Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

STUDY QUESTION: How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER: Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY: The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION: This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE: A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA: All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION: Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

The model of "genetic compartments": a new insight into reproductive genetics.

Currently, we are witnessing revolutionary advances in the analytical power of genetic tools. An enormous quantity of data can now be obtained from samples; however, the translation of genetic findings to the general status of individuals, or their offspring, should be done with caution. This is especially relevant in the reproductive context, where the concepts of "transmission" and "inheritability" of a trait are crucial. Against this background, we offer new insight based on a systemic view of genetic constitution in the compartmentalized organism, that is, the human body. This model considers the coexistence of "different" genomes in the same individual and the repercussion of this on reproductive efficacy and offspring. Herein, we review the major differences between somatic, germinal, embryonic, and fetal/placental genomes and their contribution to the next generation and its reproductive efficacy. The major novelty of our approach is the holistic interaction between microsystems within a macrosystem (i.e., the reproductive system). This panoramic model allows us to sketch the future implications of genetic results in function of the origin (compartment) of the sample: peripheral blood or other somatic tissues, gametes, zygotes, preimplantation embryos, fetus, or placenta. We believe this perspective can be of great use in the context of reproductive genetic counseling.

Do immature and mature sibling oocytes recovered from stimulated cycles have the same reproductive potential?

RESEARCH QUESTION: How can laboratory and clinical outcomes of spontaneously, early maturing germinal-vesicle oocytes and sibling in-vivo-matured (metaphase II [MII]) oocytes be quantified and compared? DESIGN: A prospective, non-randomized intra-cohort study of oocytes from women aged 38 years or younger, with six or fewer MII oocytes and four or more germinal vesicles retrieved. No indication was identified for genetic tests or oocyte or embryo cryopreservation. The study was carried out at IVIRMA-Valencia. Early maturing germinal vesicles were selected for reproductive purposes. In vitro- and in-vivo MII oocytes were fertilized. After time-lapse culture, hatching blastocysts from germinal vesicles were biopsied for aneuploidy screening and vitrified. Laboratory and clinical outcomes were compared according to oocyte origin. RESULTS: Almost 70% of germinal vesicles had matured early and spontaneously, and had comparable in vitro-outcomes and morphokinetics to sibling in vivo-matured oocytes. Fifty per cent of biopsied blastocysts were euploid. Germinal-vesicle rescue increased the number of MII oocytes per cycle to 3.9, finally adding one extra-blastocyst per cycle. A live birth confirmed the feasibility of this approach. Further data, however, are needed to quantify its real contribution to standard intracytoplasmic sperm injection cycles. Nevertheless, 40% of patients obtained either an immediate advantage (reduction of cancellation rate) or long-term benefit (availability of extra blastocysts of attempts). CONCLUSIONS: Germinal-vesicle rescue can be considered as a complementary approach when folliculometry (expected) and number of MII (observed) are unequal.

New techniques on embryo manipulation.

For many years, experience has been accumulated on embryo and gamete manipulation in livestock animals. The present work is a review of these techniques and their possible application in human embryology in specific cases. It is possible to manipulate gametes at different levels, producing paternal or maternal haploid embryos (hemicloning), using different techniques including nuclear transfer. At the embryonic stage, considering practical, ethical and legal issues, techniques will be reviewed that include cloning and embryo splitting at the cleavage stage, morula, or blastocyst stage.

Influence of sequence duration and number of electrical pulses upon rabbit oocyte activation and parthenogenetic in vitro development.

Electroactivation of in vivo mature young rabbit oocytes was investigated here. The effects of four or eight electrical pulse treatment over 90, 150 or 270 min upon oocyte activation frequency and type, and even upon their subsequent in vitro development, were studied. The lowest activation frequency was observed after applying four-pulses over 90 min (54%). However, extending four-pulse treatment duration over 150 or 270 min induced more oocytes to activate (from 84% to 100%), as did the eight pulsing treatments (from 91% to 97%). With eight pulses, extending treatment duration improved the normal activation rates (from 47% to 76%; P<0.05). Nevertheless, the haploid morulae and blastocyst rates decreased significantly with extended eight pulsing treatment duration (morulae: from 94% to 41% and blastocysts: from 31% to 0%).

Vitrification of in vitro cultured rabbit morulae.

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO-S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG-20DMSO+S) and two-step (20EG-20DMSO-S) groups (0-6% versus 36-50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham's F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG-20DMSO+S) group in s-PBS (35-45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO-S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG-20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG-20DMSO-S), regardless of percentage of FCS in the PBS-based media (81% versus 41-45%; P<0.05). In conclusion, we propose either the one step (20EG-20DMSO+S) or two steps (20EG-20DMSO-S) prepared in PBS plus 20% serum for use in future works.

Reconstruction of the heteroparental diploid condition in rabbit zygotes by nuclear transfer.

Studies on genomic imprinting showed that parental genomes have complementary roles during embryogenesis, are both essential and need to be synchronized in their embryonic stage for successful development to term. To our knowledge, these studies have not been performed in species other than mice. We studied the in vitro and in vivo development of reconstructed zygotes by combining female haploid nuclear donors and androgenetic hemizygous recipients. Haploid donor embryos at the 8- or 32-cell stage were obtained from electroactivated young rabbit ova (eight pulses maximum, consisting of 0 6 kVcm(-1) for 60 microsec each, 38 min apart) which were further cultured for 24 h or 32 h. Couplets formed by both the haploid male hemizygous recipients and haploid female donor cells were electrofused (2.2 kVcm(-1) for 60 microsec duration each, 30 min apart) and their nuclear configuration determined 122 of those fused (43%: 122/286) were diploid. Reconstructed diploid zygotes developed in vitro up to the compacted morula, blastocyst and hatched stages (1/8-nuclei x 50%, 18% and 9% vs. 1/32-nuclei: 47%, 25% and 19%; P > 0.05), respectively. In embryo transfer assays, both 1/32-reconstructed zygotes and control, non-manipulated zygotes were transferred to synchronized does Four live reconstructed fetuses (4/49: 8 1% survival rate) and five in regression stage (9/49: 18% implantation rate) were observed on Day 21 post-ovulation, whereas from control zygotes, 11 fetuses were alive (11/53 21% fetal survival rate) and 2 degenerated (13/53 x 24 5% implantation rate). Similar results were obtained from a final experiment, in which development was allowed to progress to term. Six live rabbit pups derived front experimentally reconstructed zygotes (11%; 6/54) and three fetuses in regression stage were obtained; values slightly lower than those derived from non-manipulated and transferred control zygotes (18% 9/50, live born rate).

Electroactivation of rabbit oocytes in an hypotonic pulsing medium and parthenogenetic in vitro development without cytochalasin B-diploidizing pretreatment.

We investigated the electroactivation frequencies, type of activation and in vitro development of rabbit oocytes. In Experiment 1, activation (8 pulses, 12 min apart, 60 microsec, 0.6 kVcm(-1)) was performed by altering osmolarity (190 vs. 320 mOsm kg(-1)) and Ca++ concentration (10, 60 or 100 microM) in mannitol pulsing media. More oocytes were activated in hypotonic pulsing medium, regardless of Ca++ concentration (96 to 100%). Both haploid and diploid parthenogenetic embryos developed to compacted morulae (57 to 92% and 63 to 100%, respectively) regardless of the activation treatment; however, the blastocyst rates were more variable (0 to 74% and 0 to 73%, respectively). In Experiment 2, the effects of pulse duration (30 or 60 microsec) and number of applied pulses (4, 8 or 12) under hypotonic conditions were studied. Activation frequencies were the lowest after four 30 microsec-pulses (58 vs. 88 to 100%, respectively). A lower haploid frequency was obtained when more than four 30 or 60 microsec-pulses were applied (from 67 to 25% and 83 to 0%, respectively). Increasing the number of 60-microsec pulses improved the compacted morula rate of haploid and diploid oocytes (47 to 83% and 57 to 96%, respectively). Overall, haploid development to morulae and blastocysts was lower than diploid development to these stages (69 and 25% vs. 74 and 44%, respectively).

Vitrification and rapid-freezing of cumulus cells from rabbits and pigs.

To use adult somatic cloning technology in animal breeding, this technology should be complemented with nuclear donor cell cryopreservation. Two different conventional nonequilibrium methods (vitrification, V: 3.58M EG and 2.82M DMSO in PBS plus 20% FCS and rapid-freezing, RF: 0.25M sucrose, 2.25M EG and 2.25M DMSO in PBS plus 20% FCS) were assayed here on different cumuli types from rabbits and pigs. In rabbits, the cell proliferation capability of fully disaggregated cumuli was not affected by cryopreservation procedures (V: 100% and RF: 82%). Vitrified samples from partially or non-disaggregated cumuli showed the lowest proliferation frequencies (4% and 0%, respectively). In pigs, differences in cell proliferation capability were only observed between vitrified non-disagreggated cumuli and vitrified or rapid-frozen, fully disaggregated cumuli (72% vs 100% or 100%, respectively; P < 0.05). In both species, in vitro cultured sub-confluent samples were able to survive to a second cryopreservation treatment, maintaining the cell proliferation capability in nearly 50% of thawed samples. In conclusion, before cryopreservation, disaggregation of cumulus cells from both species into small clusters of cells improved their viability after thawing. These results allow us to efficiently, easily and rapidly store rabbit and pig cumulus cells, from selected high-merit females.

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death.

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.

Vitrification and rapid freezing of rabbit fetal tissues and skin samples from rabbits and pigs.

Vitrification (3.58 M EG and 2.82 M DMSO in PBS with 20% FCS) and rapid-freezing (0.25 M sucrose, 2.25 M EG, and 2.25 M DMSO in PBS with 20% FCS) procedures were assayed to cryopreserve rabbit tissue samples from 12-day fetuses, and skin samples from live born pups and adult rabbits. These methods were also assayed to cryopreserve pig skin samples obtained from abattoir animals. The ability of rabbit tissue samples to attach and colonize the substratum by cell proliferation was not affected by the assayed cryopreservation procedures, regardless of specimen age. In porcines, sample attachment and cell proliferation capability of primary cultures were not affected by applied cryopreservation procedures. Almost all primary cultures from cryopreserved skin samples reached confluency (from 92 to 100%). Results reported here allow us to establish in both species, rabbit and pig, a cryobank of skin samples from adult specimens classified as outliers for longevity (in rabbits) and prolificacy (in pigs).

Use of a variable electrical pulsing sequence in rabbit oocyte activation.

Variability in oocyte activation sensitivity to electrical stimuli was shown in two types of oocytes (i.e., oocytes with a whole first polar body: w-PB 1 and those with a fragmented PB 1: f-PB 1), of a similar post-ovulatory age. In order to initiate the normal activation display (i.e., extrusion of the second polar body), the w-PB 1 oocytes required, on average, 3.6 +/- 0.2 pulses and the f-PB 1 oocytes 2.9 +/- 0.1 pulses (p = 0.18). From both experimental series carried out in this work, the average haploid activation rates were 68% and 70% for w-PB1 and f-PB1 oocytes, respectively. Oocyte type did not affect the haploid embryo developmental ability observed at 24 h of culture (8-cell stage: 33-35% in Series 1 and 23-26% in Series 2), nor at 32 h of culture (16-cell stage: 77-93% and morula stage: 34-41%; Series 2). Therefore, in further experiments, the f-PB 1 oocytes may also be used as potential forerunners of haploid embryos, almost up to the morula stage.

Comparison of the effect of two different handling media on rabbit zygote developmental ability.

Despite the attention paid to culture media, the relevance of the handling medium at egg recovery/transfer is frequently overlooked. In the present work, we compare the effect of two different handling media (PBS and HEPES-buffered Ham F10, both supplemented with 20% (v/v) FCS), upon in vitro and in vivo developmental ability of in vivo fertilised rabbit zygotes. Zygotes recovered in HEPES-buffered medium (permanence 1 h as maximum) and subsequently cultured in vitro developed more efficiently to the compacted morula (100%) and blastocyst stage (92%) than those recovered in PBS (83% and 76%, respectively, P < 0.05). Zygotes recovered in such media were then further bilaterally transferred to recipient does following a brief in vitro culture period (for 4 hours). At caesarean section (day 28 of pregnancy), significant differences were observed in both the percentage of pregnant uterine horns (PBS: 60% vs. HEPES-buffered Ham F10: 100%) and live birth rates (PBS: 14% vs. HEPES-buffered Ham F10: 34%). Thus when early rabbit zygotes must be handled, even for short incubation periods, the medium is not innocuous.