Amount of maternal factor within the male pronucleus affects the reprogramming and developmental efficiency of androgenetic embryos

Uniparental embryos have uniparental genomes and are very useful models for studying parental specific gene expression or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram the nuclei of sperm after fertilization to guarantee embryonic development by maternal derived reprogramming factors, which accumulate during oogenesis. However, importance of maternal materials in the efficiency of reprogramming the pronucleus of androgenetic embryos has not been ascertained.Results Androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS) in our experiments. Compared with the androgenetic embryos constructed artificially by DS, those constructed by PT, which derived from two zygotes, contained more maternal material (like Tet3 and H3.3). This study confirmed the better developmental potential of PT embryos, with higher blastocyst rates, the stronger expression of pluripotent genes, the lower expression of apoptotic genes, and superior blastocyst quality.Conclusions

[10] is a substitute protamine in the sperm nucleosome that facilitates paternal genome reprogramming and maintains the decondensation state of chromatin. It is an important reprogramming-related factor for activating various pluripotent genes. Knocking out H3.3 in oocytes impairs the formation of the paternal pronucleus, DNA replication, and rDNA transcription [11]. After fertilization, the oxidation of 5-methylcytosine (5mC) mediated by Tet3 (ten-eleven translocation DNA dioxygenase 3) facilitates DNA demethylation and activation of the paternal genome [12,13].
The developmental rate of androgenetic embryos constructed by DS (double sperm injection) to form enucleated oocytes is quite low, with just 20% of embryos developing to blastocysts [14]. In comparison, androgenetic embryos derived from the PT (pronucleus transfer) of two zygotes exhibit higher developmental rates to blastocysts (55%) [4]. Liu et al. [15] found that the reprogramming capacity of the parental pronuclei varies, with crucial reprogramming factors being primarily located in paternal pronuclei.
Based on existing studies, in our study, we chose H3.3 and Tet3 as the detection indicators. we hypothesized that PT androgenetic embryos contain more maternal factors (such as H3. 3 and Tet3) that are required for reprogramming compared to DS androgenetic embryos, resulting in much higher developmental rate and better quality PT embryos. This study will advances our understanding of the effect of maternal factors on the activation of sperm-derived paternal genome.

Results
Differences in developmental efficiency between two types of androgenetic embryos For DS embryos, two sperm heads were injected into the cytoplasm of one oocyte, from which the spindle-chromosome complex was removed, after artificial activation. As a result, one integrated pronucleus (rather than two individual pronuclei) developed in the DS embryo (Fig. 1A). However, in a PT embryo derived from two zygotes, one maternal pronucleus became depleted, and one paternal pronucleus from the other zygote was translocated to the zona pellucida with an unbroken plasma membrane to ensure successful electrofusion ( Figure. 1B).
Firstly, we examined the developmental rates of PT and DS preimplantation embryos in vitro. The fertilized embryos derived from ICSI (normal control) had the highest blastocyst rate (68.9%). For the androgenetic embryos, PT embryos had significantly higher 4-cell and blastocyst rates (86.4% and 50.0%) compared to DS embryos (72.0% and 7.6%) ( Table   2). Thus, androgenetic embryos had lower developmental rates compared to ICSI embryos; however, PT embryos were more similar to ICSI embryos.
To compare the quality of androgenetic blastocysts, we first observed morphology of two types of androgenetic blastocysts at 108 h post fertilization. Expanded blastocyst cavities or hatched blastocysts were detected in the PT blastocyst. In comparison, the extension of DS blastocysts was not obvious (Fig. 2). In addition, we examined the number of cells in the inner cell mass (ICM) and trophoblast cells (TE) of androgenetic blastocysts using immunofluorescence. ICSI blastocysts were treated as the normal control. The average total number of cells and the ICM and TE ratios in PT blastocysts were much lower compared to those in ICSI blastocysts; however, the ratio of ICM to TE between PT and ICSI was comparative (0.42 vs 0.45) (Table 3). Moreover, the number of TE cells in PT and ICSI blastocysts far exceeded those in DS blastocysts (Table 3). Thus, the developmental efficiency of PT androgenetic embryos was better than that of DS embryos.

Expression of H3.3 and Tet3 in pronucleus of androgenetic embryos
To confirm whether more reprogramming related maternal factors accumulated in the paternal pronucleus of PT embryos, we examined H3.3 protein content in PT and DS embryos at 10 h post fertilization by western blotting. The paternal pronuclei of PT embryos contained the highest H3.3 levels compared to the DS group (Fig. 3). H3.3 levels in the maternal pronucleus transfer (MT) group showed that H3.3 accumulates in the paternal pronucleus, rather than the maternal pronucleus.
The expression level of the other maternal factor, Tet3, was also examined by immunofluorescence. Tet3 expression was similar to that obtained for H3.3 (Fig. 3). The PT group had the highest florescence intensity, while the MT group had the lowest (Fig. 4).
Thus, reprogramming related maternal factors were mainly located in the paternal pronucleus.

Demethylation level in two kinds of androgenetic embryos
To investigate the degree of demethylation of androgenetic embryos, we examined 5mC and 5hmC(5-hydroxymethylcytosin) levels in PT and DS embryos at 10 h post fertilization.
MT embryos served as the control. The fluorescence intensity of 5hmC in PT embryos significantly increased, while the intensity of 5mC in PT noticeably decreased, when compared with DS embryos. Moreover, there was no obvious discrepancy to signal intensity between MT and DS embryos. Thus, the extent of epigenetic reprogramming was higher in PT embryos, which might explain the better developmental efficiency (Fig. 5).
Expression of pluripotent and apoptotic genes in the two types of androgenetic embryos To verify the reprogramming efficiency and apoptosis in androgenetic embryos, PT and DS embryos were collected at 78, 96, 108 h post fertilization as morula, early blastocysts, and late blastocyst, respectively. We used ICSI embryos as the control. Next, we examined the expression of pluripotent markers Oct4, Nanog, Cdx2 and Gata4, and the apoptotic marker Casp3 through qPCR. In the DS group, there was a significant decline in the expression of Oct4, Nanog, Cdx2, and Gata4 in the androgenetic morulae (Fig. 6A).
However, there was no obvious difference in the PT group compared to the ICSI group; however, Nanog mRNA decreased and Casp3 mRNA increased. At the early blastocyst stage, the levels of all pluripotent markers (except Oct4) were much lower in the DS group. In comparison, only Oct4 and Nanog were abnormally expressed in the PT group ( Fig. 6B). For late blastocysts, the expression of Oct4 and Nanog notably increased in the DS group. Furthermore, the expression of Cdx2 and Gata4 was still lower in the DS group compared to the ICSI group. However, only Oct4 expression was slightly lower, while other pluripotent genes showed no obvious changes (Fig. 6C). Regardless of stage (i.e., morula stage, early and late blastocyst), DS embryos had the highest Casp3 expression levels, which might drive their low developmental efficiency. Thus, the expression of pluripotent and apoptotic genes was aberrant in DS embryos, while the genes of PT embryos were more similar to those of ICSI embryos.

Discussion
This study demonstrated that the pronuclei of PT embryos contain more maternal material and more efficient reprogramming compared with DS embryos, leading to significantly higher developmental efficiency.Furthermore, we confirmed that maternal materials are vital for reprogramming based on comparisons of two types of androgenetic embryos.
After fertilization, maternal materials accumulated in the cytoplasm of oocytes, contributing to the process of pronucleus formation by entering the pronucleus.
Furthermore, in one DS embryo, two sets of paternal genomes shared maternal materials in a single enucleated oocyte. To generate a PT embryo, two paternal pronuclei (which were reprogrammed by adequate maternal materials in a single zygote, respectively) were placed in the same zygote in which the maternal pronucleus had been eliminated. After fertilization, maternal reprogramming-related materials preferentially entrance into the sperm-derived paternal nucleus, as previously demonstrated by Liu et al. [15].Therefore, we speculated that the maternal material that accumulates during oogenesis in an oocyte is not sufficient to reprogram two sets of paternal genomes. PT embryos clearly had superior blastocyst rates compared to DS embryos, confirming that maternal material is required to reprogram zygotes.
Moreover, previous studies have confirmed that maternal factors have diverse roles using mice gene-modified models. These factors are involved in several aspects of early embryonic development, including the degradation of maternal mRNA, chromatin remodeling, and epigenetic modification. H3.3 and Tet3 are key maternal materials that are involved in the reprogramming and demethylation of the paternal genome, respectively. By examining H3.3 and Tet3 content in androgenetic embryo pronuclei, we showed that DS embryos had lower H3.3 and Tet3 protein levels. Epigenetic reprogramming (such as DNA demethylation) was closely associated with the regulation of gene expression during preimplantation development. Active and rapid DNA demethylation was documented in the paternal pronucleus of the zygote. In comparison, replicationdependent passive DNA demethylation in the maternal pronucleus took place. 5hmC was the intermediate state of demethylation, which was oxidized from 5mC, and serves as a key indicator for demethylation. In addition, 5hmC and Tet3 have significant roles in reprogramming after fertilization [11]. In the current study, 5hmC levels were lower in the DS group, similar to that of Tet3, indicating insufficient demethylation in DS embryos. This phenomenon might give rise to the much lower development efficiency directly compared with PT in this study. Previous studies of mice also indicated that the developmental rate of PT androgenetic embryos is significantly higher than that of DS embryos [4,14].
Incidentally, parthenogenetic embryos had extremely high preimplantation developmental rates, probably because of the low methylation level in maternal pronuclei, which easily reached reprogramming destinations.
The extent of reprogramming influences the activation of the embryonic genome and embryonic development directly and, thus, the expression of pluripotent genes. Four pluripotent markers (Oct4, Nanog, Cdx2, and Gata4) were selected for this study. Oct4 and Nanog are involved in regulating the gene expression of embryonic stem cells, and impacts pluripotency and the self-renewal capacity of cells [16]. The transcription of Oct4 begins at the 4-cell stage in embryos, at which point expression peaks in the inner cell mass [17]. In comparison, the morula stage is the earliest phase at which transcription is detected for Nanog [18]. Cdx2 is a critical transcriptional factor that is involved in inducing differentiation in blastocyst trophectoderm cells, which are mainly expressed in the outer cells of the morula and the nuclei of trophectoderm cells [19]. Gata4 is the key factor associated with the formation of endocardium and the regulation of cardiogenesis, which is expressed in the primitive endoderm [20]. The similar expression of the pluripotent genes of PT embryos with those of ICSI embryos in our study might explain the higher developmental efficiency of PT embryos. However, the abnormal expression of Oct4 and Nanog might be caused by delayed development and gene expression; in other words, the gene expression of DS embryos is in a growing phase, whereas those in PT embryos has already reached a high and steady level.
This study focused on the preimplantation development of mice embryos. However, the postimplantation development of these two types of androgenetic embryo also needs to be elucidated. This study showed that the number of TE cells in PT embryos exceed those in DS embryos, allowing us to proceed to this next step. Furthermore, mechanisms, other than the imprinting defects identified by Li et al. [2] might contribute to androgenetic reproduction, and need to be identified. In future, knockdown H3.3 or Tet3 in androgenetic embryos could help confirm the function of these two maternal materials. How other maternal factors function in the reprogramming process should also be explored. On the other side, this study did not focus on the regulation of imprinted regions, next step we will attempt to produce androgenetic mice by controlling the expression of maternal or paternal imprinted genes.
In the future, this research advances our understanding of the regulation and function of maternal factors on zygotic genome activation, providing a basis for developing new methods of examining reproduction.

Conclusion
We revealed that the pronuclei in PT embryos possessed more maternal materials and more sufficient reprogramming, consequently obtained significantly higher developmental efficiency.Further, maternal materials were proved to be vital for reprogramming through comparing two kinds of androgenetic embryos.

Oocyte and zygote collection and spermatozoa preparation
Female B6D2F1 mice were superovulated by injecting 5IU pregnant mare serum gonadotropin (PMSG, NSH, China), followed by 5IU human chorionic gonadotropin (hCG, NSH, China) 48 h later. To obtain zygotes, female mice were mated with male B6D2F1 mice after hCG injection. Approximately 14 h and 17 h after HCG injection, the oocytes and zygotes were collected from the fallopian tubes. Mice were anesthetized by 2,2,2tribromoethanol (125mg/kg), and then euthanized by carbon dioxide suffocation after collection. Corpse were frozen for uniform disposal. Cumulus-free oocytes with appropriately sized perivitelline space and homogeneous ooplasm were selected, while only zygotes with two distinct parental pronuclei were selected. They were placed in 20 μldroplets of CZBG, and covered by sterile mineral oil (Fisher, O121-20). Then, the oocytes were placed in an incubator set to 37℃ and 5% CO 2 until use. Spermatozoa were collected from the cauda epididymis of male B6D2F1 mice at 8-10 weeks in age. The spermatozoa were then placed in CZB-HEPES medium until injection.
Generation of ICSI, PT, and DS embryos ICSI, implemented by a piezo-driven unit following a previously described method [21], was treated as control; however, our experiments were performed in HEPES-CZB containing 5 μg/ml cytochalasin B (Sigma, C6762) at room temperature. The ICSIgenerated embryos were washed at least three times, and were placed in KSOM in an incubator set to 37℃ and 5% CO 2 , after the sperm head was injected into the oocyte. PT androgenetic embryos were constructed following a previously described method [22]; however, our study used different electroporation parameters. We used two direct-currentpulses of 1.8KV/cm for 10 μsec each to complete cytoplast-karyoplasm fusion. In addition, DS androgenetic embryos were constructed following previously described methods [14,23].     White arrows indicate blastocysts from the double sperm injection).