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Hum. Reprod. Advance Access originally published online on April 11, 2007
Human Reproduction 2007 22(6):1612-1616; doi:10.1093/humrep/dem064
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Generation of progeny via ICSI following enrichment of elongated spermatids from mouse testis by flow-cytometric cell sorting

Hiroshi Ohta1, Yuko Sakaide and Teruhiko Wakayama

Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe 650-0047, Japan

1 Correspondence address: Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. Tel: +81-78-306-3049; Fax: +81-78-306-3095; E-mail: ohta{at}cdb.riken.go.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Although ICSI is a useful technique, low elongated spermatid numbers frequently causes technical difficulties, especially in the case of azoospermic patients. Enrichment of elongated spermatids from the testis prior to ICSI may solve this problem.

METHODS: To determine whether elongated spermatids had a characteristic phenotype suitable for purification, testicular cells prepared from 25-day-old mice (from spermatogonia to round spermatids) and adult mice (from spermatogonia to elongated spermatids) were compared by flow cytometry. After flow-cytometric cell sorting (FCS) based on their side (SSC) and forward scatter (FSC), purity of the elongated spermatids in the fractionated population was microscopically examined, and functional ability of purified elongated spermatids was assessed by ICSI.

RESULTS: Elongated spermatids in testicular cells showed characteristic SSC and FSC phenotypes. In the purified population, ~70–80% of the cells were morphologically determined as elongated spermatids, in contrast to only 10% before sorting. Using ICSI, purified elongated spermatids supported full-term development similar to that of unsorted elongated spermatids. Furthermore, we succeeded in enriching the elongated spermatids from the infertile testis model by ~10-fold.

CONCLUSIONS: Elongated spermatids with normal developmental ability can be efficiently purified by FCS based on SSC and FSC characteristics.

Key words: flow-cytometric cell sorting/elongated spermatids/ICSI/male infertility/sperm


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Intracytoplasmic sperm injection (ICSI), first reported in 1992 (Palermo et al., 1992Go), is a powerful method for overcoming male factor infertility and is offered to many couples who were unable to conceive through in vitro fertilization (IVF). Soon after its development, ICSI was applied in combination with testicular sperm extraction (TESE) to treat obstructive and non-obstructive azoospermia (Devroey et al., 1995Go; Silber et al., 1995aGo,b,c, 1996; Mansour et al., 1996Go). Although ICSI combined with TESE is now becoming the first-line treatment of non-obstructive azoospermia, a low sperm retrieval rate often results in treatment failure. Recently, the new procedure of microsurgical operation for TESE (microdissection TESE) was reported in which individual seminiferous tubules are retrieved under the operating microscope (Schlegel and Li, 1998Go; Schlegel, 1999Go; Amer et al., 2000Go; Silber, 2000Go). However, even using microdissection TESE, sperm retrieval rate is still only around 60% (Schlegel, 1999Go; Tsujimura et al., 2002Go). Thus, the retrieval of elongated spermatids by testicular biopsy has its limitations. An enrichment procedure for obtaining elongated spermatids from testicular cells would increase the retrieval rate and might effectively improve ICSI outcome.

Flow-cytometric cell sorting (FCS) or fluorescence-activated cell sorting (FACS) allow purification of the target cell population based on several characteristics, including cell size [forward scatter (FSC)] and complexity [side scatter (SSC)], or surface antigens in FACS. FACS has recently been applied to the isolation of spermatogonial stem cells using stem cell specific antibodies (Shinohara et al., 1999Go, 2000Go; Kanatsu-Shinohara et al., 2004Go). The sperm differentiated from purified spermatogonia were proven to have the ability for normal development following ICSI (Fujita et al., 2005Go). Although purification by FACS is useful for isolating specific germ cell types, it remains unclear whether elongated spermatids can be directly purified from testicular cells. In addition, the toxicity of the sorting procedure per se (e.g. laser exposure, electric charge and high pressure; Suh et al., 2005Go) still requires examination, as the genome integrity of testicular sperm is more sensitive to exogenous damage than fully differentiated epididymal sperm (Bedford and Calvin, 1974Go).

In this study, we examined whether elongated spermatids can be purified from testis by FCS based on FSC and SSC characteristics, and the functional ability of sorted sperm was determined by generating progeny via ICSI.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Mice
C57BL/6, BDF1 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 testicular cells and FCS
Testicular cells were prepared from C57BL/6, BDF1 and ICR mice. Briefly, the testis was decapsulated, then minced with fine scissors in phosphate-buffered saline. The resultant tissue was transferred to a 15 ml tube. Two to four testes were used per experiment [average weight of one testis was 0.11 ± 0.005 g (mean ± SD), n = 4]. Supernatant was transferred to a FACS tube, gently agitated by a micropipette, and used as testicular cell suspension, which was kept on ice and filtered (30 µm pore size) before cell sorting. By this procedure, we could obtain 1.1 ± 0.2 x 108 cells (mean ± SD, n = 4) per testis. FACSaria (BD Biosciences, San Jose, CA, USA) was used to analyse testicular cell population. The types of laser used were two solid-state lasers (488 nm blue laser, 13 mW output; 407 nm violet laser, 10 mW output) and a HeNe laser (633 nm red laser, 11 mM output). The sorted cells were exposed to these three types of laser beam during our sorting procedure, although FSC and SSC signals can be obtained with only the blue laser. The sheath fluid used was BD FACSFlowTM (BD Biosciences) and was sorted with a 70 µm nozzle. The operating pressure under this condition was 70 psi and the event number was adjusted to 2000–5000/s by changing the flow rate (1–11, corresponding to ~10–120 µl/min). To examine the characteristics of elongated spermatids in testicular cells, comparison was made between testicular cell populations prepared from 25-day-old mice (from spermatogonia to round spermatids) and adult mice (from spermatogonia to elongated spermatids). The characteristics of elongated spermatids were determined by adjusting the voltages for SSC and FSC (voltages of SSC and FSC were determined as 456 and 187, respectively). The collected fractions were kept in FACSFlowTM at 4°C overnight until ICSI. For ICSI, sorted cells were centrifuged (2300g, 2 min, 4°C) and resuspended in ~20 µl of CZB medium (Chatot et al., 1990Go). After ICSI, elongated spermatid content in each fractioned population was examined microscopically. In each population, over 200 cells were assessed.

Intracytoplasmic sperm injection
BDF1 females were induced to superovulate by consecutive injections of eCG (5 IU) and hCG (5 IU) 48 h apart. At 14 h post hCG injection, cumulus–oocyte complexes (COCs) were collected from oviducts. Oocytes were freed from the cumulus cells by adding 0.1% bovine testicular hyaluronidase (ICN Biochemicals, Costa Mesa, CA, USA) to COC-containing medium. After cumulus cells had dissociated from the oocytes, the oocytes were rinsed twice with CZB medium. Approximately 2 µl of sperm suspension was mixed with a drop of Hepes-CZB medium containing 12% (w/v) polyvinylpyrrolidone (PVP; Mr 360 000; Wako Pure Chemical Industries, Tokyo, Japan). The sperm head was separated from the tail by applying a few piezo-pulses to the sperm neck region, then injected into the oocyte as described by Kimura and Yanagimachi (1995)Go. Exploded oocytes caused by injection damage were deemed dead. Surviving oocytes that successfully received ICSI were incubated in CZB medium at 37°C under 5% CO2 in air. When embryos reached the 2-cell stage, they were transferred to oviducts of 0.5-days post coitum pseudopregnant ICR females.

Statistical analysis
Means and SD were evaluated using Student's t-tests, and P < 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Characterization of elongated spermatids in testicular cells by flow cytometry analysis
To enrich the elongated spermatids from testicular cells, testicular cells prepared from 25-day-old and adult mice were examined by flow cytometry. As shown in Figure 1A and B, the majority of haploid germ cells in 25-day-old mice were round spermatids (Fig. 1A), whereas the adult testis contained all differentiated germ cells including elongated spermatids (Fig. 1B). This suggests that the comparison of testicular cells between the two age groups might allow the detection of elongated spermatids in whole testicular cells if the elongated spermatids have characteristic phenotype. Flow cytometry analysis revealed that the adult testicular cells exhibited the characteristic SSC and FSC phenotypes (Fig. 1C–E), suggesting the possibility that elongated spermatids can be isolated from testicular cells by cell sorting.


Figure 1
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  		Figure 1: Comparison of testicular cells between 25-day-old mice and adult mice by flow cytometry. (A, B) Testicular cross sections of 25-day-old (A) and adult (B) mice. Elongated spermatids were not found in (A), whereas all the differentiated germ cells including elongated spermatids were found in (B). (C, D) Flow cytometry analysis of testicular cells prepared from 25-day-old (C) and adult (D) mice. (E) Distribution profile of testicular cells characteristically found in adult testicular cells. Distribution profile of 25-day-old testicular cells (C) was subtracted from that of adult testicular cells (D). Characteristic SSC and FSC phenotypes were found in the adult mouse testis (red dashed circles in E). Scale bars, (A, B) 100 µm.

 
Enrichment of elongated spermatids from testicular cells by FCS
We attempted to collect elongated spermatids from adult testis. Testicular cells were fractioned into three populations (P1–P3 in Fig. 2A) including predicted elongated spermatid population (P1 and P2 in Fig. 2A), and the elongated spermatid content of each population was microscopically examined. In this study, we examined testicular cells from three different mouse strains, C57BL/6 (inbred strain), BDF1 (hybrid strain) and ICR (outbred strain). In the testicular cell suspension before sorting, ~10% of the cells were identified as elongated spermatids in each strain (Table 1; Fig. 2B). Distribution patterns of testicular cells based on SSC and FSC characteristics were also similar among strains (data not shown). FCS experiments revealed that ~3% and 10% of the cells were elongated spermatids in the P2 and P3 fractions, respectively (Table 1; Fig. 2D and E). In contrast, ~70–80% of cells were morphologically identified as elongated spermatids in the P1 fraction (Table 1; Fig. 2C), demonstrating that elongated spermatids can be enriched efficiently (7–8 fold) from testicular cells by FCS based on the SSC and FSC phenotypes.


Figure 2
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  		Figure 2: Fractionation of adult testicular cells by FCS. (A) Testicular cells were fractioned into P1, P2 and P3 by FCS based on SSC and FSC characteristics. (B–E) Light microscopic photographs of cells in each population are shown. (B) Before sorting, (C) P1, (D) P2 and (E) P3. Scale bars (B–E) 50 µm.

 

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  		Table 1: Quantitative analysis of elongated spermatids in each population

 
Next, we roughly estimated the recovery rate of elongated spermatids in the P1 fraction during our FCS procedure. To this end, 1 x 106 of testicular cells were sorted and a count in the P1 fraction (nearly the same as the cell number) was made. As ~10% of cells were elongated spermatids in testicular cells before sorting (Table 1), we roughly estimated that 1 x 106 of testicular cells would contain 1 x 105 of elongated spermatids. In total, independent experiments were conducted four times and the mean count in the P1 fraction was 5.9 ± 1.9 x 104 (mean ± SD). As ~80% of the cells were elongated spermatids in the P1 fraction (Table 1), the number of spermatids collected in the P1 fraction was estimated as 4.7 ± 1.5 x 104. Thus, approximately half (4.7 ± 1.5 x 104/1 x 105) of the elongated spermatids present before sorting were collected in the P1 fraction.

Functional analysis of purified elongated spermatids by ICSI
To examine the functional ability of purified elongated spermatids, they were injected into unfertilized oocytes, and the developmental rate was compared with the unsorted elongated spermatids. Pronuclear formation and cleavage rate of oocytes that received purified elongated spermatids were similar to those of oocytes that received unsorted elongated spermatids (Table 2), indicating that purified elongated spermatids maintain activation activity for unfertilized oocytes even after FCS. When the 2-cell stage embryos were transferred to the oviducts of pseudopregnant females, normal progeny could be delivered from both embryos with no difference between purified or unsorted elongated spermatids (Table 2). These results clearly demonstrate that FCS does not affect the developmental ability of elongated spermatids.


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  		Table 2: Generation of progeny via ICSI using elongated spermatids from P1 and unsorted testicular cells

 
Enrichment of elongated spermatids from infertile testis model
Although we succeeded in enriching the elongated spermatids from normal testis 7-fold by FCS (Table 1), it remains unclear whether this technique is also applicable to the testis in the cases of male infertility. Thus, we attempted to enrich the elongated spermatids in the infertile testis model. Adult testicular cells were mixed with prepubertal testicular cells (25–26 days old) at a rate of 1:10. In this model, there should be ~1% elongated spermatids, as estimated from the percentage of elongated spermatids before sorting (~10%) given in Table 1. FCS was carried out under the same conditions and the P1 fraction was collected. Microscopic observation revealed that ~10% of cells were elongated spermatids in the P1 fraction (Table 1), indicating the elongated spermatids were enriched 10-fold. Thus, the enrichment of elongated spermatids by FCS was demonstrated to be effective even in the infertile testis model.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we determined that elongated spermatids show the characteristic SSC and FSC patterns in testicular cells and that purified elongated spermatids have the ability to produce progeny when used for ICSI. Fertilization rates as well as in vitro and in vivo development of embryos derived from purified elongated spermatids were nearly the same as those of unsorted elongated spermatids, indicating that the sorting procedure did not affect the functional ability of elongated spermatids. Furthermore, we succeeded in enriching elongated spermatids by 10-fold in the infertile testis model, suggesting the possible application of our method to human infertility treatment. Our study thus clearly demonstrates that enrichment of elongated spermatids is possible using cell sorting based on SSC and FSC characteristics.

FACS allowed us to purify the desired cell population based on several characteristics, including cell size (FSC), complexity (SSC) and surface antigens. Recent progress in the purification of germ cells revealed that spermatogonia purified by FACS can differentiate into sperm following transplantation and that differentiated sperm from the spermatogonia have the ability to develop to full-term (Fujita et al., 2005Go). FCS is also applicable for selection of male or female sperm for progeny production via IVF (for review see Garner, 2006Go). These results indicate that neither the FACS nor FCS procedure causes developmental damage to spermatogonia or fully differentiated epididymal sperm. However, it remains unclear whether the sorting procedure damages elongated spermatids, which are considered to be more sensitive to exogenous damage than epididymal sperm (Bedford and Calvin, 1974Go). Our results demonstrate that the FCS procedure per se does not affect the developmental potential of testicular sperm.

Although in our study the sorted cells were kept overnight in sheath fluid (FACSFlowTM; BD Biosciences), an alternative solution is also available. We could not obtain the precise concentration of each compound contained in FACSFlowTM, because this information is confidential (BD Biosciences). However, the company indicated that FACSFlowTM contains NaCl, Na2-EDTA, KCL, K2HPO4, NaH2PO4 and preservatives, and the pH is adjusted to 7.4. A buffer similar to this sheath fluid, i.e. nucleus isolation medium (NIM), is also useful in maintaining sperm function for ICSI (Kuretake et al., 1996Go). Thus, we think that other buffers such as NIM could be used for sperm preservation after FCS.

In the present study, we found that elongated spermatids enriched from the whole testicular cells were FSClow and SSCmiddle to high (Fig. 2; Table 1). Since FSC and SSC represent cell size and complexity, respectively, FSClow and SSCmiddle to high mean small cell size. Consistent with this notion, the sperm head is relatively small in comparison with other cell types. Our results therefore suggest that elongated spermatids can be enriched as they are relatively small cells in comparison with other cells in the testis. If this is true, the method described here may be applicable to other species including humans, as sperm head size is generally smaller than other cells in mammalian species (Cummins and Woodall, 1985Go). Furthermore, cell sorting based on FSC and SSC does not require any staining such as Hoechst or immunofluorescence, indicating purification of elongated spermatids based on cell size by FCS is safer than DNA-amount-based germ cell purification (Bastos et al., 2005Go) or cell sorting based on antigen labeling with fluorescent conjugates.

Even in the infertile testis model, we succeeded in enriching elongated spermatids by ~10-fold (Table 1). This enrichment procedure may also be applicable in the testis after transplantation experiments. For example, sperm retrieval is difficult in the testis after transplantation of testicular tissue or spermatogonial stem cells, especially when the testicular donor does not have genetic markers such as green fluorescent protein (GFP). We previously failed to find elongated spermatids for ICSI from grafted testicular tissue, even though the grafts contained elongated spermatids as identified histologically (Ohta and Wakayama, 2004Go). Thus, the enrichment procedure of elongated spermatids is also applicable for progeny generation from other testicular sources.

Although our results indicate that elongated spermatids can be enriched by FCS, it appears that not all elongated spermatids present in the testis were collected in our experiments. In fact, the recovery rate of elongated spermatids in the P1 fraction was estimated to be ~50%. Consistent with this estimation, elongated spermatids were also found in the P2 and P3 fractions, albeit at a relatively low frequency compared with the P1 fraction (Table 1). This loss of elongated spermatids may result in failure of sperm retrieval from azoospermic patients or testis post transplantation. In such cases, further improvement of current methods or the development of new strategies might be necessary. To increase the recovery rate, it might be effective to change the sorting mode, such as the ‘yield mode’ in the case of FACSaria (all the cell sorting experiments in this study were carried out using the ‘purity mode’). Alternatively, immunological isolation using antibody that recognizes cell surface antigen of elongated spermatids is also another possible method for collecting elongated spermatids more efficiently, though the toxicity of immunolabeling with, for example, immunoglobulin or fluorochrome, should be examined.

It must be noted that our technique does not increase sperm function per se but concentrates elongated spermatids. Therefore, if our technique is applied to human testicular biopsy, the technique should be used only in the case of azoospermic patients whose sperm could not be found by routine methods. In addition, if the starting materials are too small, it may cause further loss of elongated spermatids, although FCS theoretically can collect a small number of cells. For example, possible causes of loss include collected elongated spermatids drying out during FCS or the attachment of collected elongated spermatids to the tube wall during FCS or centrifugation before ICSI. These problems may be mitigated by the addition of a small amount of buffer to the FCS tube before sorting, or the use of a siliconized tube for the latter. Thus, more careful handling and pilot experiments examining the amount of starting material are required before our technique is available for clinical use.

In the present study, we demonstrated that functional elongated spermatids can be enriched using FCS based on FSC and SSC characteristics. Although we were able to enrich mouse elongated spermatids here, further study should determine whether the FSC and SSC phenotype of elongated spermatids used in our procedure can be successfully applied to other species. There might be a possibility that the characteristic features of mouse elongated spermatids, such as asymmetrical hooked head, may make them more easily distinguishable from other testicular cells. Further research would serve to improve the reliability of our method for use in difficult cases and in other species.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank the Laboratory for Animal Resources and Genetic Engineering for housing the mice. This work was supported by grants for Scientific Research in Priority Areas (15080211) and the Project for the Realization of Regenerative Medicine (research field: technical development of stem cell manipulation) to T.W. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 24, 2006; resubmitted on February 14, 2007; accepted on February 20, 2007.


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