Effects of multi-gradient equilibration during vitrification on oocyte survival and embryo development in mice.

Vitrification has been widely used for oocyte cryopreservation, but there is still a need for optimization to improve clinical outcomes. In this study, we compared the routine droplet merge protocol with modified multi-gradient equilibration vitrification for cryopreservation of mouse oocytes at metaphase II. Subsequently, the oocytes were thawed and subjected to intracytoplasmic sperm injection (ICSI). Oocyte survival and spindle status were evaluated by morphology and immunofluorescence staining. Moreover, the fertilization rates and blastocyst development were examined in vitro. The results showed that multi-gradient equilibration vitrification outperformed droplet merge vitrification in terms of oocyte survival, spindle morphology, blastocyst formation, and embryo quality. In contrast, droplet merge vitrification exhibited decreasing survival rates, a reduced proportion of oocytes with normal spindle morphology, and lower blastocyst rates as the number of loaded oocytes increased. Notably, when more than six oocytes were loaded, reduced oocyte survival rates, abnormal oocyte spindle morphology, and poor embryo quality were observed. These findings highlight that the vitrification of mouse metaphase II oocytes by the modified multi-gradient equilibration vitrification has the advantage of maintaining oocyte survival, spindle morphology, and subsequent embryonic development.

Evaluation of an automated dish preparation system for IVF and embryo culture using a mouse mode.

Manual dish preparation for IVF in human fertility clinics or animal laboratories heavily relies on embryologists' experience, which can lead to occupational illness due to long-term and monotonous operation. Therefore, introducing an automated technique to replace traditional methods is crucial for improving working efficiency and reducing work burden for embryologists. In the current study in the mouse, both manual and automated methods were used to prepare IVF or embryo culture dishes. A one-way analysis of variance was conducted to compare several factors, including preparation time, qualified rates, media osmolality of dishes, fertilization rates, and embryonic development to assess the efficiency and potential of automated preparation. The results showed that automation system significantly reduced the required time and increased the efficiencies and qualified rates of dish preparation, especially for embryo culture dishes, without significantly altering medium osmolalities. There were no significant differences between two preparations in fertilization rates and embryo development in mice. Thus, automated dish preparation can improve working efficiency and qualified rates while maintaining fertilization rates and subsequent embryonic development without compromising osmolality stability of medium. It presents a superior alternative to manual preparation, reducing the workload of embryologists and facilitating the standardization of operational procedures.

The pregnancy outcomes of day-5 poor-quality and day-6 high-quality blastocysts in single blastocyst transfer cycles.

OBJECTIVE: This study compared the outcomes of single blastocyst transfer cycles, using day- 5 poor-quality blastocysts and day-6 high-quality blastocysts. METHODS: We analyzed 462 frozen-thawed embryo transfer (FET) cycles performed at our center from January 2014 to December 2019. The cycles were divided into two groups: a day-5 poor-quality blastocyst transfer group (group A) and a day-6 high-quality blastocyst transfer group (group B). The clinical outcomes were tested. RESULTS: In groups A and B, respectively, the clinical pregnancy rate (CPR; 61.65% vs. 67.17%, p=0.258), implantation rate (IR; 61.65% vs. 67.17%, p=0.258), and live birth rate (LBR; 69.51% vs. 77.83%, p=0.134) showed no significant differences. Moreover, when day-3 embryo quality was considered, the CPR, IR, and LBR were also similar in group A and group B (p>0.05). CONCLUSION: The clinical outcomes of day-5 poor-quality blastocysts and day-6 high-quality blastocysts were similar, suggesting that the developmental speed of the embryo might be more important than embryo quality for the clinical outcomes of single blastocyst transfer in FET cycles.

Automation in vitrification and thawing of mouse oocytes and embryos.

Vitrification is a common technique for cryopreserving oocytes or embryos. However, manual vitrification is tedious and labor-intensive, and can be subject to variations caused by human factors. To address these challenges, we developed an automated vitrification-thawing system (AVTS) based on a cryo-handle. Our study firstly assessed the efficiency of cryoprotectant exchange through comparing the osmolalities of fresh and collected solutions during automated vitrification and thawing, and evaluated the cooling and warming rates of the cryo-handle. We also compared mouse oocyte survival, fertilization and embryo development after thawing and ICSI, and the development of re-frozen cleavage embryos between manual operation and automated system. The results showed that the osmolalities of collected samples were within normal range and comparable to fresh solutions. Furthermore, the automated system could obtain the reliable cooling and warming rates. Particularly, there were no significant differences in oocyte survival rates, fertilization rates, and subsequent embryo development and its quality between two procedures. Our findings suggest that AVTS has no impact on osmolalities of vitrification and thawing solutions, ensuring the proper exchange of cryoprotectants. The cryo-handle also shows the ability to achieve reliable cooling and warming rates, which benefits for the cryopreservation and thawing process. Moreover, the results from mouse oocytes and embryos indicate that automated system has effectively maintained the survival and fertilization of frozen oocytes and supported subsequent embryo development. Therefore, the automated vitrification and thawing system will inevitably represent a superior alternative to manual operation.

Stage-specific H3K9me3 occupancy ensures retrotransposon silencing in human pre-implantation embryos.

H3K9me3, as a hallmark of heterochromatin, is important for cell-fate specification. However, it remains unknown how H3K9me3 is reprogrammed during human early embryo development. Here, we profiled genome-wide H3K9me3 in human oocytes and early embryos and discovered stage-specific H3K9me3 deposition on long terminal repeats (LTRs) at the 8-cell and blastocyst stages. We found that 8-cell-specific H3K9me3 was temporarily established in enhancer-like regions, whereas blastocyst-specific H3K9me3 was more stable. DUX and multiple Krüppel-associated box domain zinc finger proteins(KRAB-ZNFs) were identified as potential factors for establishing 8C- and blastocyst-specific H3K9me3, respectively. Intriguingly, our analysis showed that stage-specific H3K9me3 allocation was attenuated by either Dux knockout or Zfp51 knockdown in mouse early embryos. Moreover, we observed the existence of H3K4me3/H3K9me3 and H3K4me3/H3K27me3 bivalent chromatin domains in human blastocysts, priming for lineage differentiation. Together, our data unveil that the epigenetic switch from DNA methylation to H3K9me3 ensures the precise regulation of retrotransposons in human pre-implantation embryos.

Mettl3 downregulation in germinal vesicle oocytes inhibits mRNA decay and the first polar body extrusion during maturation†.

In oocytes, mRNA decay is essential for maturation and subsequent events, such as maternal-zygotic transition, zygotic genomic activation, and embryo development. Reversible N6-methyladenosine RNA methylation directly regulates transcription, pre-mRNA splicing, mRNA export, mRNA stability, and translation. Here, we identified that downregulation of N6-methyladenosine modification by microinjecting a methyltransferase-like 3 (Mettl3)-specific small interfering RNA into mouse germinal vesicle oocytes led to defects in meiotic spindles and the first polar body extrusion during maturation in vitro. By further quantitative real-time polymerase chain reaction and Poly(A)-tail assay analysis, we found that N6-methyladenosine methylation mainly acts by reducing deadenylation of mRNAs mediated by the carbon catabolite repression 4-negative on TATA less system, thereby causing mRNA accumulation in oocytes. Meanwhile, transcriptome analysis of germinal vesicle oocytes revealed the downregulation of transcripts of several genes encoding ribosomal subunits proteins in the Mettl3 small interfering RNA-treated group, suggesting that N6-methyladenosine modification might affect translation. Together, our results indicate that RNA methylation accelerates mRNA decay, confirming the critical role of RNA clearance in oocyte maturation.

A Robotic System With Embedded Open Microfluidic Chip for Automatic Embryo Vitrification.

Embryo vitrification is a fundamental technology utilized in assisted reproduction and fertility preservation. Vitrification involves sequential loading and unloading of cryoprotectants (CPAs) with strict time control, and transferring the embryo in a minimum CPA droplet to the vitrification straw. However, manual operation still cannot effectively avoid embryo loss, and the existing automatic vitrification systems have insufficient system reliability, and operate differently from clinical vitrification protocol. Through collaboration with in vitro fertilization (IVF) clinics, we are in the process realizing a robotic system that can automatically conduct the embryo vitrification process, including the pretreatment with CPAs, transfer of embryo to the vitrification straw, and cryopreservation with liquid nitrogen ( LN2). An open microfluidic chip (OMC) was designed to accommodate the embryo during the automatic CPAs pretreatment process. The design of two chambers connected by a capillary gap facilitated solution exchange around the embryo, and simultaneously reduced the risk of embryo loss in the flow field. In accordance to the well-accepted procedure and medical devices in manual operation, we designed the entire vitrification protocol, as well as the robotic prototype. In a practical experiment using mouse embryos, our robotic system showed a 100 % success rate in transferring and vitrifying the embryos, achieved comparable embryo survival rates (90.9 % versus 94.4 %) and development rates (90.0 % versus 94.1 %), when compared with the manual group conducted by the senior embryologist. With this study, we aim to facilitate the standardization of clinical vitrification from manual operation to a more efficient and reliable automated process.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy.

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

MFN2 Plays a Distinct Role from MFN1 in Regulating Spermatogonial Differentiation.

Although mitochondrial morphology is well-known for its role in cellular homeostasis, there is surprisingly little knowledge on whether mitochondrial remodeling is required for postnatal germ cell development. In this study, we investigated the functions of MFN1 and MFN2, two GTPases in mitochondrial fusion, during early spermatogenesis. We observed increased MFN expressions along with increased mitochondrial and endoplasmic reticulum (ER) activities during spermatogonial differentiation. Deletion of either Mfn led to DNA oxidation and apoptosis specifically in differentiating spermatogonia and spermatocytes, which in turn caused male infertility. We further found MFN2 regulated spermatogenesis by modulating both mitochondrial and ER functions, a mechanism distinct from that of MFN1. Defects of germ cell development in MFN2 mutants were corrected by MFN2 at either mitochondria or ER but not by MFN1. Our study thus reveals an essential requirement of MFN-mediated mitochondrial and ER coordination in spermatogenesis, providing critical insights into mitochondrial determinants of male fertility.

Chromosomal microarray analysis of infertile men with azoospermia factor microdeletions.

Azoospermia factors, located in the long arm of the Y chromosome, are critical for spermatogenesis, the microdeletions of AZF are considered to be associated with male infertility. In addition to complete deletion, several AZFc partial deletions were also detected in infertile men with wide phenotypic heterogeneity. In this study, we investigated the relevance of Y chromosome deletions, Y-linked CNVs and variable phenotypes in infertile men. To clarify the relationship between phenotypic heterogeneity and Y chromosome deletion in male infertility, we performed chromosomal microarray analysis (CMA) capable of analyzing thousands of loci simultaneously to investigate Y-linked copy number variations (CNVs). Firstly, we reviewed the results of Y chromosome screening in 554 infertile patients and then compared the results of CMA to routine Y chromosome screening in 29 patients with Y chromosomal microdeletions. Then, the Y-linked CNVs associated with oligoasthenospermia were identified according to ACMG standards and guidelines. The results indicated that the prevalence of Yq microdeletions was 5.23% (29/554), with 93% (27/29) of the deletions in the AZFc region among 554 infertile men recruited in this study. The results of CMA and multiplex-PCR-based AZFc deletion analysis were generally concordant, but CMA provided more details about location, size and OMIM genes involved in deletion fragments of the AZF region. Of 29 clinically infertile phenotype-related CNVs detected by CMA, nine were pathogenic and the remaining 20 CNVs were OVUS. Except for a 15.69 Mb loss CNV in AZFa + b + c and an 8.25 Mb loss CNV in AZFb + c, others were located in the AZFc region. Based on a combination of the clinical symptoms and loss CNVs, we concluded that the CNV size and the involvement of spermatogenesis critical genes are two important factors that determine the relevance of a CNV in the AZFc region to the presence or absence of a clinically infertile phenotype.

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice, and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro.

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross-talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G-protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G-protein activation, plays an important role in embryo implantation by interfering in the cross-talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17ß-estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17ß-estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17ß-estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.

Histone methyltransferase SETD2 is required for meiotic maturation in mouse oocyte.

SET-domain-containing 2 (SETD2), a member of the histone lysine methyltransferase family, has been reported to be involved in multiple biological processes. However, the function of SETD2 during oocyte maturation has not been addressed. In this study, we find that mouse oocytes are incapable of progressing through meiosis completely once SETD2 is specifically depleted. These oocytes present an abnormal spindle morphology and deficient chromosome movement, with disrupted kinetochore-microtubule attachments, consequently producing aneuploidy eggs. In line with this, the BubR1 signal is markedly elevated in metaphase kinetochores of oocytes with SETD2 depletion, indicative of the activation of spindle assembly checkpoint. In addition, we note that loss of SETD2 results in a drastic decrease in the trimethylation level of H3K36 in oocytes. Collectively, our data demonstrate that SETD2 is required for oocyte maturation and indicate a novel mechanism controlling the meiotic apparatus.

SIRT4 is essential for metabolic control and meiotic structure during mouse oocyte maturation.

SIRT4 modulates energy homeostasis in multiple cell types and tissues. However, its role in meiotic oocytes remains unknown. Here, we report that mouse oocytes overexpressing SIRT4 are unable to completely progress through meiosis, showing the inadequate mitochondrial redistribution, lowered ATP content, elevated reactive oxygen species (ROS) level, with the severely disrupted spindle/chromosome organization. Moreover, we find that phosphorylation of Ser293-PDHE1α mediates the effects of SIRT4 overexpression on metabolic activity and meiotic events in oocytes by performing functional rescue experiments. By chance, we discover the SIRT4 upregulation in oocytes from aged mice; and importantly, the maternal age-associated deficient phenotypes in oocytes can be partly rescued through the knockdown of SIRT4. These findings reveal the critical role for SIRT4 in the control of energy metabolism and meiotic apparatus during oocyte maturation and indicate that SIRT4 is an essential factor determining oocyte quality.

Motility, progressive motility score and DNA integrity of spermatozoa from cold-preserved mouse cauda epididymis.

This study attempted to investigate and validate whether epididymis cold storage could be a suitable alternative for short-term preservation of spermatozoa. Mouse cauda epididymides and spermatozoa were preserved at 4-8°C from 1 day to 6 weeks. From days 1 to 10, motility and fertility were daily examined when motility loss occurred. From week 1, spermatozoa were used for intracytoplasmic sperm injection (ICSI) at weekly intervals to test their fertility, and spermatozoa DNA integrity was determined by comet assay. We found that motility and progressive motility scores gradually decreased with storage time. In nearly all spermatozoa, DNA integrity was maintained from days 1 to 10, but the percentage of spermatozoa with damaged DNA significantly increased from week 2 to week 6. Spermatozoa retained fertility until day 6, although fertility gradually decreased after day 3. From week 1 to week 5, fertilization rates by ICSI were more than 82.69% but decreased gradually after week 3. We found that spermatozoa preserved in the epididymis at 4-8°C had progressively lower motility, fertility and proportion of undamaged DNA, but could still fertilize oocytes. However, all the parameters of cold-preserved spermatozoa were completely inferior to that from cold-preserved cauda epididymides. The results imply that cold storage of cauda epididymides could be conducive to short-term preservation of spermatozoa, and the cold-stored spermatozoa can resist DNA denaturation, which is necessary for maintaining reproductive ability.

Mitogen-activated protein kinase-activated protein kinase 2 is a critical regulator of pig oocyte meiotic maturation.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple cellular processes. In the present study, we showed that MK2 affected not only cumulus expansion, but also the oocyte meiotic cell cycle in porcine oocytes. Inhibition of MK2 arrested oocytes at the germinal vesicle (GV) stage or the prometaphase I/metaphase I stage. Unlike in mouse oocytes, where phosphorylated (p-) MK2 was localised at the minus end of spindle microtubules and close to the spindle poles, in porcine oocytes p-MK2 was concentrated at the spindle equator and localised at the plus end of spindle microtubules. Knockdown or inhibition of MK2 resulted in spindle defects: spindles were surrounded by irregular chromosome non-disjunction or by chromosomes detached from the spindles. MK2 regulated spindle organisation and chromosome alignment by connecting microtubules with kinetochores. In addition, unlike in mitotic cells and meiotic mouse oocytes, the MK2-p38 MAPK pathway may not play an important role during meiotic cell cycle in porcine oocytes. In conclusion, MK2 is an important regulator of porcine oocyte meiotic maturation.

Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.

STUDY QUESTION: Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? SUMMARY ANSWER: Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. WHAT IS KNOWN ALREADY: The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. STUDY DESIGN, SIZE, DURATION: Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. MAIN RESULTS AND THE ROLE OF CHANCE: The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. WIDER IMPLICATIONS OF THE FINDINGS: Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. STUDY FUNDING/COMPETING INTERESTS: The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.

Inhibition of the Binding between RGS2 and ß-Tubulin Interferes with Spindle Formation and Chromosome Segregation during Mouse Oocyte Maturation In Vitro.

RGS2 is a negative regulator of G protein signaling that contains a GTPase-activating domain and a ß-tubulin binding region. This study aimed to determine the localization and function of RGS2 during mouse oocyte maturation in vitro. Immunofluorescent staining revealed that RGS2 was widely expressed in the cytoplasm with a greater abundance on both meiotic spindles and first/second polar bodies from the fully-grown germinal vesicle (GV) stage to the MII stages. Co-expression of RGS2 and ß-tubulin could also be detected in the spindle and polar body of mouse oocytes at the MI, AI, and MII stages. Inhibition of the binding site between RGS2 and ß-tubulin was accomplished by injecting anti-RGS2 antibody into GV-stage oocytes, which could result in oocytes arrest at the MI or AI stage during in vitro maturation, but it did not affect germinal vesicle breakdown. Moreover, injecting anti-RGS2 antibody into oocytes resulted in a significant reduction in the rate of first polar body extrusion and abnormal spindle formation. Additionally, levels of phosphorylated MEK1/2 were significantly reduced in anti-RGS2 antibody injected oocytes compared with control oocytes. These findings suggest that RGS2 might play a critical role in mouse oocyte meiotic maturation by affecting ß-tubulin polymerization and chromosome segregation.

GASZ and mitofusin-mediated mitochondrial functions are crucial for spermatogenesis.

Nuage is an electron-dense cytoplasmic structure in germ cells that contains ribonucleoproteins and participates in piRNA biosynthesis. Despite the observation that clustered mitochondria are associated with a specific type of nuage called intermitochondrial cement (pi-body), the importance of mitochondrial functions in nuage formation and spermatogenesis is yet to be determined. We show that a germ cell-specific protein GASZ contains a functional mitochondrial targeting signal and is largely localized at mitochondria both endogenously in germ cells and in somatic cells when ectopically expressed. In addition, GASZ interacts with itself at the outer membrane of mitochondria and promotes mitofusion in a mitofusin/MFN-dependent manner. In mice, deletion of the mitochondrial targeting signal reveals that mitochondrial localization of GASZ is essential for nuage formation, mitochondrial clustering, transposon repression, and spermatogenesis. MFN1 deficiency also leads to defects in mitochondrial activity and male infertility. Our data thus reveal a requirement for GASZ and MFN-mediated mitofusion during spermatogenesis.

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro.

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

FACS selection of valuable mutant mouse round spermatids and strain rescue via round spermatid injection.

Round spermatid injection (ROSI) into mammalian oocytes can result in the development of viable embryos and offspring. One current limitation to this technique is the identification of suitable round spermatids. In the current paper, round spermatids were selected from testicular cells with phase contrast microscopy (PCM) and fluorescence-activated cell sorting (FACS), and ROSI was performed in two strains of mice. The rates of fertilization, embryonic development and offspring achieved were the same in all strains. Significantly, round spermatids selected by PCM and FACS were effectively used to rescue the infertile Pten-null mouse. The current results indicate that FACS selection of round spermatids can not only provide high-purity and viable round spermatids for use in ROSI, but also has no harmful effects on the developmental capacity of subsequently fertilized embryos. It was concluded that round spermatids selected by FACS are useful for mouse strain rederivation and rescue of infertile males; ROSI should be considered as a powerful addition to the armamentarium of assisted reproduction techniques applicable in the mouse.