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Development of golden hamster embryos effectively produced by injection of sperm heads sonicated in Tris‐HCl buffer with EGTA

Abstract Purpose To investigate the effects of sperm treatment medium—TCM199 or EGTA in Tris‐HCl buffer (TBS + EGTA)―for sonication of frozen‐thawed hamster spermatozoa in terms of sperm chromosome integrity and development of hamster oocytes injected with the sperm heads (ICSI). Methods Frozen‐thawed hamster spermatozoa were separated into heads and tails by sonication in TCM199 or TBS + EGTA. Sperm heads were injected into mouse oocytes to assess hamster sperm chromosomes. We further compared the development of hamster ICSI embryos produced by injecting sonicated sperm heads in TCM199 vs TBS + EGTA. Results Sperm chromosome integrity was greater following sonication of frozen‐thawed hamster spermatozoa in TBS + EGTA than in TCM199 (89.7% vs 69.0%). Embryonic development was improved following hamster oocyte injection with sperm heads sonicated in TBS + EGTA compared to in TCM199 (8‐cell: 84.1% vs 65.4%; morula: 78.4% vs 43.2%; blastocyst: 42.0% vs 17.3%). Gene expression of zygotic genome activation in 2‐cell embryos was significantly higher with TBS + EGTA than with TCM199. We transferred 43 morulae/blastocysts from the TBS + EGTA group to foster mothers, and 4 (9.3%) developed into live offspring. Conclusion These results showed that the rapid injection of hamster sperm heads separated by sonication in TBS + EGTA effectively produced more ICSI embryos during a short time.

reported that injection of oocytes with freeze-dried spermatozoa produced hamster embryos that could develop into live offspring after embryo transfer. However, only a low percentage of ICSI embryos exhibit in vitro and in vivo development.
Hamster oocytes are sensitive to short-wave visible light emitted from ordinary fluorescence light sources, such that oocytes injected with spermatozoa under fluorescence light never develop beyond the 2-cell stage. 4,[7][8][9] In hamster zygotes, fluorescent light reportedly increases reactive oxygen species (ROS). 9 Moreover, intracytoplasmic injection of fresh sperm heads with intact acrosomes dissolves the cytoplasm of hamster oocytes, leading to oocyte death. 4,10 Successful hamster ICSI requires the removal of acrosomes from spermatozoa. After injection of hamster or rabbit acrosome-intact sperm heads into mouse oocytes, extrusion of the second polar body is followed by degeneration of the mouse oocyte cytoplasm, and cleavage never occurs. 10 On the other hand, when a single acrosome-intact spermatozoon is injected during human and mouse ICSI, the released acrosome enzymes do not cause any serious problem. 11 Another complicating factor is that golden hamster oocytes show a high rate of spontaneous activation. [12][13][14] Therefore, in hamsters, ICSI should be performed as soon as possible after ovulated oocyte collection. Yamauchi et al 4 produced fertilized eggs by injecting acrosome-free sperm heads that were separated from frozen-thawed spermatozoa using a piezo pulse. However, compared to mouse spermatozoa, hamster spermatozoa show a relatively strong "neck" connection between the head and midpiece, such that it takes several minutes to detach sperm heads using a piezo pulse.
Sonication is an easy method for separating sperm heads from tails prior to ICSI. In early studies of hamster ICSI, Yanagida et al and Katayose et al used sperm heads detached by sonication. 15,16 They observed pronuclear formation following sperm head injection after heating or freeze-drying, but did not demonstrate the developmental competence of zygotes following sonicated sperm head injection.
In contrast, Kuretake et al 17 performed mouse ICSI and showed higher percentages of blastocysts and normal offspring development following intracytoplasmic injection of sperm heads that were sonicated in nuclear isolation medium (NIM; a K-rich and EDTA-containing medium). Tateno et al demonstrated that sonication per se is not deleterious to sperm chromosomes, but that sperm suspension medium is easily damaged by sonication and chromosomal abnormalities can be caused by activation of Ca 2+ -dependent DNase adjacent to the sperm chromosome. 18 The solution used to freeze-dry spermatozoa without chromosomal aberrations is also an attractive choice of solution for sonication. Suspension of mouse spermatozoa in a simple Tris-HClbuffered solution containing 50 mM EGTA and 50 mM NaCl enables maintenance of chromosome integrity during freeze-drying or freezing without cryoprotection. 19 Furthermore, Kusakabe et al 20 showed that prior incubation of mouse spermatozoa in Tris-HCl-buffered solution containing only 50 mM EGTA (without NaCl) increases the chromosomal normality after freeze-drying and that these spermatozoa can support embryo development. However, it is presently unclear whether this solution can reduce chromosomal aberrations in sperm heads separated by sonication.
In the present study, we aimed to examine how the solution used during frozen-thawing and sonication influenced hamster spermatozoa integrity, and the development of hamster zygotes produced by injecting sonicated sperm heads into oocytes. We also analyzed the expression of maternal effect genes (MEGs) and zygotic gene activation (ZGA) genes in the ICSI embryos.

| Chemicals and animals
Inorganic salts were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Nacalai Tesque Inc (Kyoto, Japan). Organic reagents were purchased from Sigma-Aldrich unless otherwise stated.
Golden hamsters (Mesocricetus auratus) with brown coats and black eyes were purchased from Japan SLC Inc (Shizuoka, Japan).
Mature females of 2-4 months of age were used for the collection of

| Oocyte collection
Golden hamster females were induced to superovulate via an im injection of 20 IU eCG (Asuka Pharmaceuticals, Tokyo, Japan) during the morning of the day of postestrus discharge. 21 At 56 hours after eCG injection, the hamsters received an im injection of 20 IU hCG (Asuka Pharmaceuticals, Tokyo, Japan). 22 At approximately 15 hours hCG injection, mature unfertilized oocytes were collected from oviducts and were freed from cumulus cells by a 1-minute treatment with 250 IU/mL hyaluronidase in M2 medium. 23 Next, the oocytes were rinsed with TCM199TE and stored in fresh medium. All experiments were performed in a dark room with a small table light, and red filters were used on the microscope light source, as previously reported. 4-6

| Sperm preparation
From mature male hamster (2-5 months of age), a dense sperm mass was collected from the cauda epididymis. A small drop of this sperm mass was placed in a 3.5-mL dish containing 5 mL of TCM199 supplemented with 0.1% bovine serum albumin (Sigma-Aldrich; A6003).
Spermatozoa were allowed to swim through this medium for 5 minutes at 37.5°C, and then 700 µL aliquots of the medium were dispensed into 1.5-mL centrifuge tubes. The collected sperm were washed by centrifugal separation and preserved with 50 mM EGTA in 100 mM Tris-HCl-buffered solution (TBS + EGTA). 20 Control sperm samples were stored in TCM199 supplemented with bovine serum albumin (1 mg/mL). As a negative control, we used TBS containing 2 mM CaCl 2 (TBS + Ca), which is the same concentration as in TCM199. Sperm samples were frozen at −196°C with liquid nitrogen.
Then, the frozen sperm were thawed in a water bath at 37°C and sonicated in the experimental or control medium using an ultra homogenizer (VP-52; TITEC, Japan) for 1 minute just prior to ICSI. After the spermatozoa were frozen-thawed and sonicated, they were immobilized, they lost their acrosomes, and the sperm heads and tails were separated.

| Chromosomal analysis of hamster spermatozoa by injection into mouse oocytes
To analyze the sperm chromosomes, we performed intracytoplasmic injection of hamster spermatozoa into mouse oocytes, following the method described by Kishikawa et al 24 B6D2F1 mouse females (CLEA Japan, Tokyo) at 8-12 weeks of age were each injected with 5 IU eCG followed by 5 IU hCG 48 later. Oocytes were collected from oviducts between 15 and 16 hours after hCG injection. They were freed from cumulus cells by a 1-minute treatment with 250 IU/ mL hyaluronidase in M2 medium. These oocytes were kept in Global Total medium (Astec, Fukuoka, Japan) at 37°C for 30 minute before sperm injection.

| Embryo transfer
About 78 hours after ICSI, we randomly selected 43 morulae and blastocysts for transferred into recipient albino females that had been naturally mated with albino males 3 days previously. We

| Statistical analysis
The chi-square test was used to compare fertilization and embryo development rates, and chromosome analysis results. Quantitative PCR data were compared using Student's t test. Differences between means were evaluated using GraphPad PRISM 5 (GraphPad Software, La Jolla, CA, USA). Differences were considered to be significant at P < 0.05.

| RE SULTS
3.1 | Chromosome analysis of hamster spermatozoa after sonication in TCM199 or TBS + EGTA Table 2 summarizes the results of chromosomal analysis of hamster spermatozoa following injection into mouse oocytes. The percentage of normal karyotypes was significantly higher in spermatozoa that were frozen-stored and sonicated in TBS + EGTA (89.7%) than in TCM199 (69.0%) or TBS + Ca (68.0%; P < 0.05). Chromosome-type breaks were the chromosome aberration most commonly observed after sonication ( Figure 1A,B).  TA B L E 2 Chromosome analysis of hamster spermatozoa that were frozenstored; separated by sonication in TCM199, TBS + EGTA, or TBS + Ca; and then injected into mouse oocytes Table 4 summarizes the result of embryo transfer. A total of 43 embryos derived from the injection of sperm heads sonicated in TBS + EGTA were transferred to recipients, of which 4 (9.3%) developed into live young. Figure 3 shows a recipient mother that gave birth to one pup (brown fur) that developed from the oocytes fertilized by injection of sonicated sperm heads in TBS + EGTA.

| D ISCUSS I ON
Our present results showed that TBS + EGTA was effective for cryopreservation and sonication of hamster spermatozoa, enabling the injection of sperm heads with a higher proportion of normal chromosomes ( Table 2). The percentages of 8-cell embryos, morulae, and blastocysts were significantly higher in TBS + EGTA than in TCM199 (Table 3). Furthermore, after transfer of 43 ICSI embryos, 4 developed into live offspring (Table 4). These results indicated that the To analyze hamster sperm chromosomes, we performed xenogeneic injection of hamster sperm heads separated by sonication into mouse oocytes. This method of interspecies sperm chromosome analysis using mouse oocytes is systematic, and the procedure is completely controlled. 18,24,25 The xenogeneic system makes it easy to identify the hamster sperm chromosomes. However, future studies should include homogeneous sperm chromosome analysis using hamster oocytes to determine the relationship between sperm chromosome normality and ICSI embryo development. It is important to be able to produce hamster ICSI embryos using sonicated sperm heads and to enhance the efficiency of sperm injection per unit time. The rapid ICSI system is necessary to reduce the time from oocyte collection to injection, since spontaneous oocyte activation is common in hamsters. To produce higher quality hamster ICSI embryos, we must improve the manipulation of sonicated sperm heads to enable smooth fertilization, optimize the culture medium for ICSI embryos, and regulate spontaneous oocyte activation.
In conclusion, here we found that the use of TBS + EGTA for sonication of hamster frozen-thawed spermatozoa reduced sperm chromosome aberrations. Upon injecting hamster oocytes with sonicated sperm heads having less chromosome aberrations, we found that the ZGA gene expression at the two-cell stage was relatively higher than with TCM199. Moreover, the ICSI zygotes produced using TBS + EGTA developed to morulae and blastocysts at a higher rate compared to with TCM199. Finally, ICSI embryos produced with TBS + EGTA successfully developed to live offspring after embryo transfer. Overall, these results suggest that rapid ICSI using sperm heads separated by sonication in TBS + EGTA leads to increased chromosome normality and effective production of live offspring.