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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008706
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BIOLOGY OF REPRODUCTION 68, 136–139 (2003)
DOI: 10.1095/biolreprod.102.008706
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Effect of pH Value of Freeze-Drying Solution on the Chromosome Integrity and Developmental Ability of Mouse Spermatozoa1

Takehito Kaneko2,a,b, David G. Whittinghama, and Ryuzo Yanagimachia

a The Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822 b Division of Reproductive Engineering, Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4–6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.

embryo, fertilization, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The recent developments in transgenesis and mutagenesis research have led to the generation of a large number of new mouse lines worldwide [1–4]. Obviously, it is not economically and practically possible to maintain all these unique stocks by conventional breeding. The cryopreservation of a large number of eggs and/or embryos is an option but is not realistic because of the involvement of large numbers of animals [5, 6]. The banking of spermatozoa would be an efficient and cost-effective approach [3, 7]. Although mouse sperm cryopreservation has become successful through the endeavors of several investigators [8–14], spermatozoa of some mouse strains (e.g., C57BL/6 [15], BALB/c [3], and 129/J [16]) are difficult to freeze. Furthermore, long-term storage of spermatozoa in liquid nitrogen is by no means inexpensive. Shipping of frozen spermatozoa in liquid nitrogen or dry ice is becoming more restrictive.

Sperm storage and shipment without liquid nitrogen or dry ice would be ideal. We previously reported that, even though freeze-dried mouse spermatozoa are all "dead" in the conventional sense, their nuclei remain capable of participating in normal embryo development when injected into oocytes [17, 18]. Advantages of freeze-drying are obvious. Freeze-dried spermatozoa can be stored at ambient temperature or in ordinary refrigerators or freezers. Shipment of sperm samples from one place to another would be easier and much less expensive. The current protocols for sperm freeze-drying are still empirical and need improvements. Here we studied whether pH values of the sperm freeze-drying solution affect chromosome integrity and the developmental potential of freeze-dried spermatozoa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Epididymal spermatozoa and matured oocytes were collected from 8- to 12-wk-old B6D2F1 hybrid mice. Recipients of two-cell embryos were 8- to 16-wk-old random-bred CD-1 females. All animals were maintained according to the guidelines of the Laboratory Animal Service at the University of Hawaii and those prepared by the Committee on Care and Use of Laboratory Animals of the National Research Council [19]. The protocol of animal handling and treatment was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.

Buffered Solution for Freeze-Drying Spermatozoa

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. The buffer for freeze-drying of spermatozoa consisted of 50 mM EGTA (ethylene glycol-bis [beta-aminoethyl ether]-N, N,N',N'-tetraacetic acid), 50 mM NaCl, and 10 mM Tris-HCl buffer [18]. This was called EGTA Tris-HCl buffered solution. The pH value of this solution was adjusted to 9.0, 8.0, 7.4, or 6.0 by adding a small quantity of 1 M hydrochloric acid (HCl) or 1 M sodium hydroxide (NaOH). The pH-adjusted solutions were stored at 4°C for less than 1 wk before use.

Media

CZB medium [20, 21] supplemented with 5.56 mM D-glucose was used for the culture of mouse oocytes after microinjection. The medium for oocyte collection and subsequent oocyte treatments, including micromanipulation, was a modified CZB medium (Hepes-CZB medium [22]) containing 20 mM Hepes-HCl, 5 mM NaHCO3, and 0.1 mg/ml polyvinyl alcohol (cold water soluble; Mr 30 000–70 000) instead of bovine serum albumin. CZB medium was used under 5% CO2 in air, and Hepes-CZB was used under air.

Freeze-Drying of Spermatozoa

Sperm freeze-drying was carried out as described previously [18]. Briefly, 1 ml of the pH-adjusted EGTA Tris-HCl-buffered solution was placed in a 1.5-ml polypropylene microcentrifuge tube (no. 05-408-10, Fisher Scientific, Pittsburgh, PA) and warmed to 37°C. Two epididymides were removed from a B6D2F1 male and a dense sperm mass was squeezed out of the caudae region of each epididymis after cutting it with a pair of sharp forceps. Sperm masses from two epididymides were gently put at the bottom of 1 ml EGTA Tris-HCl-buffered solution in the microcentrifuge tube and kept at 37°C for 10 min to allow spermatozoa to disperse into the solution. The upper 800 µl of the sperm suspension was collected and 100-µl aliquots were transferred into long-necked glass ampoules for freeze-drying (no. 651506; Wheaton, Millville, NJ). Ampoules were plunged into liquid nitrogen for 20 sec and then were connected to the freeze-drying machine (Freeze-Dry Systems, Labconco, Kansas City, MO). Four hours later, ampoules were flame-sealed. The pressure inside the ampoules was 30–33 x 10-3 mbar at the time of sealing. Ampoules were stored at 4°C until use.

Collection of Oocytes

B6D2F1 females were induced to superovulate by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Calbiochem, La Jolla, CA) followed by injection of 5 IU hCG (Calbiochem) 48 h later. Matured oocytes were collected from the ampullary region of oviducts 13–15 h after the hCG injection. Oocytes were freed from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (359 units/mg solid) in Hepes-CZB medium. Oocytes were rinsed and kept at room temperature (26°C) in fresh Hepes-CZB medium before sperm injection.

Intracytoplasmic Sperm Injection

An ampoule with freeze-dried spermatozoa was opened and spermatozoa were rehydrated by adding 100 µl of sterile distilled water. A drop (about 3 µl) of the sperm suspension was mixed thoroughly with Hepes-CZB medium containing 12% (w/v) polyvinylpyrrolidone (PVP; Mr 360 000; ICN Pharmaceuticals, Costa Mesa, CA). Rehydrated spermatozoa often had separated heads and broken tails. Those with normal structure were selected and transferred to another droplet of 12% PVP solution to remove EGTA Tris-HCl. A single spermatozoa was drawn, tail first, into the injection pipette in such a way that its neck (the junction between the head and tail) was at the opening of the pipette. The head was separated from the tail by applying a few piezo-pulses to the neck region and was injected into an oocyte as described by Kimura and Yanagimachi [22]. Intracytoplasmic sperm injection (ICSI) oocytes were incubated in CZB medium at 37°C under 5% CO2 in air.

Chromosome Analysis

Chromosome analysis of ICSI oocytes was carried out with the procedures described previously [23, 24]. Briefly, oocytes with two well-developed pronuclei and a distinct second polar body about 5 h after ICSI were transferred to CZB medium containing 0.006 µg/ml vinblastine. Those arrested at the first cleavage metaphase after 19–21 h of culture in the vinblastine solution were treated a few minutes with 0.5% pronase (1000 tyrosine units/mg; Kaken Pharmaceuticals, Tokyo, Japan) in PBS to remove the zonae pellucidae. The eggs were then treated with hypotonic solution (1:1 mixture of 30% fetal bovine serum and 1% sodium citrate) for a few minutes, fixed, and air-spread on a glass slide for chromosome analysis [25]. An egg with two groups of 20 chromosomes without any structural and numerical abnormalities was recorded as karyotypically normal. Uncountable number of chromosomal aberrations such as fragmentation or multiple exchanges was defined as >10 breaks per oocyte. The number of aberrations per oocyte was recorded without discriminating between paternal and maternal chromosomes. As our preliminary experiments showed that maternal chromosomes in parthenogenetically activated mouse oocytes were almost always normal (>99%) at the first cleavage, abnormal chromosomes in the first cleavage metaphase of ICSI oocytes were likely of paternal (sperm) origin. The chromosome status of the oocytes injected with spermatozoa suspended in EGTA Tris-HCl buffered solution but without freeze-drying served as the control.

Embryo Culture and Embryo Transfer

Oocytes with two well-developed pronuclei and a distinct second polar body 5 h after ICSI were recorded as being activated and were cultured in CZB medium until they reached the morula/blastocyst stage. Some embryos at the two-cell stage (20–24 h after ICSI) were transferred into oviducts of surrogate CD-1 females that were mated with vasectomized males of the same strain on the day before embryo transfer. Numbers of implantation sites and normal developing fetuses were counted on Day 15 of gestation. We examined fetuses on Day 15 of gestation because this provides information on the extent of early embryonic loss after implantation and normal fetuses at Day 15 of gestation very rarely fail to develop to full term [18].

Analysis of Data

The data from different treatments were compared with chi-square test analysis using Yates correction for continuity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chromosome analysis of ICSI oocytes, summarized in Table 1, showed that 77–88% of the oocytes injected with fresh (control) spermatozoa had normal chromosomes regardless of the pH value of the EGTA Tris-HCl buffered solution. When spermatozoa were freeze-dried, the incidence of eggs with chromosome abnormalities increased. Obviously, freeze-drying damages sperm chromosomes. However, damage was less when sperm-suspending solutions were alkaline (pH 8.0–9.0) rather than neutral or acidic (pH 6.0–7.4) (P < 0.05). A slightly alkaline (pH 8.0) solution was the best in maintaining chromosome integrity of freeze-dried spermatozoa.


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  		TABLE 1. Chromosome analysis of oocytes injected with spermatozoa freeze dried in EGTA Tris-HCl-buffered solution with various pH values

Almost all ICSI oocytes were activated and the majority reached the two-cell stage regardless of the pH value of the EGTA Tris-HCl buffered solution used for freeze-drying (Table 2). Development of two-cell embryos to blastocysts was better when spermatozoa were freeze-dried in solutions with higher pH values (pH 8.0–9.0) than those with lower pH values (pH 6.0–7.4) (P < 0.05).


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  		TABLE 2. In vitro development of oocytes injected with freeze-dried spermatozoa. Spermatozoa were freeze-dried in EGTA Tris-HCl-buffered solution with various pH values

When ICSI-produced two-cell embryos were transferred to surrogate females, the majority implanted (Table 3). More embryos developed normally when spermatozoa were freeze-dried with slightly alkaline (pH 8.0–9.0) solutions than with neutral (pH 7.4) solution (P < 0.05). All live fetuses were normal in size and morphology regardless of the pH values of the EGTA Tris-HCl buffered solution.


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  		TABLE 3. In vivo development of embryos derived from the oocytes injected with spermatozoa freeze-dried in EGTA Tris-HCl buffered solution with various pH values

No differences were obtained on chromosome integrity and developmental ability of freeze-dried spermatozoa stored at 4°C between short-term (<8 days) and long-term (26–56 days) storage groups of each pH values (Tables 1–3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To optimize the conditions for freeze-drying mouse spermatozoa, we examined how pH values of freeze-drying solutions affect sperm chromosomes and developmental ability. We found that a slightly alkaline (pH 8.0) solution best maintains chromosome integrity and the developmental ability of spermatozoa.

Freeze-drying does damage structural components of spermatozoa. All freeze-dried spermatozoa placed in a live/dead cell stain [18] took up dye, indicating that the plasma membranes of all spermatozoa were extensively damaged. Separation of sperm heads from tails and the broken tails were commonly observed among spermatozoa that were freeze-dried and rehydrated. Perhaps tails of freeze-dried spermatozoa were very brittle and broken even by slight agitation. Nevertheless, many, if not all, nuclei of freeze-dried spermatozoa well retained their genetic integrity, as evidenced by their normal chromosome constitution and their ability to initiate embryo development.

In this study, we injected only sperm heads into oocytes, as tail components are not essential for embryo development, at least in the mouse [26]. Sperm cytoplasmic components that activate the oocyte at fertilization reside within the sperm head, not in the tail [26, 27]. In fact, the majority of the oocytes injected with the heads of freeze-dried spermatozoa were activated (Tables 2 and 3), confirming the notion that freeze-drying does not damage the sperm-borne oocyte-activating components [17, 18].

In this study, we examined fetuses at Day 15 of gestation because this allowed us to determine precisely how many of the transferred embryos implanted and how many of the implanted embryos developed normally. Those that are normal at this time of development are most likely to reach term [18]. It was somewhat unexpected that the spermatozoa freeze-dried in the solution with physiological pH (7.4) were inferior to those freeze-dried in solutions at pH 8.0–9.0 with respect to the ability of sperm heads (nuclei) to participate in development (Table 3). A high pH may prevent or slow down the action of substances such as endonucleases. The optimal pH for DNase I, for example, is 7.0, and it is most stable at pH 5.0–6.0 [28]. As divalent cations are essential for DNase activity [29], chelating agents such as EDTA and EGTA would suppress their activity. The combination of EGTA and an alkaline pH may synergistically suppress DNase activity when freeze-dried spermatozoa no longer have intact plasma membranes.

We are aiming at developing a better sperm freeze-drying protocol. Many factors other than the pH of the freeze-drying solution must be considered. We are studying whether the moisture content of freeze-dried spermatozoa affect chromosome integrity. It is our goal to store freeze-dried spermatozoa indefinitely at ambient temperature without "losing" the sperm's genetic integrity.


   ACKNOWLEDGMENTS
 
We thank Dr. H. Kusakabe for invaluable technical advice and Dr. R. Tasca, Dr. J. Shaw, Dr. S. Moisyadi, and Mrs. C. Oser for reviewing the original manuscript.


   FOOTNOTES
 
1 This study was conducted as part of the National Cooperative Program on Mouse Sperm Cryopreservation sponsored by the National Institute of Child Health and Human Development and the National Center for Research Resources (U01HD38205). Back

2 Correspondence: Takehito Kaneko, The Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 7316; takehito{at}hawaii.edu Back

Received: 21 June 2002.

First decision: 8 July 2002.

Accepted: 5 August 2002.


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 DISCUSSION
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