Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure.

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans.

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.

Variant PNLDC1, Defective piRNA Processing, and Azoospermia.

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.

A multicentre double-blinded randomized controlled trial on the efficacy of laser-assisted hatching in patients with repeated implantation failure undergoing IVF or ICSI.

STUDY QUESTION: Does assisted hatching increase the cumulative live birth rate in subfertile couples with repeated implantation failure? SUMMARY ANSWER: This study showed no evidence of effect for assisted hatching as an add-on in subfertile couples with repeated implantation failure. WHAT IS KNOWN ALREADY: The efficacy of assisted hatching, with regard to the live birth rate has not been convincingly demonstrated in randomized trials nor meta-analyses. It is suggested though that especially poor prognosis women, e.g. women with repeated implantation failure, might benefit most from assisted hatching. STUDY DESIGN, SIZE, DURATION: The study was designed as a double-blinded, multicentre randomized controlled superiority trial. In order to demonstrate a statistically significant absolute increase in live birth rate of 10% after assisted hatching, 294 participants needed to be included per treatment arm, being a total of 588 subfertile couples. Participants were included and randomized from November 2012 until November 2017, 297 were allocated to the assisted hatching arm of the study and 295 to the control arm. Block randomization in blocks of 20 participants was applied and randomization was concealed from participants, treating physicians, and laboratory staff involved in the embryo transfer procedure. Ovarian hyperstimulation, oocyte retrieval, laboratory procedures, embryo selection for transfer and cryopreservation, the transfer itself, and luteal support were performed according to local protocols and were identical in both the intervention and control arm of the study with the exception of the assisted hatching procedure which was only performed in the intervention group. The laboratory staff performing the assisted hatching procedure was not involved in the embryo transfer itself. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were eligible for inclusion in the study after having had either at least two consecutive fresh IVF or ICSI embryo transfers, including the transfer of frozen and thawed embryos originating from those fresh cycles, and which did not result in a pregnancy or as having had at least one fresh IVF or ICSI transfer and at least two frozen embryo transfers with embryos originating from that fresh cycle which did not result in a pregnancy. The study was performed at the laboratory sites of three tertiary referral hospitals and two university medical centres in the Netherlands. MAIN RESULTS AND THE ROLE OF CHANCE: The cumulative live birth rate per started cycle, including the transfer of fresh and subsequent frozen/thawed embryos if applicable, resulted in 77 live births in the assisted hatching group (n = 297, 25.9%) and 68 live births in the control group (n = 295, 23.1%). This proved to be statistically not significantly different (relative risk: 1.125, 95% CI: 0.847 to 1.494, P = 0.416). LIMITATIONS, REASONS FOR CAUTION: There was a small cohort of subfertile couples that after not achieving an ongoing pregnancy, still had cryopreserved embryos in storage at the endpoint of the trial, i.e. 1 year after the last randomization. It cannot be excluded that the future transfer of these frozen/thawed embryos increases the cumulative live birth rate in either or both study arms. Next, at the start of this study, there was no international consensus on the definition of repeated implantation failure. Therefore, it cannot be excluded that assisted hatching might be effective in higher order repeated implantation failures. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrated no evidence of a statistically significant effect for assisted hatching by increasing live birth rates in subfertile couples with repeated implantation failure, i.e. the couples which, based on meta-analyses, are suggested to benefit most from assisted hatching. It is therefore suggested that assisted hatching should only be offered if information on the absence of evidence of effect is provided, at no extra costs and preferably only in the setting of a clinical trial taking cost-effectiveness into account. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NTR 3387, NL 3235, https://www.clinicaltrialregister.nl/nl/trial/26138). TRIAL REGISTRATION DATE: 6 April 2012. DATE OF FIRST PATIENT'S ENROLMENT: 28 November 2012.

Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice.

Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129-null mutant testes possess low levels of piRNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.

Chromosome-wide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation.

In mammalian males, the first meiotic prophase is characterized by formation of a separate chromatin domain called the sex body. In this domain, the X and Y chromosomes are partially synapsed and transcriptionally silenced, a process termed meiotic sex-chromosome inactivation (MSCI). Likewise, unsynapsed autosomal chromatin present during pachytene is also silenced (meiotic silencing of unsynapsed chromatin, MSUC). Although it is known that MSCI and MSUC are both dependent on histone H2A.X phosphorylation mediated by the kinase ATR, and cause repressive H3 Lys9 dimethylation, the mechanisms underlying silencing are largely unidentified. Here, we demonstrate an extensive replacement of nucleosomes within unsynapsed chromatin, depending on and initiated shortly after induction of MSCI and MSUC. Nucleosomal eviction results in the exclusive incorporation of the H3.3 variant, which to date has primarily been associated with transcriptional activity. Nucleosomal exchange causes loss and subsequent selective reacquisition of specific histone modifications. This process therefore provides a means for epigenetic reprogramming of sex chromatin presumably required for gene silencing in the male mammalian germ line.

Incomplete meiotic sex chromosome inactivation in the domestic dog.

BACKGROUND: In mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes. Asynapsis of these arms and the persistent DSBs then trigger transcriptional silencing through meiotic sex chromosome inactivation (MSCI), resulting in formation of the XY body. This inactive state is partially maintained in post-meiotic haploid spermatids (postmeiotic sex chromatin repression, PSCR). For the human, establishment of MSCI and PSCR have also been reported, but X-linked gene silencing appears to be more variable compared to mouse. To gain more insight into the regulation and significance of MSCI and PSCR among different eutherian species, we have performed a global analysis of XY pairing dynamics, DSB repair, MSCI and PSCR in the domestic dog (Canis lupus familiaris), for which the complete genome sequence has recently become available, allowing a thorough comparative analyses. RESULTS: In addition to PAR synapsis between X and Y, we observed extensive self-synapsis of part of the dog X chromosome, and rapid loss of known markers of DSB repair from that part of the X. Sequencing of RNA from purified spermatocytes and spermatids revealed establishment of MSCI. However, the self-synapsing region of the X displayed higher X-linked gene expression compared to the unsynapsed area in spermatocytes, and was post-meiotically reactivated in spermatids. In contrast, genes in the PAR, which are expected to escape MSCI, were expressed at very low levels in both spermatocytes and spermatids. Our comparative analysis was then used to identify two X-linked genes that may escape MSCI in spermatocytes, and 21 that are specifically re-activated in spermatids of human, mouse and dog. CONCLUSIONS: Our data indicate that MSCI is incomplete in the dog. This may be partially explained by extensive, but transient, self-synapsis of the X chromosome, in association with rapid completion of meiotic DSB repair. In addition, our comparative analysis identifies novel candidate male fertility genes.

Optimized flow cytometry isolation of murine spermatocytes.

Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis, and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment, and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells' similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult mouse testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P, and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying MPI.

Improved phenotypic classification of male infertility to promote discovery of genetic causes.

An increasing number of genes are being described in the context of non-syndromic male infertility. Linking the underlying genetic causes of non-syndromic male infertility with clinical data from patients is important to establish new genotype-phenotype correlations. This process can be facilitated by using universal nomenclature, but no standardized vocabulary is available in the field of non-syndromic male infertility. The International Male Infertility Genomics Consortium aimed at filling this gap, providing a standardized vocabulary containing nomenclature based on the Human Phenotype Ontology (HPO). The "HPO tree" was substantially revised compared with the previous version and is based on the clinical work-up of infertile men, including physical examination and hormonal assessment. Some causes of male infertility can already be suspected based on the patient's clinical history, whereas in other instances, a testicular biopsy is needed for diagnosis. We assembled 49 HPO terms that are linked in a logical hierarchy and showed examples of morphological features of spermatozoa and testicular histology of infertile men with identified genetic diagnoses to describe the phenotypes. This work will help to record patients' phenotypes systematically and facilitate communication between geneticists and andrologists. Collaboration across institutions will improve the identification of patients with the same phenotypes, which will promote the discovery of novel genetic causes for non-syndromic male infertility.

Cytoplasmic compartmentalization of the fetal piRNA pathway in mice.

Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic "cementing material" first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function.

Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2'-deoxycytidine.

5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo(-) and DNA polymerase ß. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.

Sperm-derived histones contribute to zygotic chromatin in humans.

BACKGROUND: about 15% to 30% of the DNA in human sperm is packed in nucleosomes and transmission of this fraction to the embryo potentially serves as a mechanism to facilitate paternal epigenetic programs during embryonic development. However, hitherto it has not been established whether these nucleosomes are removed like the protamines or indeed contribute to paternal zygotic chromatin, thereby potentially contributing to the epigenome of the embryo. RESULTS: to clarify the fate of sperm-derived nucleosomes we have used the deposition characteristics of histone H3 variants from which follows that H3 replication variants present in zygotic paternal chromatin prior to S-phase originate from sperm. We have performed heterologous ICSI by injecting human sperm into mouse oocytes. Probing these zygotes with an antibody highly specific for the H3.1/H3.2 replication variants showed a clear signal in the decondensed human sperm chromatin prior to S-phase. In addition, staining of human multipronuclear zygotes also showed the H3.1/H3.2 replication variants in paternal chromatin prior to DNA replication. CONCLUSION: these findings reveal that sperm-derived nucleosomal chromatin contributes to paternal zygotic chromatin, potentially serving as a template for replication, when epigenetic information can be copied. Hence, the execution of epigenetic programs originating from transmitted paternal chromatin during subsequent embryonic development is a logical consequence of this observation.

Transient reduction of DNA methylation at the onset of meiosis in male mice.

BACKGROUND: Meiosis is a specialized germ cell cycle that generates haploid gametes. In the initial stage of meiosis, meiotic prophase I (MPI), homologous chromosomes pair and recombine. Extensive changes in chromatin in MPI raise an important question concerning the contribution of epigenetic mechanisms such as DNA methylation to meiosis. Interestingly, previous studies concluded that in male mice, genome-wide DNA methylation patters are set in place prior to meiosis and remain constant subsequently. However, no prior studies examined DNA methylation during MPI in a systematic manner necessitating its further investigation. RESULTS: In this study, we used genome-wide bisulfite sequencing to determine DNA methylation of adult mouse spermatocytes at all MPI substages, spermatogonia and haploid sperm. This analysis uncovered transient reduction of DNA methylation (TRDM) of spermatocyte genomes. The genome-wide scope of TRDM, its onset in the meiotic S phase and presence of hemimethylated DNA in MPI are all consistent with a DNA replication-dependent DNA demethylation. Following DNA replication, spermatocytes regain DNA methylation gradually but unevenly, suggesting that key MPI events occur in the context of hemimethylated genome. TRDM also uncovers the prior deficit of DNA methylation of LINE-1 retrotransposons in spermatogonia resulting in their full demethylation during TRDM and likely contributing to the observed mRNA and protein expression of some LINE-1 elements in early MPI. CONCLUSIONS: Our results suggest that contrary to the prevailing view, chromosomes exhibit dynamic changes in DNA methylation in MPI. We propose that TRDM facilitates meiotic prophase processes and gamete quality control.

gammaH2AX signalling during sperm chromatin remodelling in the mouse zygote.

In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions induced thereafter will have to be restored upon gamete fusion in the zygote. Misrepair of such lesions often results in chromosome type aberrations at the first cleavage division, suggesting that the repair event takes place prior to S-phase. During this stage of the zygotic cell cycle, the paternal chromatin transits from a protamine- to a nucleosome-based state. We addressed the question whether the canonical signalling pathway to DNA double strand breaks (DSBs), the phosphorylated form of histone H2AX (gammaH2AX) is active during chromatin restructuring of the male genetic complement in the zygote. Here, we describe the detailed characterization of gammaH2AX signalling in the early stages of zygotic development up to the appearance of the pronuclei. We have found the gammaH2AX signalling pathway to be already active during sperm chromatin remodelling after gamete fusion in a dose dependent manner, reflecting the amount of DSBs present in the sperm nucleus after in vivo male irradiation. Using DNA damaging compounds to induce lesions in the early zygote, differences in DSB sensitivity and gammaH2AX processing between paternal and maternal chromatin were found, suggesting differences in DNA repair capacity between the parental chromatin sets.

Fen1 does not control somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat in a knock-in mouse model for DM1.

The mechanism of trinucleotide repeat expansion, an important cause of neuromuscular and neurodegenerative diseases, is poorly understood. We report here on the study of the role of flap endonuclease 1 (Fen1), a structure-specific nuclease with both 5' flap endonuclease and 5'-3' exonuclease activity, in the somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat of the DMPK gene in a mouse model for myotonic dystrophy type 1 (DM1). By intercrossing mice with Fen1 deficiency with transgenics with a DM1 (CTG)(n)*(CAG)(n) repeat (where 104n110), we demonstrate that Fen1 is not essential for faithful maintenance of this repeat in early embryonic cleavage divisions until the blastocyst stage. Additionally, we found that the frequency of somatic DM1 (CTG)(n)*(CAG)(n) repeat instability was essentially unaltered in mice with Fen1 haploinsufficiency up to 1.5 years of age. Based on these findings, we propose that Fen1, despite its role in DNA repair and replication, is not primarily involved in maintaining stability at the DM1 locus.

Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote.

In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. We elaborate on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, we performed a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage and show that asymmetries reported earlier are systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process which is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and presence of loosely anchored HP1-beta combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary the male, in contrast to female G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant.

A role for retrotransposon LINE-1 in fetal oocyte attrition in mice.

Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition. We demonstrate that experimental elevation of L1 expression correlates with increased MPI defects, FOA, oocyte aneuploidy, and embryonic lethality. Conversely, reverse transcriptase (RT) inhibitor AZT has a profound effect on the FOA dynamics and meiotic recombination, and it implicates an RT-dependent trigger in oocyte elimination in early MPI. We propose that FOA serves to select oocytes with limited L1 activity that are therefore best suited for the next generation.

Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications.

The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos.