HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 69/6/1859    most recent biolreprod.103.019729v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaneko, T. Articles by Yanagimachi, R. Search for Related Content PubMed PubMed Citation Articles by Kaneko, T. Articles by Yanagimachi, R. Agricola Articles by Kaneko, T. Articles by Yanagimachi, R.

Tolerance of the Mouse Sperm Nuclei to Freeze-Drying Depends on Their Disulfide Status¹

Institute for Biogenesis Research,³ Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822 Division of Reproductive Engineering,⁴ Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan Division of Reproductive Biology,⁵ Department of Obstetrics and Gynecology, University of California, Davis, California 95616

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mouse spermatozoa from the caudae epididymides could be freeze-dried without losing their ability to support normal development. Immature spermatozoa from the testes, in contrast, were damaged by freeze-drying. However, immature spermatozoa became resistant to freeze-drying after their treatment with diamide, which oxidizes free -SH groups. Conversely, epididymal spermatozoa were damaged by freeze-drying if first treated with dithiothreitol (DTT), which reduces -SS- bonds. The potential for freeze-drying damage seems likely to relate to the -SS- status of sperm proteins, in particular its protamines.

epididymis, fertilization, gamete biology, sperm, testis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sperm cryopreservation has greatly facilitated assisted reproduction in livestock and laboratory animals as well as humans [1, 2]. Freezing in liquid nitrogen (-196°C) is the gold standard for long-term storage of spermatozoa. However, this approach is by no means problem free. The shipping of liquid nitrogen is considered hazardous, requires a special container, and is not accepted by certain airlines. It is expected that in the future, regulation of the shipment of frozen spermatozoa in liquid nitrogen or with dry ice will increase rather than decrease. On the other hand, the storage of spermatozoa in a freeze-dried state at ambient temperature would eliminate many problems inherent in conventional sperm storage and shipment.

Although attempts to freeze-dry mammalian spermatozoa are not new [3–6], not until 1998 was it shown that mammalian (mouse) spermatozoa can be freeze-dried without losing their ability to initiate and support normal development [7]. This necessarily involves intracytoplasmic sperm injection (ICSI), however, since the spermatozoa are no longer "alive and motile." We are improving the freeze-drying technique [8, 9], and our ultimate goal is to store spermatozoa indefinitely at ambient temperature without damaging the genome.

The conventional method of sperm cryopreservation obviously requires fully mature spermatozoa, from either semen [10] or the caudae epididymides [11], and our previous studies with freeze-drying also have involved mature spermatozoa [7–9]. However, if males do not have mature spermatozoa in their semen or in the epididymides, can we freeze-dry spermatozoa recovered from the testes? One prominent difference between immature and mature spermatozoa is the thiol status of their protamines and perinuclear theca proteins with those of immature spermatozoa lacking the extensive -SS- cross-linking seen in mature spermatozoa [12–17]. This lack of cross-linking makes the nucleus of immature spermatozoa vulnerable to physical and chemical disruption. We report here that immature spermatozoa are indeed vulnerable to damage from freeze-drying but become resistant to this when their free thiols are oxidized to disulfides by diamide. Conversely, epididymal spermatozoa become vulnerable to damage by freeze-drying when treated with the disulfide-reducing agent, dithiothreitol (DTT).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals

Hybrid mice B6D2F1 (C57BL/6 x DBA/2) were used as oocyte and sperm donors. Females and males were 2–3 mo and 3–5 mo old, respectively, when used. Out-bred CD-1 female mice, 2–4 mo old, were used as recipients of two-cell embryos. All animals were maintained in an air-conditioned (22°C) and light-controlled room (14L:10D with light starting from 0700 h) 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 (DHEW publication no. 80-23, 1985).

Media

All organic and inorganic chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. CZB medium [18, 19] supplemented with 5.56 mM D-glucose was used for the culture of oocytes after sperm injection. A modified CZB medium, called Hepes-CZB medium, contained 20 mM Hepes-HCl, 5 mM NaHCO₃, and 0.1 mg/ml polyvinyl alcohol (PVA; cold-water soluble; M_r 30 000–70 000) instead of bovine serum albumin [20]. This medium was used for oocyte collection and sperm injection. CZB and Hepes-CZB media were used in atmospheres of 5% CO₂ in air and air, respectively.

The medium for sperm freeze-drying, called EGTA Tris-HCl-buffered solution, 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 [8]. Its pH value was adjusted to 8.0 by adding a small quantity of 1 M HCl [9]. This medium was kept at 4°C for less than 1 wk and was warmed to 37°C before use.

Collection, Treatment, and Freeze-Drying of Spermatozoa

For each experiment, spermatozoa were released from two caudae epididymides. The distal end of each caudae was cut with a pair of sharp forceps, and the dense masses of spermatozoa emerging were placed at the bottom of 1 ml EGTA Tris-HCl-buffered solution in a 1.5-ml polypropylene microcentrifuge tube (no. 05-408-10, Fisher Scientific, Pittsburgh, PA). This tube was kept at 37°C for 10 min, allowing spermatozoa to disperse into the buffer. The upper 800 µl of the buffer with spermatozoa was carefully transferred to another tube. In some experiments, drops of 1-M stock solution of diamide or DTT were mixed thoroughly with the sperm suspension such that the final concentration of diamide and DTT were 1 or 10 mM, respectively. Diamide is a thiol-oxidizing agent, and DTT is a disulfide-reducing agent [21]. The buffer containing with spermatozoa was allowed to stand for 30 min at room temperature (24–26°C) before it was divided into eight 100-µl aliquots in long-neck glass ampules for freeze-drying (no. 651506, Wheaton, Millville, NJ).

Testicular spermatozoa were prepared as follows. A testis was held under finger pressure, the tunica albuginea was punctured with a pair of sharp forceps, and a small mass of seminiferous tubules was removed by grasping and lifting the tubules until they broke into 4–6-mm fragments. This procedure was repeated several times until a bolus of the tubules accumulated within the tip of the forceps. The bolus was then transferred into 1 ml of EGTA Tris-HCl-buffered solution with or without 1 or 3 mM diamide or DTT in a culture dish (no. 3037, Falcon, Franklin Lakes, NJ). After gentle mixing, large tissue debris was removed. This buffer, containing testicular spermatozoa (free and Sertoli-cell bound), spermatogenic cells, and other cells, was allowed to stand for 30 min at room temperature before it was divided into 10 100-µl aliquots in glass ampules.

Freeze-drying was carried out using the procedure described previously [8, 9]. Briefly, the ampules containing 100-µl aliquots of sperm suspension with or without diamide or DTT were plunged into liquid nitrogen for 20 sec and then connected to the freeze-drying machine (Freeze-Dry Systems 77530, Labconco, Kansas City, MO). After 4 h of freeze-drying, each ampule was flame-sealed. The pressure inside the ampules was 30–39 x 10^-3 mbar during sealing. Ampules were stored at 4°C for up to 5 mo.

Preparation of Oocytes and ICSI

Females were induced to superovulate by intraperitoneal injection of 5 IU eCG (Calbiochem, La Jolla, CA) followed by injection of 5 IU hCG (Calbiochem) 48 h later. Cumulus-oocyte complexes were collected from oviducts at 13–15 h after hCG injection. Oocytes were freed from cumulus cells by treatment for 3 min with 0.1% bovine testicular hyaluronidase (359 units/mg solid) in Hepes-CZB medium and then rinsed and kept at room temperature in fresh Hepes-CZB medium for less than 10 min before use.

An ampule of freeze-dried spermatozoa was opened, and spermatozoa were rehydrated by adding 100 µl of sterile distilled water. A small volume (3 µl) of the sperm suspension was mixed with 10 µl of Hepes-CZB medium containing 12% (w/v) polyvinylpyrrolidone (PVP; M_r 360,000, ICN Pharmaceuticals, Costa Mesa, CA). Rehydrated spermatozoa often had separated heads and broken tails. Those with visibly "intact" heads and tails were selected and transferred to another droplet of Hepes-CZB medium with 12% PVP to remove EGTA, diamide, and DTT in the freeze-drying medium. A single spermatozoon 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 only the head was injected into each oocyte, as described previously [20, 22]. Injected oocytes were cultured in CZB medium. Spermatozoa that were not freeze-dried were also injected into oocytes to serve as controls.

Analysis of Sperm Chromosomes

Sperm chromosomes can be examined by injecting a single spermatozoon into an enucleated oocyte, the sperm chromosomes that appear to be arrested on the metaphase of the first cleavage. In this study, however, we did not enucleate oocytes, as our preliminary studies revealed that less than 1% of normal mouse oocytes have abnormal chromosomes [8, 9]. Therefore, virtually all aberrant chromosomes on the metaphase of the first cleavage following ICSI were assumed to be of sperm origin. The procedures were as follows. About 5 h after ICSI, oocytes with two distinct pronuclei and a second polar body were placed in CZB medium containing 0.006 µg/ml vinblastine to arrest eggs at the metaphase of the first cleavage. Between 19 and 21 h after ICSI, the eggs were freed from the zona pellucida by treatment for 5 min with 0.5% pronase (1000 tyrosine units/mg, Kaken Pharmaceuticals, Tokyo, Japan) in phosphate-buffered saline (PBS). Zona-free eggs were then treated with hypotonic solution (1:1 mixture of 30% fetal bovine serum and 1% sodium citrate) and fixed by air drying on glass slides [23]. Chromosomes on slides were stained with 5% Giemsa (Merck, Darmstadt, Germany) in PBS (pH 6.8) for 10 min. Two groups of metaphase chromosomes were seen, one of which always consisted of a normal haploid set and the second of which had either a normal or an abnormal set of chromosomes. It was assumed that eggs with two normal haploid chromosome sets had been fertilized by normal spermatozoa (Fig. 1A) and those with aberrant chromosomes in one of the two chromosome sets by chromosomally abnormal spermatozoa (Fig. 1B).

View larger version (49K): [in this window] [in a new window]   FIG. 1. Normal (A) and abnormal (B) sperm chromosomes (arrows) at the metaphase of the first cleavage after ICSI. Magnification x 1000

Embryo Transfer

At about 5 h after ICSI, eggs with two distinct pronuclei and a second polar body were cultured in CZB medium. Within 20–24 h after ICSI, embryos that had developed to the two-cell stage were transferred into oviducts of CD-1 females made pseudopregnant by mating with vasectomized males of the same strain during the previous night. Females were killed on Day 19.5 of pregnancy to determine the numbers of live, dead, and absorbed fetuses. Live fetuses were fostered to lactating CD-1.

Analysis of Data

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

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We assessed the normality of sperm chromosomes by examining chromosomes of fertilized eggs at the metaphase of the first cleavage following ICSI. When fresh testicular spermatozoa with or without diamide treatment were injected, the vast majority of the fertilized eggs had normal chromosomes, indicating that most testicular spermatozoa had intact chromosomes (experiments 1 and 3 in Table 1). After freeze-drying, only 3% of the testicular spermatozoa were chromosomally normal, indicating that they were vulnerable to damage by freeze-drying (experiment 2). When testicular spermatozoa were treated with 1 or 3 mM diamide before and during freeze-drying, the number of spermatozoa with normal chromosomes increased significantly (experiments 4 and 5). DTT had an opposite effect (experiment 6).

Table 2 summarizes the results of experiments in which spermatozoa from the caudae epididymides were freeze-dried with or without diamide or DTT treatment. The majority of untreated epididymal spermatozoa had normal chromosomes before and after freeze-drying (experiments 1 and 2), as did those treated with diamide (experiment 3). DTT, on the other hand, reduced the incidence of chromosomally normal spermatozoa after freeze-drying (experiments 4 and 5), though it had no detrimental effect on the chromosomes of epididymal spermatozoa (experiment 6).

The development of two-cell embryos derived from oocytes injected with testicular spermatozoa is summarized in Table 3. Only two normal living term fetuses were obtained from two-cell embryos created with testicular spermatozoa freeze-dried without diamide (experiment 2). By contrast, we obtained 24 normal term fetuses with freeze-dried testicular spermatozoa treated with diamide (experiments 3 and 4). When two female and two male offspring from experiment 3 were randomly selected and allowed to mature and mate, they all produced offspring of normal litter sizes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study shows that the chromosomes of testicular spermatozoa are vulnerable to damage from freeze-drying and become resistant to it after treatment with the -SH oxidizing agent, diamide. In contrast, DTT, which reduces protein -SS- to -SH, made epididymal spermatozoa susceptible to damage from freeze-drying. Sperm proteins that would be affected by DTT and diamide include cysteine-rich protamines and lamin B in the nucleus and a basic protein, calicin, in the perinuclear theca [13, 15, 17, 24]. Of these, protamines are most likely to be responsible for genetic (chromosomal) integrity of sperm nucleus in face of an artificial insult such as freeze-drying. The fact that some testicular spermatozoa could withstand freeze-drying without diamide treatment and produce live offspring (experiment 2 in Table 1 and experiment 2 in Table 3) may be explained by the fact that the protamines are partially cross-linked in about 10% of mouse testicular spermatozoa [21]. The fact that testicular spermatozoa completely lost their tolerance to freeze-drying after DTT treatment (experiment 6 in Table 1) supports the view that the -SS- cross-links of the sperm head, and most probably those of the nucleus, make the sperm nuclei resistant to damage by freeze-drying.

Diamide, which oxidizes protein -SH to -SS-, did not make epididymal spermatozoa more resistant to damage by freeze-drying (experiment 3 in Table 2) because free thiols of the sperm head are almost fully cross-linked by the time the spermatozoa reach the caudae epididymides [13, 21]. The finding that a high concentration of DTT, which reduces -SS- to -SH, rendered epididymal spermatozoa vulnerable to freeze-drying (experiments 4 and 5 in Table 2) again supports the view that protein -SS- makes spermatozoa resistant to harsh physical treatment such as freeze-drying. We now show that testicular spermatozoa can be made resistant to freeze-drying damage and can produce live offspring (experiments 3 and 4 in Table 3), although there is room for technical improvement to make these procedures more efficient.

Because the protamines of noneutherian vertebrates (from most fish to marsupials) have little or no cysteine [13], it may be that the spermatozoa of these species cannot be freeze-dried successfully. However, it is possible that spermatozoa of some invertebrates (such as grasshoppers) that have nuclear protamines rich in -SH [25, 26] may become resistant to freeze-drying damage after diamide treatment. Recently, Bhowmick et al. [27] reported successful "drying" (without freezing) of mouse epididymal spermatozoa. Whether mammalian testicular spermatozoa and spermatozoa of noneutherian mammals can be dried and kept for many days without losing their genetic integrity remains to be determined.

	ACKNOWLEDGMENTS

 
We thank Drs. J.M. Bedford, M. Meistrich, and S. Moisyadi for their valuable comments and help in the preparation of the 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).

² Correspondence: Takehito Kaneko, 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

Received: 26 May 2003.

First decision: 15 June 2003.

Accepted: 29 July 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Leibo SP, Songsasen N. Cryopreservation of gametes and embryos of non-domestic species. Theriogenology 2002 57:303-326[CrossRef][Medline]
2. Agca Y, Critser JK. Cryopreservation of spermatozoa in assisted reproduction. Semin Reprod Med 2002 20:15-23[CrossRef][Medline]
3. Sherman JK. Freezing and freeze-drying of human spermatozoa. Fert Steril 1954 5:357-371[Medline]
4. Bialy G, Smith VR. Freeze-drying of bovine spermatozoa. J Dairy Sci 1957 40:739-745[Abstract/Free Full Text]
5. Nei T, Nagase H. Attempts to freeze-dry bull spermatozoa. Low Temp 1961 19:107-115
6. Singh SG, Roy DJ. Freeze-drying of bovine semen. Indian J Vet Sci 1967 37:1-7
7. Wakayama T, Yanagimachi R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat Biotechnol 1998 16:639-641[CrossRef][Medline]
8. Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci U S A 2001 98:13501-13506[Abstract/Free Full Text]
9. Kaneko T, Whittingham DG, Yanagimachi R. Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa. Biol Reprod 2003 68:136-139[Abstract/Free Full Text]
10. Songsasen N, Leibo SP. Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa. Lab Anim Sci 1998 48:275-281[Medline]
11. Nakagata N. Cryopreservation of mouse spermatozoa. Mamm Genome 2000 11:572-576[CrossRef][Medline]
12. Calvin HI, Bedford JM. Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J Reprod Fertil Suppl 1971 13:suppl 1365-75
13. Bedford JM, Calvin HI. The occurrence and possible functional significance of -S-S- crosslinks in sperm heads, with particular reference to eutherian mammals. J Exp Zool 1974 188:137-155[CrossRef][Medline]
14. Marushige Y, Marushige K. Transformation of sperm histone during formation and maturation of rat spermatozoa. J Biol Chem 1975 250:39-45[Abstract/Free Full Text]
15. Longo FJ, Krohne G, Franke WW. Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements. J Cell Biol 1987 105:1105-1120[Abstract/Free Full Text]
16. Balhorn R, Corzett M, Mazrimas J, Watkins B. Identification of bull protamine disulfides. Biochemistry 1991 30:175-181[CrossRef][Medline]
17. Korley R, Pouresmaeili F, Oko R. Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent. Biol Reprod 1997 57:1426-1432[Abstract]
18. Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989 86:679-688[Abstract]
19. Chatot CL, Lewis JL, Torres I, Ziomek CA. Development of 1-cell embryos from different strains of mice in CZB medium. Biol Reprod 1990 42:432-440[Abstract]
20. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995 52:709-720[Abstract]
21. Kosower NS, Katayose H, Yanagimachi R. Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei. J Androl 1992 13:342-348[Abstract/Free Full Text]
22. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol Reprod 1996 55:789-795[Abstract]
23. Kamiguchi Y, Mikamo K. An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova. Am J Hum Genet 1986 38:724-740[Medline]
24. Moss SB, Burnham BL, Bellve AR. The differential expression of lamin epitopes during mouse spermatogenesis. Mol Reprod Dev 1993 34:164-174[CrossRef][Medline]
25. Rodman TC, Litwin SD, Romani M, Vidali G. Life history of mouse sperm protein. Intratesticular stages. J Cell Biol 1979 80:605-620[Abstract/Free Full Text]
26. Ausio J. Histone H1 and evolution of sperm nuclear basic proteins. J Biol Chem 1999 274:31115-31118[Free Full Text]
27. Bhowmick S, Zhu L, McGinnis L, Lawitts J, Nath BD, Toner M, Biggers J. Desiccation tolerance of spermatozoa dried at ambient temperature: production of fetal mice. Biol Reprod 2003 68:1779-1786[Abstract/Free Full Text]

This article has been cited by other articles:

				N. Nakamura, A. Miranda-Vizuete, K. Miki, C. Mori, and E. M. Eddy Cleavage of Disulfide Bonds in Mouse Spermatogenic Cell-Specific Type 1 Hexokinase Isozyme Is Associated with Increased Hexokinase Activity and Initiation of Sperm Motility Biol Reprod, September 1, 2008; 79(3): 537 - 545. [Abstract] [Full Text] [PDF]

				Y. Kawase, T. Hani, N. Kamada, K.-i. Jishage, and H. Suzuki Effect of pressure at primary drying of freeze-drying mouse sperm reproduction ability and preservation potential Reproduction, April 1, 2007; 133(4): 841 - 846. [Abstract] [Full Text] [PDF]

				N. Ogonuki, K. Mochida, H. Miki, K. Inoue, M. Fray, T. Iwaki, K. Moriwaki, Y. Obata, K. Morozumi, R. Yanagimachi, et al. Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring PNAS, August 29, 2006; 103(35): 13098 - 13103. [Abstract] [Full Text] [PDF]

				K.-B. Lee and K. Niwa Fertilization and Development In Vitro of Bovine Oocytes Following Intracytoplasmic Injection of Heat-Dried Sperm Heads Biol Reprod, January 1, 2006; 74(1): 146 - 152. [Abstract] [Full Text] [PDF]

				J. Seligman, G. L. Newton, R. C. Fahey, R. Shalgi, and N. S. Kosower Nonprotein Thiols and Disulfides in Rat Epididymal Spermatozoa and Epididymal Fluid: Role of {gamma}-Glutamyl-Transpeptidase in Sperm Maturation J Androl, September 1, 2005; 26(5): 629 - 637. [Abstract] [Full Text] [PDF]

				R. Suganuma, C. M. Walden, T. D. Butters, F. M. Platt, R. A. Dwek, R. Yanagimachi, and A. C. van der Spoel Alkylated Imino Sugars, Reversible Male Infertility-Inducing Agents, Do Not Affect the Genetic Integrity of Male Mouse Germ Cells During Short-Term Treatment Despite Induction of Sperm Deformities Biol Reprod, April 1, 2005; 72(4): 805 - 813. [Abstract] [Full Text] [PDF]

				Y. Kawase, H. Araya, N. Kamada, K.-i. Jishage, and H. Suzuki Possibility of Long-Term Preservation of Freeze-Dried Mouse Spermatozoa Biol Reprod, March 1, 2005; 72(3): 568 - 573. [Abstract] [Full Text] [PDF]

				I.-K. Kwon, K.-E. Park, and K. Niwa Activation, Pronuclear Formation, and Development In Vitro of Pig Oocytes Following Intracytoplasmic Injection of Freeze-Dried Spermatozoa Biol Reprod, November 1, 2004; 71(5): 1430 - 1436. [Abstract] [Full Text] [PDF]

				J. Seligman, Y. Zipser, and N. S. Kosower Tyrosine Phosphorylation, Thiol Status, and Protein Tyrosine Phosphatase in Rat Epididymal Spermatozoa Biol Reprod, September 1, 2004; 71(3): 1009 - 1015. [Abstract] [Full Text] [PDF]

				J.-L. Liu, H. Kusakabe, C.-C. Chang, H. Suzuki, D. W. Schmidt, M. Julian, R. Pfeffer, C. L. Bormann, X. C. Tian, R. Yanagimachi, et al. Freeze-Dried Sperm Fertilization Leads to Full-Term Development in Rabbits Biol Reprod, June 1, 2004; 70(6): 1776 - 1781. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 69/6/1859    most recent biolreprod.103.019729v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaneko, T. Articles by Yanagimachi, R. Search for Related Content PubMed PubMed Citation Articles by Kaneko, T. Articles by Yanagimachi, R. Agricola Articles by Kaneko, T. Articles by Yanagimachi, R.