Resumption Of Mitosis Research Articles

P-143 Comparison of clinical outcomes between overnight warmed and embryo transfer day warmed blastocysts in frozen embryo transfer cycles

Abstract Study question Is there any difference between clinical outcomes of overnight warmed (ONW) and embryo transfer day warmed (ETDW) blastocysts in frozen embryo transfer (FET) cycles? Summary answer ETDW blastocysts had better clinical outcomes compared to ONW blastocysts in FET cycles. What is known already Some studies reported that the resumption of mitosis in thawed cleavage-stage embryos resulted in significantly better clinical outcomes in FET cycles. Unnecessary transfer of non-functioning embryos can be avoided by incubating thawed embryos overnight to deselect embryos that developed abnormally or failed to undergo additional cleavage. The synchronisation between a viable embryo and a receptive endometrium is vital for successful implantation to take place. The optimal implantation rates occur with embryo-endometrial developmental asynchrony of ± 1.5 days or less. The implantation rates are progressively reduced when embryo-endometrial development asynchrony is greater than 3 days. Study design, size, duration This was a retrospective study involving 2368 FET cycles in KKIVF Centre, KK Women’s and Children’s Hospital, Singapore from Jan 2021 to Dec 2022. The transfer of embryos or blastocysts derived from thawed oocytes, cleavage-stage embryos, double blastocysts and cultured blastocysts from thawed cleavage-stage embryos were excluded. Only single vitrified-warmed blastocyst transfers (n = 1445) were included in this study. Participants/materials, setting, methods Cryopreserved blastocysts were routinely warmed one day before embryo transfer (ET) using Vit Kit - Warm (FUJIFILM Irvine Scientific®) and incubated overnight (ONW group). However, when ETs fell on Monday or post public holiday, vitrified blastocysts were warmed on ET day (ETDW group). Regardless of blastocyst age, surviving blastocysts were transferred into day 5 endometrium. Statistical analysis was performed using t-test or Chi-square test. Differences with P < 0.05 were considered as significant. Main results and the role of chance No statistical significant difference was observed in terms of maternal age distribution at the time of oocyte retrieval between ONW and ETDW groups (34.26 ± 3.29 vs. 34.49 ± 3.22, P = 0.221). Overall, clinical pregnancy rates (CPR) and live birth rates (LBR) were significantly higher in the ETDW group (CPR: 47.47% vs. 40.47%, P < 0.01; LBR: 34.99% vs. 28.78%, P < 0.05). Clinical outcomes were comparable in both ONW and ETDW groups for vitrified-warmed day 5 blastocyst transfers (CPR: 51.27% vs. 58.44%, P = 0.11; LBR: 37.06% vs. 44.81%, P = 0.07). CPR (45.02% vs. 25.71%, P < 0.0001) and LBR (32.03% vs. 17.45%, P < 0.0001) were significantly increased when vitrified day 6 blastocysts were warmed and transferred on the actual day of ET. 18.42% CPR and 13.16% LBR were achieved when vitrified-warmed day 7 blastocysts were transferred on the ET day itself. However, transfer of vitrified-warmed day 7 blastocysts in the ONW group did not result in any pregnancy. Limitations, reasons for caution Study subjects were not stratified according to patient’s age band and blastocyst quality. Age itself is an independent determinant of blastocyst quality which can affect clinical outcomes. Wider implications of the findings Warming of a vitrified blastocyst on ET day is preferred regardless of blastocyst age. However, to facilitate seamless operation in laboratory, overnight warming for vitrified day 5 blastocysts is feasible without compromising clinical outcomes. Trial registration number Not applicable

Does Overnight Culture of Cleaved Embryos Improve Pregnancy Rate in Vitrified-Warmed Embryo Transfer Programme?

BackgroundVitrification is a routine procedure in assisted reproductive technique (ART) lab. However, there is widespread variability between protocols of different centres. The aim of this study was to compare the chemical pregnancy, clinical pregnancy and live birth rates between one-day embryo culture and immediate transfer for frozen-thawed embryo transfer (FET) cycles.MethodsIn this cohort retrospective study, 366 FET cycles were divided into two groups: Group A, the embryos were warmed one day before transfer, and were cultured overnight; Group B, the embryos were warmed on the same day of transfer, at least were cultured 1 h before embryo transfer (ET). Chemical and clinical pregnancy and live birth rates were compared between two groups.ResultsThe chemical pregnancy was higher in group A than B (37.9% versus 28.9%), but this difference was not significant (P = 0.07). Clinical pregnancy (30.8% versus 24.1%) and live birth (19.8% versus 22.05%) were similar in group A and B, (P = 0.15), and (P = 0.8). Conclusion: In conclusion, overnight culture and confirmation of mitosis resumption was not essential for FET cycles in vitrification method.

Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study

BackgroundMorphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming.MethodsDuring fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing.ResultsVitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones.ConclusionsInterestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies.Trial registrationRetrospectively registered on December 15, 2015 NCT02639715.

Analysis of embryo intactness and developmental potential following slow freezing and vitrification

ABSTRACTThe aim of this study was to identify the parameters that are related to intactness and developmental potential of a day 3 embryo after warming to improve the selection criteria used to cryopreserve and transfer embryos. We also sought to compare slow freezing and vitrification methods of cryopreservation and to evaluate the viability of non-intact embryos. Embryos warmed following slow freezing (n=220) or vitrification (n=522) were divided into 3 groups according to the proportion of surviving blastomeres (I<50%; II=50-99%; and III=100%). The developmental potential of embryos, including the mitosis resumption rate, blastocyst formation rate, and formation rate of grade A blastocysts (i.e., fully expanded blastocysts with an inner cell mass and grade A or B trophectoderm) were retrospectively assessed in embryos. Cleavage-stage embryos with <50% blastomere survival were analyzed using next-generation sequencing (NGS). Logistic regression analysis showed that vitrification and grade 1 were independent predictive factors of embryo intactness and developmental potential (all p<0.05). On day 3, embryos with 4-6 cells or blastomere damage had lower developmental potential than those with 7-9 cells or intact blastomeres (all p<0.05). NGS results showed that the chromosomal status was completely normal in 8 embryos that developed into expanded blastocysts, whereas 4 out of 5 embryos in which development was arrested were abnormal. The results of this study suggest that vitrification is a better choice than slow freezing for embryo cryopreservation. Embryos showing poor quality (fragmentation >30% and/or a non-stage-specific cell size) and lower cell numbers (4-6 cells) on day 3 should be cultured to the blastocyst stage and then vitrified if they develop into good-quality blastocysts. The developmental potential of non-intact embryos is lower than that of intact embryos; however, after they are cultured to the fully expanded blastocyst stage, embryos with <50% blastomere survival appear to be better candidates for transfer.Abbreviations ART: assisted reproductive technology; grade A blastocyst: fully expanded blastocyst with an inner cell mass and grade A or B trophectoderm; NGS: next-generation sequencing; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; FET: frozen-thawed embryo transfer

Peripheral Blood Monocytes as Adult Stem Cells: Molecular Characterization and Improvements in Culture Conditions to Enhance Stem Cell Features and Proliferative Potential.

Adult stem or programmable cells hold great promise in diseases in which damaged or nonfunctional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into cells resembling specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion, the monocytes downregulate monocyte/macrophage differentiation markers, being indicative of partial dedifferentiation, and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency and resumption of mitosis. Upregulation of stem cell markers and mitotic activity in the cultures was shown to be controlled by autocrine production/secretion of activin A and transforming growth factor-beta (TGF-β). These reprogrammed monocyte derivatives were termed “programmable cells of monocytic origin” (PCMO). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMO in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies.

Predictors of twin live birth following cryopreserved double embryo transfer on day 3

To determine embryo and cycle-specific parameters associated with twin live birth in day 3 cryopreserved double embryo transfer (DET) cycles, and to propose a new prediction model for external validation. All cycles with autologous or donor cryopreserved DET of day 3 embryos from 2002 to 2011 at a single academic institution with a singleton or twin live birth were included (n = 207). Patient characteristics, post-thaw embryo morphology and freeze-thaw parameters were compared between patients with a single and twin live birth. Demographic characteristics were similar, except that patients delivering twins were younger at age of cryopreserved embryo transfer (CET), fewer were parous and more were anovulatory. Duration of embryo storage, time in culture post-thaw, endometrial thickness and use of assisted hatching were comparable. Six predictors of twin delivery were identified: patient age <35 year at CET, intact survival of the lead embryo, resumption of mitosis, 7-8 viable cells in the non-lead embryo, transfer of a lead embryo with ≥7 cells and a sum of ≥14 viable cells in the two transferred embryos. Regression modeling predicted a step-wise increase in the probability of twins with addition of each predictor; with all six present, the risk of twins was predicted to be 53 % and with none present, the risk decreased to 6 %. The six identified variables associated with twin live birth following day 3 cryopreserved DET have been applied to derive a prediction model for estimating the risk of twin delivery. External validation of the model is required prior to clinical application.

Accumulation and loss of asymmetric non-CpG methylation during male germ-cell development

DNA methylation is a well-characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. It mainly occurs on cytosines at CpG sites but methylation at non-CpG sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. The biological significance of non-CpG methylation is unknown. Here, we show that non-CpG methylation is also present in male germ cells, within and around B1 retrotransposon sequences interspersed in the mouse genome. It accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a Dnmt3l-dependent manner. The preferential site of non-CpG methylation is CpA, especially CpApG and CpApC. Although CpApG (and CpTpG) sites contain cytosines at symmetrical positions, hairpin-bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template-dependent copying mechanism. Indeed, the level of non-CpG methylation decreases after the resumption of mitosis in the neonatal period, whereas that of CpG methylation does not. The cells eventually lose non-CpG methylation by the time they become spermatogonia. Our results show that non-CpG methylation accumulates in non-replicating, arrested cells but is not maintained in mitotically dividing cells during male germ-cell development.

The fate of frozen human embryos when transferred either on the day of thawing or after overnight culture

Abstract Objective To study the performance of thawed zygotes and cleavage stage embryos transferred either on the day of thaw or after overnight culture. Methods A retrospective study of 864 frozen embryo transfer cycles. Cryosurvival rates per thawed embryo and implantation rates were analysed for embryos frozen on Day 1, Day 2 or Day 3 relative to oocyte collection (Day 0) and transferred on the day of thaw or after overnight culture, together with clinical pregnancy rates and prevalence of multiple gestations. Results Survival of Day 3 embryos was significantly lower than those frozen on Day 1 (P=0.017) or Day 2 (P=0.015). Following overnight culture, resumption of mitosis of zygotes was more frequent than Day 2 (P=0.000) which are in turn higher than Day 3 (P=0.000) embryos. The implantation rate for Day 2 embryos dividing overnight was significantly higher than those that did not divide for women Conclusions Later cleavage stage post-thaw embryos survive and resume mitosis less frequently compared to earlier stages. Embryos not resuming mitosis after culture overnight can implant, particularly Day 3 embryos, suggesting that they can further increase the cumulative pregnancy rate per oocyte collection and that discarding them is wasteful. Overnight culture is best used for logistical reasons rather than a strategy to improve pregnancy rates.

Gonocyte development in rats: proliferation, distribution and death revisited

Spermatogonial stem cells are responsible for the constant production of spermatozoa. These cells differentiate from the gonocytes, but little is known about these cells and their differentiation into spermatogonia. This study analyzed rat gonocyte proliferation, death and distribution as well as their differentiation into spermatogonia. Rat testes were collected at 19 dpc and at 1, 3, 5, 8, 11 and 15 dpp and submitted to apoptosis investigation through morphological analysis and TUNEL, p53 and cleaved caspase 3 labeling. Ki67 and MVH labeling was used to check gonocyte proliferation and quantification, respectively. OCT4 and DBA labeling were used to check gonocyte differentiation. Seminiferous cord length and gonocyte numerical density were measured to check gonocyte distribution along the seminiferous cords. Although a reduction of gonocyte number per testicular section has been observed from 1 to 5 dpp, the total number of these cells did not change. Apoptotic gonocytes were not detected at these ages, suggesting that the reduction in gonocyte number per testicular section was due to their redistribution along the seminiferous cords, which showed continuous growth from 19 dpc to 5 dpp. The first proliferating germ cells were observed at 8 dpp, coinciding with OCT4 upregulation and with the emergence of the first spermatogonia. In conclusion, this study suggests that (a) gonocytes do not die in the first week after birth, but are rather redistributed along the seminiferous cords just before their differentiation into spermatogonia; (b) mitosis resumption and the emergence of the first spermatogonia are coincident with OCT4 upregulation.

Natural cycle is superior to hormone replacement therapy cycle for vitrificated-preserved frozen-thawed embryo transfer

We undertook this retrospective variables-control analysis to compare the reproductive outcomes of frozen-thawed embryo transfer using endometrial preparation with either natural cycle or hormone replacement therapy cycle. Patients were divided into three subgroups. Subgroup A (n = 32) consisted of patients having three 8-cell post-thawed embryos transferred. Subgroup B (n = 404) consisted of patients having three good quality post-thawed embryos transferred. Subgroup C (n = 578) consisted of patients having two or three all intact and mitosis resumption post-thawed embryos transferred. Implantation rate, biochemical pregnancy rate, and clinical pregnancy rate were measured. In subgroup A, significantly higher implantation rate, clinical pregnancy rate ongoing pregnancy rate, and lower biochemical pregnancy rate were observed in the natural cycle compared with hormone replacement therapy (HRT) cycle. Subgroup B, had a significantly higher rate of implantation, ongoing pregnancy, and a significantly lower rate of biochemical pregnancy in natural cycle compared with HRT cycle. The natural cycle had a higher trend of clinical pregnancy rate without reaching statistical significance. No statistical difference in reproductive outcomes between natural cycle and HRT cycle was observed in subgroup C. The results suggest the superiority of the natural cycle as compared with the HRT cycle under certain circumstances in a selected population of patients.

Correlation between embryological factors and pregnancy rate: development of an embryo score in a cryopreservation programme

To establish which embryo parameters, in frozen thawed embryo transfers, have the highest prognosis value in the establishment of pregnancy. The relative importance of different embryo parameters is used to develop an embryo score. Retrospective analysis of the implantation rate in 356 frozen/thawed single embryo transfers. A logistic regression model is used to establish an embryo score. A direct correlation is established between the implantation rate and fresh embryo development (number of blastomeres and their symmetry), survival rate after thawing and mitosis resumption after overnight culture. An embryo score is developed to determine the implantation potential of frozen/thawed embryos.

Increasing dehydration of human cleavage-stage embryos prior to slow cooling significantly increases cryosurvival

Increasing the proportions of embryos and blastomeres which survive cryopreservation would be expected to make a significant contribution to the outcome of assisted reproduction treatment. Despite this, the methodology used for slow cooling of human cleavage-stage embryos has remained largely unchanged for over two decades. Previous studies have demonstrated the value, in terms of cryosurvival, of increasing the extent of intracellular dehydration by increasing the concentration of non-permeating cryoprotectant prior to slow cooling of oocytes and embryos which have been biopsied for preimplantation genetic diagnosis. The present study extends the use of this approach to the slow cooling of non-biopsied day-2 embryos. Dehydration in the presence of 0.2 mol/l sucrose significantly increased the proportions of surviving embryos, surviving blastomeres and fully intact embryos (92.6%, 91.1%, and 80.4%, respectively) relative to those observed after dehydration in 0.1 mol/l sucrose (78.5%, 74.1%, and 54.6%, respectively, all P < 0.001). Post-thaw resumption of mitosis in vitro and implantation were not adversely affected by the increased prefreeze dehydration. This improved method for slow cooling of cleavage-stage embryos should have a major impact on clinical outcome.

Resumption of mitosis in frozen–thawed embryos is not related to the chromosomal constitution

To study the relation between the resumption of mitosis after thaw and chromosomal constitution in frozen-thawed embryos. In addition, to evaluate the correlation among the three parameters of resumption of mitosis after thaw, postthaw blastomere loss, and multinucleation. Frozen-thawed embryos were morphologically evaluated at thaw and after 24 hours of culture. Then, fluorescence in situ hybridization (FISH) analysis, including enumeration of 13 chromosomes, was performed by using a combination of peptide nucleic acid and DNA probes. In vitro fertilization laboratory. Forty IVF and/or intracytoplasmic sperm injection patients. Embryo thawing, morphological evaluation, and fluorescence in situ hybridization analysis for aneuploidy screening. Resumption of mitosis, blastomere loss, multinucleation, and chromosome enumeration. No difference was observed in the chromosomal constitution of embryos with and without resumption of mitosis. Neither was the postthaw blastomere loss connected to the chromosomal constitution. The resumption of mitosis was not associated with postthaw loss of blastomeres or with multinuclearity. Resumption of mitosis and blastomere loss of frozen-thawed embryos is not related to chromosome aberrations in the embryo. Further, the resumption of mitosis is not correlated with multinucleation. However, the high incidence of multinucleated embryos after thawing indicates that the freezing and thawing procedure may affect this condition.

Parameters predicting the implantation rate of thawed IVF/ICSI embryos: a retrospective study

Single embryo transfer is becoming increasingly popular in IVF/ICSI. More IVF/ICSI cycles therefore include freezing of high quality embryos, and the cumulative effect of such cycles becomes more important. To improve the results obtained using frozen–thawed embryos, the predictive value of embryo and patient characteristics was analysed retrospectively. Excess embryos were frozen on day 2 according to routine procedures and thawed 24 h before transfer. The women were either treated with oestradiol/progesterone, or the transfer took place after a natural cycle. Of 1777 embryos thawed, 1360 survived (survival rate 76.5%). In the 410 transfer cycles, 819 embryos were transferred, giving 109 positive serum HCG tests and 69 clinical pregnancies. The implantation rate was 10.4%. Five parameters were positively related to implantation: (i) four or more cells at the freezing time (day 2); (ii) resumption of mitosis after thawing; (iii) more than six cells at transfer (day 3); (iv) assisted hatching and (v) child in a previous fresh cycle. The relative predictive value of these parameters may be a guideline for the selection of embryos used for transfer. Assisted hatching may become routine in freeze–thaw cycles if its utility is confirmed in subsequent studies.

The application and impact of cryopreservation of early cleavage stage embryos in assisted reproduction

The contribution of cryopreserved embryos to the overall outcomes achieved by a clinical assisted reproduction programme has increased in importance with the trend towards reducing the numbers of fresh embryos transferred following in vitro fertilisation. Although cryopreservation appears to fully preserve developmental potential in early cleavage stage embryos that survive intact, it results in a reduction in potential when blastomere loss occurs during freezing and thawing. Overall, it can be estimated that cryopreservation results in approximately a 30% reduction in the potential for pregnancy in a population of embryos. Both blastomere survival and post-thaw resumption of mitosis can act as markers of implantation potential in frozen/thawed embryos. Application of strict criteria for freezing embryos and transferring thawed embryos may enhance apparent success rates, but may also result in some pregnancy potential being discarded. The role of embryo cryopreservation in minimising the incidence of multiple pregnancy must be balanced with the need for efficiency in the quest to establish pregnancy.

Cell cycle regulator Cdc14 is expressed during sporulation but not hyphal growth in the fungus-like oomycete Phytophthora infestans.

Cdc14 proteins are important regulators of mitosis and the cell cycle. These phosphatases have been studied previously only in yeasts and metazoans, which grow by fission or budding. Here we describe a homologue (piCdc14) from the oomycete Phytophthora infestans, a primitive eukaryote lacking a classical cell cycle. PiCdc14 complements a cdc14ts mutant of Saccharomyces cerevisiae and may function like other Cdc14 proteins, but displays a strikingly different pattern of expression. Whereas previously studied Cdc14 genes are constitutively transcribed, piCdc14 is not expressed during normal growth but instead only during asexual sporulation. In transformants of P. infestans expressing a fusion between the piCdc14 promoter and the -glucuronidase reporter, expression was first detected in sporangiophore initials, persisted in sporangiophores bearing immature sporangia, and later became restricted to mature sporangia. After germination, expression ended a few hours before the resumption of mitosis in hyphae emerged from the spores. Homology-dependent silencing experiments supported an essential role of piCdc14 in sporulation. It is proposed that the function of piCdc14 may be to synchronise nuclear behaviour during sporulation and maintain dormancy in spores until germination. These results help illuminate the process of sporulation in oomycetes and the evolution of the cell cycle in eukaryotes.

Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal.

The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos. A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation. A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells. The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).

Peroxisome-proliferator-activated receptor δ mediates the effects of long-chain fatty acids on post-confluent cell proliferation

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation. PPARgamma, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis. We have recently demonstrated that PPARdelta, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARgamma, thereby inducing adipose differentiation. Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown. We show here that PPARdelta is directly implicated in fatty acid-induced cell proliferation. Ectopic expression of PPARdelta renders 3T3C2 cells capable of responding to treatment with long-chain fatty acids by a resumption of mitosis, and this effect is limited to a few days after confluence. This response is restricted to PPARdelta activators and, for fatty acids, takes place within the range of concentrations found to trigger differentiation of preadipocytes both in vitro and in vivo. Furthermore, the use of a mutated inactive PPARdelta demonstrated that transcriptional activity of the nuclear receptor is required to mediate fatty acid-induced proliferation. These data demonstrate that PPARdelta, as a transcription factor, is directly implicated in fatty acid-induced proliferation, and this could explain the hyperplastic development of adipose tissue that occurs in high-fat-fed animals.

Acquisition of theH19Methylation Imprint Occurs Differentially on the Parental Alleles during Spermatogenesis

The imprinted mouseH19gene is hypomethylated on the expressed maternal allele and hypermethylated on the silent paternal allele. A 2-kb region of differential methylation located from −2 to −4 kb relative to theH19transcriptional start site has been proposed to act as the imprinting mark since hypermethylation in this region is inherited from sperm and retained on the paternal allele throughout development. Here, we describe a temporal analysis of the methylation patterns at theH19locus during postnatal male germ cell development. The 2-kb region is methylated on the paternal allele throughout spermatogenesis, suggesting that methylation is acquired in this region prior to the resumption of mitosis in postnatal male mice. Likewise, more than half of the maternal alleles are hypermethylated prior to the resumption of mitosis. However, the remaining maternal alleles are not hypermethylated until the completion of meiosis I, indicating thatde novomethylation in this region is a continuous process. Sequences proximal to theH19promoter, which are methylated in spermatozoa and on the paternal allele in somatic cells, are differentially methylated in diploid, mitotic spermatogonia. The maternal allele becomes hypermethylated in this region during meiotic prophase. Thus, the parentalH19alleles acquire methylation differentially in the male germline.

Transforming growth factor beta1 and beta2 reduce the number of gonocytes by increasing apoptosis.

Transforming growth factors beta1 and beta2 (TGFbetas) have recently been detected by immunohistochemistry in the fetal and neonatal rat testis, and the aim of the present study was to determine whether these factors can act as local regulators to control the number of gonocytes. Testes were kept in organ culture, and TGFbeta1 was found to have dose-dependent inhibitory effect on the number of gonocytes in testes explanted on fetal day 13.5. Either TGFbeta1 or beta2 at 10 ng/ml reduced the number of gonocytes by half after 2 days culture. TGFbetas did not decrease the BrdU labeling index of gonocytes or Sertoli cells, whereas these factors significantly increased the DNA fragmentation in gonocytes (TUNEL method). The other testicular cell types showed no positive TUNEL reaction. TGFbeta1 did not reduce the number of gonocytes in testes explanted on fetal day 17.5 (i.e. during the quiescent phase), but it did so in testes explanted on postnatal day 3 (i.e. stage of resumption of mitosis). To determine the potential cell type targets for TGFbetas, type I and type II TGFbeta receptors were immunolocalized in developing testis from fetal day 13.5 to postnatal day 3. Both receptors were present in the gonocytes throughout the whole period studied, and in the Leydig cells from fetal day 16.5 onward, but they were not detected in the Sertoli cells. Taken together, these results suggest that TGFbetas directly increase apoptosis in gonocytes without changing their mitotic activity during the developmental phases of proliferation.