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Reproductive Technology; |
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe 650-0047, Japan
ABSTRACT
Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 µm thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at –30°C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-µm sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.
fertilization, frozen testicular section, ICSI, spermatid, spermatogenesis, spermatozoa, testicular, testis
The mouse is the primary research animal used in mammalian genetics, providing many different models for the analysis of embryonic development and human genetic diseases. Transgenic or mutant mice are routinely produced to elucidate gene function, which has resulted in an abundance of valuable mouse lines that need to be conserved for future use. Efficient and dependable methods for gamete and embryo cryopreservation are needed to avoid inadvertent loss of these unique materials through disease or other hazards. In addition, these methods can provide an effective means of distributing novel genetic models among the biomedical research community. As spermatozoa are produced in much larger numbers than oocytes and embryos, sperm cryopreservation is less labor intensive than embryo freezing. Thus, the development of a simple, inexpensive, and space-effective means to preserve mouse sperm would be an effective way to store transgenic and mutant stocks. Although several reliable methods have been established to cryopreserve male gametes [1–3], all involve the use of cryostraws [1], cryotubes [2], or ampules [3], which require storage space.
The preparation of frozen sections of male reproductive tissue may offer a more efficient alternative to these techniques, as tissue sectioning using a cryostat is relatively easy and does not require expensive reagents. Furthermore, large numbers of male gamete samples can be produced from a single testis and can be stored in a much more space-efficient manner because the tissue sections are very thin. Thus, the production of progeny via frozen testicular sections may offer a novel alternative to the current methods of preservation. In addition to these advantages, sperm retrieval from testicular sections allows observation of spermatogenesis in a histological section before performing micro-insemination. This would lead to increased odds of finding male gametes within the testis, since it is much easier to identify testicular spermatozoa in histological sections than it is to identify them in testicular cell suspensions. Thus, sperm retrieval from testicular sections may be effective in cases of severe male sterility.
In the present study, we investigated whether testicular spermatozoa could be retrieved from frozen testicular sections, and assessed the functional viability of these gametes after cryopreservation by intracytoplasmic sperm injection (ICSI).
C57BL/6-GFP transgenic mice [TgN(acro/act-EGFP)OsbC3-N01-FJ002] were kindly provided by Dr. Masaru Okabe (Osaka University) [4, 5]. BDF1, C57BL/6, and ICR mice were purchased from SLC (Hamamatsu, Japan). All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of the RIKEN Kobe Institute.
Preparation of Frozen Testicular Sections
Frozen testicular sections were prepared from the testes of adult BDF1 or C57BL/6-GFP transgenic mice. Briefly, the testis was removed and embedded in Optimal Cutting Temperature compound (Tissue-Tek; Sakura Finetechnical Co., Tokyo, Japan). The specimen was then frozen at –30°C and sectioned to a thickness of 10 or 25 µm using a cryostat. Sections were mounted on glass slides (10 sections per slide), placed into slide cases, and stored at –30°C for future use. To prepare a testicular cell suspension, the entire glass slide was incubated with PBS or potassium-rich buffer (nucleus isolation medium [NIM] [6]; 123 mM KCl, 2.6 mM NaCl, 7.8 mM NaH2PO4, 1.4 mM KH2PO4, 3 mM EDTA; pH 7.2 adjusted by 1M KOH) at 4°C for 2 min. The sections were washed by gently pipetting with approximately 1 ml of PBS or NIM via a micropipette. The detached cells were centrifuged at 2300 x g for 2 min at 4°C and then resuspended in PBS or NIM. Testicular cell suspensions were kept at 4°C until ICSI. To assess the viability of testicular spermatozoa, propidium iodide staining was performed using the LIVE/DEAD Sperm Viability Kit (Molecular Probes, Eugene, OR). Three independent replicates using three different males were performed, and approximately 100 testicular spermatozoa were assessed in each experiment.
Intracytoplasmic Sperm Injection
Superovulation was induced in BDF1 or C57BL/6 females by an injection of 5 IU eCG followed by a second injection of 5 IU hCG 48 h later. At 14 h post-hCG injection, the cumulus–oocyte complexes (COCs) were collected from the oviducts. Oocytes were freed from the cumulus cells by adding 0.1% bovine testicular hyaluronidase (ICN Biochemicals, Costa Mesa, CA) to the COC-containing medium. After the cumulus cells had dissociated, the oocytes were rinsed twice with Chatot, Ziomet, and Bavister (CZB) medium [7]. Approximately 2 µl of the sperm suspension was mixed with a drop of Hepes-human tubal fluid (HTF) medium containing 10% (w/v) polyvinylpyrrolidone (IrvineScientific, Santa Ana, CA). The sperm head was separated from the tail by applying several piezo pulses to the neck region of the spermatid, and the head was then injected into the oocyte according to the method described by Kimura and Yanagimachi [8]. The testicular cell suspension was replaced every 30 min during the ICSI experiment. The oocytes that survived ICSI were incubated in CZB medium at 37°C under an atmosphere of 5% CO2. When the embryos reached the 2-cell stage, they were transferred to the oviducts of 0.5-dpc pseudopregnant ICR females.
The efficiency of embryo development was analyzed by using arcsine transformation followed by two-way ANOVA. A P-value of <0.05 was considered statistically significant.
Preparation and Characterization of Testicular Spermatozoa from Frozen Testicular Sections for the Use of ICSI
Testicular cross sections were cut at a thickness of 10 or 25 µm and mounted on glass slides (Fig. 1A). Microscopic observation revealed evidence of spermatogenesis within the seminiferous tubules (Fig. 1, B and C). After preservation overnight at –30°C, a testicular cell suspension was prepared by gently agitating the sections with a micropipette. Approximately 2–3 x 106 testicular cells were successfully retrieved from 10 testicular sections of both thicknesses (Fig. 1D and Table 1). A single testicular section yielded a sufficient number of gametes for ICSI. To estimate the physical damage caused by sectioning, testicular spermatozoa were classified into three groups according to tail length (less than 30%, approximately 50%, or over 80%), which was measured as the percent length of a normal tail (Fig. 1, E–G). Thirty-six percent of the testicular spermatozoa in the 25-µm testicular sections had short tails, whereas 66% had short tails in the 10-µm sections (Table 1). This suggests that the heads of testicular spermatozoa may also be damaged by sectioning, especially in the 10-µm sections. In addition, propidium iodide staining indicated that the plasma membrane was not intact in almost all of the testicular spermatozoa (99 ± 0.6%; n = 3), probably as a result of cryodamage [9] (Fig. 1H). Thus, although many testicular spermatozoa had been damaged by sectioning and freezing, an adequate number of male gametes could still be obtained from the frozen testicular sections.
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Functional Ability of Testicular Spermatozoa Prepared from Frozen Testicular Sections
To assess functional viability, the spermatids retrieved from testicular sections were injected into oocytes. Previously, Ogonuki et al. demonstrated that the cell-suspension medium used to freeze male haploid germ cells can affect developmental capability after ICSI [2]. Therefore, we tested two physiological buffers: PBS and NIM [6]. Testicular spermatozoa were isolated from 10-µm or 25-µm testicular sections that had been frozen overnight at –30°C, and were suspended in PBS or NIM. After ICSI, more than 90% of the oocytes formed pronuclei (Table 2), indicating that the activating factor(s) remained functional in these prepared spermatids. Furthermore, we succeeded in generating normal progeny from the ICSI embryos after transferring them into pseudopregnant females (Fig. 2A and Table 2). Statistical analysis (two-way ANOVA) did not reveal any significant interaction between the two factors (suspension medium and section thickness) with respect to birth rate (P > 0.05), indicating that these factors are independent of each other. In addition, we also confirmed that NIM is more effective than PBS in obtaining progeny from frozen testicular sections (P < 0.05). Furthermore, there was no significant difference between section thicknesses (P > 0.05). These results demonstrate that testicular spermatozoa prepared in this manner were able to produce full-term progeny.
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ICSI Using Testicular Spermatozoa Prepared from Frozen Testicular Sections Preserved for 1 and 3 mo at –30°C
Next, we assessed whether our technique would be useful for preserving male gametes. Testicular sections were frozen for 1 or 3 mo at –30°C, and then the developmental capability of the frozen testicular spermatozoa was assessed using ICSI. After 1 mo of preservation, male gametes were injected into oocytes. Pronuclear formation, cleavage rate, and the birthrate of progeny were found to be similar to those of testicular spermatozoa preserved overnight (Table 3). Furthermore, we succeeded in generating progeny from sections preserved for 3 mo (Table 3). The success rate using spermatids from 10-µm sections may have been slightly lower than that of the other preservation times examined, although there was no significant difference found between section thicknesses (P = 0.15). This may indicate that the preservation of the thinnest sections (i.e., 10 µm thick) affects the developmental capability of testicular spermatozoa. Importantly, although nearly 200 mice were generated and assessed in this study (Tables 2 and 3), malformations such as overgrowth or growth retardation were not observed in the neonatal mice, indicating that normal progeny can be produced from oocytes fertilized with testicular spermatozoa previously preserved as frozen testicular sections.
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Production of Inbred Mice (C57BL/6) via ICSI Using Testicular Spermatozoa Prepared from Frozen Testicular Sections Preserved for 3 mo at –30°C
Although we generated progeny from preserved testicular spermatozoa, it remained unclear whether this technique would be feasible for large-scale preservation, especially in large animal facilities. In such a setting, it is important to be able to produce progeny even from inbred strains; ICSI, however, has previously been ineffective in producing inbred mice [10, 11]. We preserved testicular sections from C57BL/6 mice carrying the green fluorescent protein (GFP) transgene and assessed the functional capability of the prepared testicular spermatozoa by injecting them into C57BL/6 oocytes. Pronuclear formation and the rate of cleavage were comparable to those of testicular spermatozoa using BDF1 mice, even when the testicular sections had been frozen for 3 mo at –30°C (Table 3). In addition, the birth rate of normal progeny expressing GFP was similar to that observed in the experiments using BDF1 mice (Fig. 2, B and C, and Table 2). To confirm the transmission of the GFP transgene to the next generation, we carried out a mating experiment using the progeny delivered from our procedure. Three males and three females, which were expected to be hemizygous for the GFP transgene, were mated with each other, and 17 progeny (10 males and 7 females) in total were obtained from the mating. Of the 17 progeny, 15 showed green fluorescence, indicating transmission of the GFP transgene, at approximate Mendelian frequency. Thus, our technique would also be useful for generating mouse colonies, even from inbred and transgenic mouse strains.
We demonstrated that male haploid germ cells can be preserved as frozen testicular sections for at least 3 mo at –30°C. Although mRNA and DNA are commonly prepared from tissue sections [12, 13], this is the first evidence to show that functional genetic material with the capability to support normal development can be retrieved from tissue sections. The cryopreservation of male gametes in frozen testicular sections is inexpensive, as there is no need for a controlled-rate cell freezer or liquid nitrogen, and space-efficient (see below), both of which are important features for use in animal facilities. Moreover, the preservation procedure using the cryostat is very simple and can be carried out by individuals who are not specialists in reproductive technology. The progeny generated showed normal growth and fertility, suggesting that no genetic or epigenetic damage resulted from sectioning or cryopreservation. Furthermore, as suggested in the Supplemental Figure (available online at www.biolreprod.org), male gametes can be preserved in the form of a book if the testicular cross sections are mounted on paper-thin sheets. Overall, our technique offers a novel approach to preserving male haploid germ cells.
In general, male gametes are stored in liquid nitrogen (–196°C), which requires space and constant replacement of liquid nitrogen for maintaining preservation. Alternative preservation techniques without liquid nitrogen are also reported, such as freezing male gametes as testicular tissue [2] or the cryopreservation of sperm suspension [9], which is similar to the preservation of cultured cells. If the space required for preservation is compared between our technique and these methods, our technique is approximately 30- and 4-fold more space efficient than the former and latter techniques, respectively. In the former method, cryotubes (2-ml volume) containing male reproductive tissues are preserved within the freezing container. One freezing container (approximate volume 650 cm3) can hold between seven and ten 2-ml cryotubes. For the preservation of male gametes as mouse genetic resources, multiple samples should be preserved for future distribution or backup. If we assume 10 samples are needed to preserve one mouse line, one freezing container could preserve one mouse line; therefore, this method requires space of approximately 650 cm3 per line. In the latter technique, sperm suspension is stored in 1.5- or 2-ml cryotubes and preserved in a cryobox. One cryobox (approximate volume 850 cm3) can hold eighty to one hundred 1.5- or 2-ml cryotubes, and thus 8–10 mouse lines can be preserved in one cryobox, since 85–100 cm3 is required for preserving one mouse line (85–100 cm3 per line). If frozen testicular sections were to be preserved in book form as suggested in the Supplemental Figure (available online at www.biolreprod.org), 60 frozen sections can be mounted on one page, which allows enough samples for one mouse line to be preserved on one page, as a single testicular section yielded a sufficient number of gametes for ICSI (Table 1). Since the book we used (approximate volume 850 cm3) contained 40 pages, 40 mouse lines can be preserved in one book (i.e., 21 cm3 per line). Thus, our technique described here will be space efficient for the preservation of male gametes.
However, it remains unclear whether testicular spermatozoa from frozen sections are stable over an extended period of time. A recent study demonstrated that the functional capability of male haploid germ cells is retained after freezing male reproductive organs, such as the testis or epididymis, for more than 10 yr without cryoprotection [2]. Thus, it may be possible to preserve testicular spermatozoa for a much longer period even in the frozen testicular sections. However, the functional capability of testicular spermatozoa may have been affected by the duration of preservation with our technique. A loss of function was observed in the testicular spermatozoa obtained from the 10-µm sections that had been preserved for 3 mo (Table 3) and could potentially also occur in preparations from 25-µm sections. The technique may, therefore, require some improvements to enable long-term preservation. One potential improvement is to avoid exposing the tissue sections to air, which may cause damage via oxidation or desiccation of the testicular spermatozoa during preservation. Air exposure may have been responsible for damaging the testicular spermatozoa from the 10-µm sections. Thus, sealing the sections, as suggested in the Supplemental Figure (available online at www.biolreprod.org) where the sections were covered with laminate film, may increase the success rate of the technique. Alternatively, it may help to store frozen testicular sections at much lower temperatures, such as –80°C or –196°C (liquid nitrogen). Further experimentation is required to clarify these issues.
Another advantage of using testicular sections is that testicular spermatozoa within the seminiferous tubules can be identified via microscopy before performing micro-insemination (Fig. 1, A–C). This is much easier than attempting to identify them in testicular cell suspensions and is effective even in cases of severe male sterility. If serial cross sections of testicular tissue were mounted on two separate glass slides, it would be possible to use one section to confirm the presence of testicular spermatozoa, and then use the second slide for micro-insemination. More directly, it may be possible to combine this technique with laser microdissection [12, 13] and specifically to collect functional testicular spermatozoa from infertile male patients in a clinical setting.
ACKNOWLEDGMENTS
We are grateful to Dr. Masaru Okabe for kindly providing EGFP transgenic mice. We also thank the Laboratory for Animal Resources and Genetic Engineering for housing the mice.
FOOTNOTES
1Supported by grants for Scientific Research in Priority Areas and the Project for the Realization of Regenerative Medicine (research field: technical development of stem cell manipulation) to T.W. by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 
Correspondence: 2Hiroshi Ohta, Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. FAX: 81 78 306 3095; e-mail: ohta{at}cdb.riken.jp
Received: 12 September 2007.
First decision: 8 November 2007.
Accepted: 4 January 2008.
REFERENCES
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