DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

The lack of Tex44 causes severe subfertility with flagellar abnormalities in male mice.

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility.

Neurotensin and Its Involvement in Reproductive Functions: An Exhaustive Review of the Literature.

Neurotensin (NTS) is a peptide discovered in 1973, which has been studied in many fields and mainly in oncology for its action in tumor growth and proliferation. In this review of the literature, we wanted to focus on its involvement in reproductive functions. NTS participates in an autocrine manner in the mechanisms of ovulation via NTS receptor 3 (NTSR3), present in granulosa cells. Spermatozoa express only its receptors, whereas in the female reproductive system (endometrial and tube epithelia and granulosa cells), we find both NTS secretion and the expression of its receptors. It consistently enhances the acrosome reaction of spermatozoa in mammals in a paracrine manner via its interaction with NTSR1 and NTSR2. Furthermore, previous results on embryonic quality and development are discordant. NTS appears to be involved in the key stages of fertilization and could improve the results of in vitro fertilization, especially through its effect on the acrosomal reaction.

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

Fertilization, but Not Post-Implantation Development, Can Occur in the Absence of Sperm and Oocyte Beta1 Integrin in Mice.

Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm ß1 integrin. Males and females with ß1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm ß1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin ß1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.

Cyclic FEE Peptide Improves Human Sperm Movement Parameters without Modification of Their Energy Metabolism.

Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor's sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.

Deletion of the Spata3 Gene Induces Sperm Alterations and In Vitro Hypofertility in Mice.

Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.

Partial Sperm beta1 Integrin Subunit Deletion Proves its Involvement in Mouse Gamete Adhesion/Fusion.

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Identification of a New QTL Region on Mouse Chromosome 1 Responsible for Male Hypofertility: Phenotype Characterization and Candidate Genes.

Male fertility disorders often have their origin in disturbed spermatogenesis, which can be induced by genetic factors. In this study, we used interspecific recombinant congenic mouse strains (IRCS) to identify genes responsible for male infertility. Using ultrasonography, in vivo and in vitro fertilization (IVF) and electron microscopy, the phenotyping of several IRCS carrying mouse chromosome 1 segments of Mus spretus origin revealed a decrease in the ability of sperm to fertilize. This teratozoospermia included the abnormal anchoring of the acrosome to the nucleus and a persistence of residual bodies at the level of epididymal sperm midpiece. We identified a quantitative trait locus (QTL) responsible for these phenotypes and we have proposed a short list of candidate genes specifically expressed in spermatids. The future functional validation of candidate genes should allow the identification of new genes and mechanisms involved in male infertility.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

Polymorphisms of human placental alkaline phosphatase are associated with in vitro fertilization success and recurrent pregnancy loss.

Fertility is a quantitative, complex character governed by a considerable number of genes. Despite clinical and scientific advances, several cases of human infertility remain unexplained. In the present study, using a positional cloning approach in a mouse model of interspecific recombinant lines, a candidate gene, ALPP, encoding the placental alkaline phosphatase, was identified as being potentially involved in recurrent spontaneous abortion. We then analyzed patients for detecting putative associations between ALPP polymorphisms, in vitro fertilization failures, and miscarriages. ALPP was sequenced in 100 controls and 100 patients affected by recurrent spontaneous abortion, from the same ethnic background. The frequency of several alleles and allelic combinations were different between recurrent spontaneous abortion and control women. One polymorphism induced a coding substitution (Ile89Leu) that was associated with a decreased risk of abortion and in vitro fertilization failure. Thereafter, the population was increased by the analysis of 92 additional controls and 612 additional patients for the coding polymorphism Ile89Leu. We finally show, by functional analysis, that the 89Leu placental alkaline phosphatase has an enhanced alkaline phosphatase activity. This study suggests that ALPP genotyping could be a strong predictor of implantation success.

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization.

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.

Plastic used in in vitro fertilization procedures induces massive placental gene expression alterations.

BACKGROUND: The exposure to plastic derivatives during human life is deleterious. Infants conceived using ART (IVF or ICSI) have twice as many risks of major birth defects compared to naturally conceived infants. Could plastic ware used during ART trigger defects in the fetal development? METHODS: Three groups of blastocysts were transferred to pseudopregnant mice. One was obtained after IVF and embryo development in plastic ware, the second in glass ware. The third, was obtained in vivo by natural mating. On day 16.5 of pregnancy, females were sacrificed and fetal organs collected for gene expression analysis. Fetal sex was determined by RT-PCR. RNA was extracted from a pool of five placental or brain samples coming from at least two litters from the same group and analyzed by hybridisation onto the mouse Affymetrix 430.2.0 GeneChips, confirmed by RT-qPCR for 22 genes. FINDINGS: This study highlights a major impact of plastic ware on placental gene expression (1121 significantly deregulated genes), while glassware was much closer to in vivo offspring (only 200 significantly deregulated genes). Gene Ontology indicated that the modified placental genes were mostly involved in stress, inflammation and detoxification. A sex specific analysis revealed in addition a more drastic effect on female than male placentas. In the brains, whatever the comparison, less than 50 genes were found deregulated. INTERPRETATION: Embryos incubated in plastic ware resulted in pregnancy with massive alterations of placental gene expression profile in concerted biological functions. There were no obvious effects on the brains. Besides other effects, this suggests that plastic ware in ART could be a cause of the increased level of pregnancy disorders observed recurrently in ART pregnancies. FUNDING: This study was funded by two grants from the Agence de la Biomedecine in 2017 and 2019.

H19 acts as a trans regulator of the imprinted gene network controlling growth in mice.

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.

Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes.

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

Sperm-egg interaction: is there a link between tetraspanin(s) and GPI-anchored protein(s)?

Both female mice deficient in CD9 tetraspanin- and oocyte-specific glycosyl-phosphatidylinositol-anchored family proteins showed severely reduced fertility due to the failure of sperm-egg fusion. This raises the question of a link between these two groups of proteins at the oocyte membrane. We propose two hypotheses to explain why the absence of one of these proteins from the oocyte membrane results in the same phenotype. The first hypothesis envisages different levels of control by these molecules of the common induced signaling cascade. The second relies on the known involvement of these molecules in the overall organization of the plasma membrane. Their disappearance could thus prevent sperm-egg fusion either by disruption of the signaling cascade and/or by an important disorganization of the oocyte membrane. In this review, describing their structural and functional characteristics, and using published results on the oocyte, we try to analyze how these two protein families could interact.

SPACA6 ectodomain structure reveals a conserved superfamily of gamete fusion-associated proteins.

SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of the SPACA6 ectodomain at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like ß-sandwich connected via a quasi-flexible linker. This structure is reminiscent of IZUMO1, another gamete fusion-associated protein, making SPACA6 and IZUMO1 founding members of a superfamily of fertilization-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion.

A fertilin-derived peptide improves in vitro maturation and ploidy of human oocytes.

OBJECTIVE: To analyze the effect of a cyclic fertilin-derived peptide (cFEE) on in vitro maturation of human oocytes. DESIGN: Randomized study. SETTING: Fertility center in an academic hospital. PATIENT(S): Not applicable. INTERVENTION(S): Human immature germinal vesicle-stage oocytes (n = 1,629) donated for research according to French bioethics laws were randomly allocated to groups treated with 1 or 100 µM of cFEE or to a control group. They were incubated at 37 °C in 6% CO2 and 5% O2, and their maturation was assessed using time-lapse microscopy over 24 hours. In vitro maturated metaphase II oocytes were analyzed for chromosomal content using microarray comparative genomic hybridization, and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. MAIN OUTCOME MEASURE(S): The percentage of oocytes undergoing maturation in vitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared with the control to obtain insights into its mechanism of action. RESULT(S): cFEE significantly increased the percentage of oocytes that matured in vitro and improved euploidy in meiosis II oocytes by the up-regulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. CONCLUSION(S): cFEE improves human oocyte maturation in vitro and reduces aneuploidy. It may prove useful for treating oocytes before fertilization in assisted reproductive technology and for in vitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

Cyclic fertilin-derived peptide stimulates in vitro human embryo development.

OBJECTIVE: To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin ß (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor. DESIGN: Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts. SETTING: The study was performed in an academic hospital and research laboratory. PATIENT(S): Human embryos donated for research. This project was approved by the French "Agence de la Biomédecine." INTERVENTION(S): Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action. MAIN OUTCOME MEASURE(S): Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 µM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the in vitro hatching rate from 14% to 45%. RESULT(S): Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport. CONCLUSION(S): Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the in vitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome.