Identification of a round, freeze-dried sperm using a Chromocenter

Round spermatids can be easily collected from fresh testis suspensions using a glass pipette and a microprocessor, depending on the chromosome center in the nucleus (Fig. 1a), which is the unique and specific structure of round spermatids28. After FD treatment and rehydration of the testis suspension (Fig. 1b,c), the membranes of many cells were destroyed, and many small debris remained. Although elongated spermatids and spermatids could be easily eliminated by their specific shapes (Fig. 1b), the chromosomal center of round spermatids was not easy to observe by light microscopy.

shape 1
shape 1

Sperm detection after FD treatment. (aFresh round sperm. (BFD Sperm Moisturizer. The middle section shows high magnification and the right section shows the fluorescent observation of the nuclei. The arrow shows a chromocentre. The arrowhead shows an elongated sperm. (c) Glass ampoules from FD mice spermatozoa. (DrAnd the e(Set of FD sperms as morphologically evaluated)Dr), fluorescent sperm monitoring (FD)e). The arrow shows cells without nuclei. (F) FD sperm were injected into fresh oocytes.

When cell nuclei were stained with Hoechst 33342, the chromosomal center of FD sperm was easy to observe, as it was in fresh sperm. Based on this observation, we observed that the FD sperm had a relatively small and clear cytoplasm and a relatively round morphology (Fig. 1b). Using these morphological markers, we attempted to collect FD sperm without fluorescent observation; As in the production of offspring, toxicity/phototoxicity of DNA-binding dye and exposure of cells to UV rays should be avoided completely. However, in the first experiment, the selected cells, presumably round sperms, were observed using Hoechst 33342 and UV light to check if we could select perfectly round sperms in this way. Of the cells observed, 73% of the round spermatocytes were classified due to the center of the chromosomes in the nucleus (Fig. 1d,e, Table 1). Some cells do not have nuclei (18%). The remaining cells had flat and round nuclei (9%), indicating that these cells were not a single round spermatid but an elongated spermatid.

Table 1. Chromocentre detection of cells collected by morphological markers using Hoechst staining.

Detection of the sperm-specific round marker H3K9me3 in the zygote after ROSI

To confirm that the selected cells were a round spermatid, they were injected into oocytes (Fig. 1F), and the male nucleus (Fig. 2a) was examined using the round epigenetic marker H3K9me3 (Fig. 2b).29. When fresh sperm or long sperm were injected into the oocytes, and the male nuclei of the inoculated animals were immunostained with the H3K9me3 antibody, none were stained. However, when fresh round spermatids were injected, most of the male prenuclear rings (93–96%) were clearly stained for H3K9me3 (Fig. 2b, Table 2). Interestingly, 4-7% of the zygotes did not show a positive signal for H3K9me3, although we injected fresh round spermatids that were confirmed to have a chromosyr center prior to injection. This indicates that some of the round sperms were epigenetically abnormal and could be one of the reasons for the lower success rate of offspring production from round sperm compared to spermatozoa.14. We next examined the male nuclei of zygotes inoculated with putative FD spermatids with or without chromocentre confirmation prior to injection into the oocytes. The results showed that 86–89% of the vaccinators showed positive signals for H3K9me3 in the prenuclear male ring, regardless of the status of the chromosomal center before injection (Fig. without Hoechst staining.

Figure 2
Figure 2

Confirm the presence of sperm after oocyte injection and production of offspring from FD sperm. (a) Six hours after injection and activation, most of the oocytes formed male and female nuclei. (B) Immunoprecipitation of sperm derived from fresh sperm (top left), fresh sperm (lower left), FD sperm (top right), and FD sperm (lower right) using anti-H3K9me3 antibody. The arrow shows the prenuclear annulus. (c) Proportion of H3K9me3-positive heterozygotes. Parthenogenetically activated zygote; Sp: sperm. El: elongated spermatid. RS: Round sperm. The chromosin center of some round spermatozoa was observed before collection using Hoechst and UV-staining. (Dr) FD sperm were injected into the oocytes without artificial stimulation. Six hours after injection, these oocytes contained pseudo-MII spindles derived from FD spermatid nuclei (bright top image; bottom, DNA under UV light). (e) Percentage of activated eggs after germ cell injection. (F) Immunostaining of FD (top) or FD spermatid (bottom) sperm-injected oocytes. The left panel showed DNA staining by DAPI, and the right panel showed histone staining using an anti-histone antibody. The arrow shows the male nucleus. (J) Percentage of fertilized eggs positive for pan-histone (PH). (h) Two-celled embryos derived from an FD sperm. (I) Offspring were obtained from FD sperm.

Table 2 Detection of H3K9me3 in the male nucleus of the zygote enriched with different germ cell stages with or without chromocentre confirmation before ROSI.

Possibility of activating eggs for injected cells

We also confirmed whether we had mistakenly injected an elongated FD sperm instead of a round spermatid. Sperm and elongated spermatids already contain spermatogenic factors, such as PLCgwhich activates the eggs after fertilization / injection30,31. Because FD treatment does not cause loss of sperm factors4, the oocytes must be activated by the spermicidal agents if an elongated FD sperm is injected instead of the round spermicides. However, if the round sperms are injected, the eggs should not be activated because the sperm factors are absent in the round spermatids. In the latter case, the nuclei of the injected spermicide will condense and form a spindle, which will show a MII-like structure inside the oocytes. As a consequence, when sperm or long sperm were injected, most of the oocytes were activated in either the fresh or FD treatments (Table 3). However, when putative FD sperm were injected into the oocytes, most of the oocytes failed to activate, and the injected round sperm nuclei formed MII-like structures within the oocytes (Fig. 2D), indicating that these cells were in fact spermatogenic. Although approximately 13% of oocytes were activated by injection of putative FD sperm, a similar rate of oocytes were activated when fresh sperm (13%) were injected (Fig. 2E, Table 3). This activation may cause the co-injection of sperm factors derived from degraded sperm32.

Table 3 Possibility of oocyte activation of male germ cells injected at different stages with or without freeze-drying treatment.

Detection of histones in FD sperm-injected oocytes

Next, we examined whether the cell nuclei injected into the oocytes contained histones. If the cells of our choice were spermatids or elongated sperms in which the histones have been replaced by protamine, then the histones will not be detected in the nuclei of the eggs immediately after injection. However, if the cells of our choice are spermatogonia, then histones can be detected in the nuclei. When somatic cells (cumulus cells) were injected as controls into oocytes and immunostained with pan-histone, all nuclei derived from cumulus cells and oocytes with MII spindles showed positive pan-histone signals (Table 4). However, when fresh sperm or elongated sperm 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. G). Then, fresh or FD sperm were injected into the oocytes and stained immediately. In this experiment, all nuclei injected into oocytes showed positive signals, regardless of fresh or FD conditions.

Table 4 Pan-histone detection in the cell nuclei injected into oocytes.

Production of offspring from a fertilized egg fertilized with sperm FD

Finally, we attempted to produce offspring from FD sperm. When FD spermicides maintained at −80 °C for 1–6 months were injected into oocytes, 76% of the fertilized embryos developed into 2-cell embryos the next day. The two-celled embryos showed a relatively normal morphology (Fig. 2H). After embryo transfer into recipient females, ten offspring (3% of the transferred two-celled embryo) were successfully obtained (Fig. 2I, Table 5). Surprisingly, even healthy offspring were obtained from FD sperm kept for 1 year at −80 °C (4%). The success rate of the offspring obtained with this method was significantly lower than that obtained from using fresh round sperm (14%).

Table 5 Production of offspring from lyophilized spermicides.

We also attempted to produce offspring of FD sperm derived from 3- to 4-week-old immature male mice. These mice started to develop sperms and had only round sperms but no sperms in the testes. Therefore, if we can generate offspring from this experiment, there will be strong evidence that the offspring was indeed produced from FD sperm. When FD sperm derived from immature males were injected into the oocytes, three (1%) offspring were obtained. Although the success rate was lower than when using fresh sperm (10%), these results clearly demonstrate that these offspring were generated from FD sperm. Body and placental weights for all offspring were within the normal range.

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