Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

doi:10.21203/rs.3.rs-1760037/v1

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Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

https://doi.org/10.21203/rs.3.rs-1760037/v1

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Recently, mice have been produced from Freeze-drying (FD) somatic cells by nuclear transfer, but the success rate was very low compared to FD spermatozoa. Since spermatozoa, unlike somatic cells, are haploid cell and their nuclei were hardened by protamine, it is unclear which is responsible for tolerance to FD treatment. Here, we attempt to produce offspring from FD spermatid, a haploid sperm progenitor cell, but nuclei were not yet replaced by protamine as same as somatic cells. We developed the method to collect FD spermatids from testicular suspension. When FD spermatids were injected into oocytes, healthy offspring were obtained, despite the significantly reduced success rate compared to FD spermatozoa. Offspring were also obtained from FD spermatids derived from immature male mice, which have not yet produced spermatozoa. These results indicate that nuclear protaminization, rather than haploid nuclei, is the key process responsible for tolerance to FD treatment.

spermatid

spermatozoa

freeze-dry

protamine

ROSI

The preservation of genetic resources is important to prepare for unknown diseases are spread, or environmental changes occur, such as global warming ¹. In mammals, spermatozoa or oocytes/embryos are cryopreserved in liquid nitrogen ^{2 3}. However, the use of liquid nitrogen has several drawbacks, such as high maintenance cost, and during a disaster, LN2 supply may be stopped, and all genetic resources will be destroyed. To solve this problem, we developed a freeze-drying (FD) technique for mouse spermatozoa ⁴. Although all spermatozoa died after the FD treatment, their DNA remained intact, and those sperm acquire strong tolerance against temperature or radiation after FD treatment ⁵. Healthy offspring were obtained from FD sperm stored in a desk drawer of laboratory ⁶ or even on the International Space Station ^7,8, as well as from transported FD sperm on the postcard ⁹. Thus, freeze drying could be the best way to preserve genetic resources for a long period in a safe, low-cost, and location-independent manner ^10,11.

However, it is difficult to collect spermatozoa from infertile animals, such as aged animals, animals with their reproductive organs accidentally damaged, or immature males. In such cases, it may be possible to collect spermatids from the testes, in which haploid male germ cells are found, before the completion of spermiogenesis. Importantly, even though spermatids cannot fertilize oocytes by themselves, when spermatids are artificially injected into oocytes, healthy offspring can be obtained ^12,13. Although the use of this method results in lower birth rates than when mature spermatozoa are used, it suggests that spermatids are important components for the preservation of the genetic material of infertile or immature male animals ¹⁴.

However, until recently, the only type of FD cells from which offspring have been successfully produced thus far are spermatozoa. Oocytes or embryos are thought to be difficult to freeze dry because of the large amount of cytoplasm that they contain ¹⁵. On the other hand, although somatic cells or spermatids have a similar size with spermatozoa, it has not been possible long time to produce offspring after subjecting them to an FD treatment ^16–19. Very recently, we succeeded to generate cloned offspring from FD somatic cell nuclei, but the success rate was very low compared to FD spermatozoa ²⁰. This could potentially be attributed to structural differences; the DNA of almost all cell nuclei consists of histones, while in spermatozoa, histones are replaced by protamine during spermiogenesis, and their nuclei become compact and hardened ^21,22. Therefore, it is thought that haploid nucleus or the compaction of the nucleus by protamine is essential to overcome the damage caused by an FD treatment ^23,24.

The aim of this study was to clarify whether haploid nucleus or nuclear hardening by protamine are important to protect the nucleus from the effects of an FD treatment. To this end, we attempted to generate offspring from FD spermatids. Although the success rate was very low as same as FD somatic cell, the results obtained in this study may provide a viable method of preserving the genetic material of infertile animals in a safe manner, even if power and liquid nitrogen supplies are interrupted during a disaster.

Round spermatids can be easily collected from fresh testicular suspensions by a glass pipette using a micromanipulator, depending on the chromocenter in the nucleus (Fig. 1A), which is the unique and specific structure of round spermatids ²⁵. After freeze-drying and rehydrating the testicular suspension (Fig. 1B, C), the membranes of many cells were destroyed, and a lot of small debris remained. Although spermatozoa and elongated spermatids could be easily eliminated by their specific morphology (Fig. 1B), the chromocenter of round spermatids were not readily observable by light microscopy.

When the nuclei of these cells were stained with Hoechst 33342, the chromocenter of FD spermatids was readily observable, as would be the case in fresh spermatids. Based on this observation, we noticed that the FD spermatid had a relatively small, clear cytoplasm and relatively round morphology (Fig 1B). Using such morphological markers, we tried to collect FD spermatids without fluorescent observation, as in offspring production the toxicity/phototoxicity of the DNA-binding dye and exposure of the cells to UV radiation must be avoided as much as possible. However, in the first experiment, the selected cells, presumed to be round spermatids, were observed using Hoechst 33342 and UV light in order to check if we could select perfectly round spermatids in this manner. Of the cells observed, 73% were classified as round spermatids owing to the chromocenter in the nucleus (Fig. 1D, E, Table S1). Some cells had no nuclei (18%). The remaining cells had flat and round nuclei (9%), suggesting that these cells were not single round spermatids, but elongated spermatids.

To confirm that the selected cells were indeed round spermatids, they were injected into oocytes (Fig. 1F), and the male pronuclei (Fig. 1G) were examined using the round spermatid-specific epigenetic marker, H3K9me3 (Fig. 1H)²⁶. When fresh spermatozoa or elongated spermatids were injected into oocytes and the male pronuclei of zygotes were immune-stained using H3K9me3 antibody, none of them stained. However, when fresh round spermatids were injected, most male prenucleolar ring (93–96%) were clearly stained with H3K9me3 (Fig. 1H, Table S2). Interestingly, 4–7% of zygotes did not show a positive signal for H3K9me3, even though we injected round spermatids that were confirmed to have chromocenter. This may suggest that some round spermatids were epigenetically not normal, and could be one of the reasons for the lower success rate of offspring production from round spermatids than from spermatozoa ¹³. Then, we examined the male pronucleus of zygotes fertilized with presumed FD spermatids with or without confirmation of the chromocenter before injection into oocytes. The results showed that 86–89% of zygotes exhibited positive signals for H3K9me3 in the male prenucleolar ring, irrespective of their chromocenter status before injection (Fig. 1H, I), which suggests that most of the selected FD cells were round spermatids, even some of those collected without Hoechst staining.

We also confirmed the possibility that we might have accidentally injected FD elongated spermatids rather than round spermatids. Spermatozoa and elongated spermatids already contain sperm factors such as PLCζ that activate the oocytes after fertilization/injection ²⁷^,²⁸. As sperm factors are not lost owing to an FD treatment ⁴, if we injected FD elongated spermatids instead of round spermatids, the oocytes should be activated by sperm factors; however, if we injected round spermatids, the oocytes should not be activated because there are no sperm factors in round spermatids. In the latter case, the nucleus of the injected spermatid will be condensed and form a spindle, which will show an MII-like structure inside oocytes. As a result, when spermatozoa or elongated spermatids were injected, most oocytes were activated in either the fresh or FD treatments. On the other hand, when presumed FD spermatids were injected into oocytes, most oocytes failed to activate, and the nuclei of the injected round spermatids formed MII-like structures inside oocytes (Fig. 1J), which suggests that these cells were actually spermatids. Although approximately 13% of oocytes were activated by the injection of presumed FD spermatids, a similar rate of oocytes was activated when fresh spermatids were injected (13%) (Fig. 1K, Table S3). This activation may cause the co-injection of sperm factors derived from lysed spermatozoa ²⁹.

Next, we examined whether the cell nucleus injected into oocytes had histones. If the cells we chose were spermatozoa or elongated spermatids, in which histones were replaced with protamine, we would not be able to detect histones in the nuclei of oocytes immediately after injection. However, if the cells we chose were spermatids, we should be able to detect histones in the nuclei. When somatic cells (cumulus cells) were injected into oocytes and immune-stained using pan-histone, all nuclei derived from cumulus cells and oocytes with MII spindles showed positive signals for pan-histones. On the other hand, when fresh spermatozoa or elongated spermatids were injected into oocytes and immediately stained, although oocytes with MII spindles showed positive signals, 0% or 12%, respectively, of injected nuclei showed positive signals (Fig. 1L, M, Table S4). Then, fresh or FD spermatids were injected into the oocytes and immediately stained. In this experiment, all injected nuclei into oocytes showed positive signals, irrespective of the fresh or FD conditions.

Finally, we attempted to produce offspring from FD spermatids. When FD spermatids preserved at -80 °C for 1–6 months were injected into oocytes, 76% of zygotes developed to 2-cell stage embryos the next day. The 2-cell embryos showed a relatively normal morphology (Fig. 1N). After transferring the embryos into recipient females, we successfully obtained 10 offspring (3% of the embryo transfers were successful) (Table 1; Fig. 1O). Surprisingly, healthy offspring were obtained even from FD spermatids preserved for one year at -80 °C (4%). The offspring success rate obtained from this method was significantly lower than that from the use of fresh round spermatids (14%).

In addition, we tried to produce offspring from FD spermatids derived from 3–4-week-old immature male mice. These mice started spermiogenesis and only had round spermatids but did not produce spermatozoa in the testes. Therefore, if we could generate offspring from this experiment, there would be strong evidence that the offspring were actually produced from FD spermatids. When FD spermatids derived from immature males were injected into oocytes, three offspring (1%) were obtained. Although the success rate was lower than when fresh spermatids (10%) were used, these results clearly demonstrate that these offspring were generated from FD spermatids. The body and placenta weight of all offspring were within the normal range (Table S5).

In the preliminary study, we attempted to select round spermatids by fluorescence-activated cell sorting (FACS) before implementing the FD treatment. However, we were unable to completely separate the elongated spermatids and round spermatids, as suggested previously (Fig. S1) ^21,30. Then, we manually identified FD round spermatids using four different methods, including morphology ¹³, epigenetic markers ^21,26, oocyte activation potential ^29,31, and the existence of pan-histones ²¹. In addition, we obtained offspring from zygotes fertilized with FD spermatids derived from immature males, which did not produce spermatozoa in the testis ^32,33. Although these experiments offered indirect evidence, the results strongly demonstrated that we used FD round spermatids rather than elongated spermatids. In a few cases, we may have selected unknown cells instead of FD spermatids. However, based on their morphology, these were either somatic cells or secondary spermatocytes and never developed to full term after being injected into oocytes. Unfortunately, the success rate of offspring production from embryos fertilized with FD spermatids was only 3–4%. It is known that the success rate of offspring production decreases significantly when FD spermatozoa are used compared to the use of intact spermatozoa (average birth rate from fresh spermatozoa were around 60%, whereas from FD spermatozoa were around 20%) ^6,34. It is also known that the success rate of offspring production decreases largely when fresh spermatids are used compared to when fresh spermatozoa are used (average birth rate from fresh spermatozoa were around 60%, whereas from fresh round spermatid were around 20%) ^12,13. Thus, the synergistic negative effect of both methods (FD treatment and the use of spermatids) would have greatly reduced the success rate of FD spermatids.

Even though, the production rate of offspring from the FD spermatid in this study was far too low. Since spermatids are haploid cells as same as spermatozoa, it is likely that the lack of protaminization of the nucleus caused to the lower success rate. Recently, we succeeded to produce cloned offspring from FD somatic cells, but the success rate was only 0.02%. Considering those result and this study, it seems clear that although it is not essential for the nucleus to be protaminization since the offspring were born, protaminization of nuclei is an important process to make the tolerant to FD treatment.

The use of FD sperm is a better solution than traditional cryopreservation for the permanent conservation of the genetic resources of mammalian species, as it facilitates preservation at room temperature. However, spermatozoa can only be collected from fertile males. In this study, we demonstrated that it is possible to preserve genetic material from infertile or juvenile males using FD spermatids. Although the production rate of offspring was largely decreased, as neither nuclear hardening nor protaminization are essential for FD treatments, in the future it may be possible to preserve oocytes or embryos under FD conditions. We have to develop a more stable and reliable method for FD spermatids, which may help develop a method for FD oocyte/embryos. The preservation of genetic resources at room temperature has to commence before a serious disaster affects Earth.

Animals. BDF1 (C57BL/6N ´ DBA/2; 4 weeks and 8–10 weeks of age) and ICR mice (8–10 weeks of age) were obtained from SLC Inc. (Hamamatsu, Japan). Surrogate pseudo-pregnant ICR females, which were used as embryo recipients, were mated with vasectomized ICR males, the sterility of which had been demonstrated previously. On the day of the experiment or after finishing all experiments, the mice were euthanized by CO₂ inhalation or cervical dislocation and used for experiments. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi (reference number: A29-24), which followed the ARRIVE guidelines.

Preparation of FD spermatids and FD spermatozoa

To collect spermatids, the seminiferous tubules of the testes were minced using sharp scissors, as described previously ¹³; the only difference was that the cells were suspended in HEPES-buffered CZB medium ¹². In order to collect spermatozoa, we first collected epididymides and cut their ducts using sharp scissors, as described previously ²⁸. Aliquots (100 mL) of the testicular suspension or sperm suspension were dispensed into glass ampoules. The ampoules were kept at 4 °C for 3 h, -30 °C for 3 h, and -80 °C for several days until use. For the FD treatment, frozen ampoules were placed in liquid nitrogen for a few minutes and then freeze-dried using an FDU-2200 freeze dryer (EYELA, Tokyo, Japan). The cork of the freeze-dryer was opened for at least 3 h until all the samples were completely dry. After drying, the ampoules were sealed by melting the ampoule necks using a gas burner under vacuum, as described previously ⁵.

Oocyte preparation. Superovulation was induced in female mice via the injection of 5 IU of equine chorionic gonadotropin, followed by 5 IU of human chorionic gonadotropin (hCG) 48 h later. Cumulus–oocyte complexes (COCs) were collected from the oviducts of females 14–16 h later and moved to a Falcon dish containing an HEPES-CZB medium ³⁵. To disperse the cumulus cells, COCs were transferred to a 50-mL droplet of HEPES-CZB medium containing 0.1% bovine testicular hyaluronidase for 3 min. Cumulus-free oocytes were washed twice and moved to 20-mL droplets of a CZB medium ³⁶ for culture.

Rehydration of FD spermatids or FD sperm and observation

Just before the round spermatid injection (ROSI) or intracytoplasmic spermatid injection (ICSI), the neck of the glass ampoule was broken, and 100 µL of sterile distilled water was immediately added and mixed using a pipette. For the observation of FD spermatids, 2 µL of suspension was mixed with Hoechst 33342 and placed in the manipulation chamber. Using microcapillaries, FD spermatids were collected and moved to different drops in the same chamber. Then, FD spermatids were observed using UV light to determine whether they contained a chromocenter.

ROSI and ICSI

One to two µL of the rehydrated suspension was moved directly to the injection chamber. ROSI ³⁷^,³⁸ and ICSI were performed as previously described ²⁸^,³⁵. The oocytes that survived the ROSI/ICSI procedures were incubated in an CZB medium at 37 °C under 5% CO₂ in humidified air. Pronuclear formation was checked 6 h after ROSI/ICSI.

Oocyte activation and embryo transfer. For the ROSI experiments, oocytes were activated 15–20 min after the FD spermatid injection using 5 mM SrCl₂ in Ca²⁺-free CZB medium for 1 h, followed by a culture in CZB medium until the embryo was used. The next day, fertilized embryos that had reached the 2-cell stage were transferred into the oviducts of pseudo-pregnant ICR female mice 0.5 days post coitum (dpc), which was when these mice had been mated with a vasectomized male the night before embryo transfer. On the day of embryo transfer, the recipients were anesthetized by an intraperitoneal injection (medetomidine, midazolam, and butorphanol). Between 5 and 8 embryos were transferred into each uterine horn, and an equal amount of atipamezole was injected. At 19.5 dpc, the offspring were delivered by caesarean section and randomly selected offspring were transferred to the cage of a foster mother who had delivered pups naturally. Three weeks later, the offspring were mated and their fertility was examined.

Immunostaining of zygotes. Ten hours after the ICSI/ROSI, the zona pellucidae of surviving oocytes were removed using an acetic Tyrode solution, and the naked oocytes were fixed for 30 min at 25 °C in 4% (w/v) paraformaldehyde. The fixed oocytes were washed three times in PBS–polyvinyl alcohol (0.1 mg/mL PVA; Sigma-Aldrich, St. Louis, MO, USA) for 10 min and stored overnight at 4°C in PBS supplemented with 1% (w/v) bovine serum albumin (BSA/PBS, Sigma-Aldrich) and 0.1% (v/v) Triton X-100 (Nacalai Tesque, Inc., Kyoto, Japan). The following procedure was previously described in ⁶. The primary antibodies used were an anti-histone H3 (trimethyl K9) mouse monoclonal antibody (1:500; Abcam, Cambridge, UK) or an anti-pan-histone mouse monoclonal antibody (1:500: Merck, Darmstadt, Germany). The secondary antibodies used were Alexa Fluor 488-labeled goat anti-mouse IgG (1:500; Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568-labeled goat anti-rabbit IgG (1:500 dilution; Molecular Probes). DNA was stained with 4¢6-diamidino-2-phenylindole (DAPI; 2 mg/mL; Molecular Probes). The brightness of the whole male pronucleus was measured using ImageJ and was then subtracted from the brightness of the zygote cytoplasm.

Statistical analysis. Birth rates were evaluated using chi-squared tests. The statistical significance of any differences between the variables was set at p < 0.05.

Acknowledgments

We thank Mr. Nakamura and Miss Yamaguchi for assistance in preparing this manuscript. This work was partially funded by the Japan Society for the Promotion of Science to D. I. (15KT0086), and M.O. (17K08134); Naito Foundation and Takahashi-Sangyo Foundation to S.W.; Asada Science Foundation, Watanabe Foundation, and the Canon Foundation (M20-0008) to T.W.

Author contributions

S.W. and T.W. designed research and wrote the manuscript. S.W., D.I., M.O. and T.W. performed research. All authors reviewed the manuscript.

Declaration of competing interests

The authors declare that they have no competing interests.

Data Availability

All data generated or analyzed during this study are included in this published article and are available from the corresponding author on reasonable request.

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Table 1 is available in the Supplemental Files section.

No competing interests reported.

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Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

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