
centrifugation and subsequent sonication to isolate sperm
heads by diluting a small sample of sperms with buffer
followed by repeated sonication (e.g. 4 x 15 seconds) [5].
If the piezo assisted method is used, described at 5.2.1,
sperms are prepared using mini swim-up [6] or density
gradient centrifugation.

2 Preparation of oocytes

The metaphase II oocytes are collected from superovulated
females and further treated as described previously [4]. The
cumulus-free oocytes are transferred to a culture dish [4].

3 Preparation of the microinjection dish

The arrangement of the drops in the microinjection dish de­pends on personal preferences. Examples for different sperm

Sperm
droplets

Sperm
suspension

Injection
droplets

Fig. 3: ICSI dish with sonicated sperm. Image adapted from [4].

ICSI dish for sperm head isolation using piezo-assisted
method

2 dishes (A; B) are prepared as shown in Figure 4. In dish
A 2 x 5 μL flat droplets of CZB-HEPES with 12 % PVP are
placed in the center of the dish. One droplet for the sperm
suspension and the other one for the isolated sperm heads
collection. In dish B, a 5 μL droplet of CZB-HEPES with
12 % PVP is placed at the center of the dish and 6 x 5 μL
injection drops of CZB-HEPES are positioned adjacent to
the center. Add approx. five eggs; one in each of the injec­tion droplets. (One drop is left without cells for cleaning the
capillary.)
First of all sperms are cut in the ‘sperm suspension droplet‘
(dish A). Then the sperm heads are transferred to the
‘sperm heads collection droplet’. Collect as many sperm
heads as possible before transferring them to dish B, where
the injection takes place.
The advantage of this preparation is to minimize the ex­posure time of oocytes out of the CO

incubator and thus

2

improve the survival rate of oocytes.

Userguide No 037 | Page 3

Injection droplets

Sperm
droplet

Sperm head
droplet

A

Fig. 4:

ICSI dishes for sperm head isolation (A) and injection (B) using piezo-assisted method

4. Equipment setup

4. 1 Installation of PiezoXpert onto TransferMan NK 2

Loosen the M3x12 cheese head screw and remove the X
head (Figure 5A). Rotate the X head 180°.
Tighten the X head again using the M3x12 cheese head
screw.
Loosen the knurled screw (Figure 5B). Remove the knurled

Place the actuator in the upper (see Figure 6A) or lower (see
Figure 6B) groove of the distance plate. Tighten the knurled
screw to secure the actuator between the distance plate
and pressure plate.
The flatter the angle of the capillary, the more direct the ef­fect of the piezo impulses. Most people use straight or low

angled capillaries for piezo-actuated injection.
screw and the pressure plate from the X head.
Place the provided spacer plate on the bore of the X head.
Secure the spacer plate using the knurled screw and pres­sure plate.

Sperm head droplet

Injection droplets

B

Fig. 5: Installation of spacer plate onto TransferMan NK 2

A

Fig. 6: Installation of piezo actuator onto TransferMan NK 2

Connect the tubing from CellTram vario to the rear end of

the capillary holder of the actuator.

B

Dispense the oil by rotating the CellTram vario knob to the

right until it is dripping from the opening of the grip head.

A

B