Sperm Nuclear Morphology Research Articles

P-028 Loss of the m6A methyltransferase METTL5 impairs spermiogenesis and male fertility

Abstract Study question Whether and how METTL5 regulates germ cell development during spermatogenesis. Summary answer METTL5 is required for spermiogenesis. Loss of METTL5 resulted in teratozoospermia and male infertility via reduced translation of acrosome and flagellum formation proteins. What is known already The roles of m6A modifications on mRNA in spermatogenesis have been extensively studied. It was reported that METTL5 knockout mice showed the brain development defect and sterility of 16-week male mice. However, the detailed mechanism of METTL5 affecting male fertility remains elusive. Study design, size, duration Mettl5 KO mice were kindly gifted from Prof. Shuibin Lin of The First Affiliated Hospital of Sun Yat-sen University. Heterozygotes of Mettl5 mice were used to generate Mettl5 homozygous knockout mice. The phenotype of KO mice was assessed. Ribosomal sequencing, proteomics analysis and further validation of protein translation were performed to explore the mechanism. Participants/materials, setting, methods Fertility assessment, sperm parameter analyses, sperm nuclear morphology analysis, Transmission electron microscopy (TEM), tissue Collection and histological analysis, protein extraction and western blot analysis, immunofluorescence studies, cDNA synthesis and qRT-PCR, in vitro fertilization (IVF) were used in this study. Main results and the role of chance Here we reported that methyltransferase-like 5 (METTL5) is involved in spermiogenesis as a methyltransferase mediating m6A modification on rRNA. Mettl5 knockout mice were infertile with teratozoospermia. The acrosome in the sperm head was absent with reduced sperm motility. Furthermore, the fertilization ability of sperm in the IVF experiment failed. Mechanistically, the level of rRNA m6A modification was significantly decreased in the testes of Mettl5 KO mice. The translational efficiency and protein levels of acrosome and flagellum formation proteins such as FSIP2, ODF2, GK2, PGK2, and AKAP4 were significantly reduced when METTL5 was depleted. Limitations, reasons for caution The METTL5 mutation in the patient with teratozoospermia was not examined in the present study Wider implications of the findings The rRNA m6A modification is also involved in regulating spermatogenesis by METTL5. This study highlights the critical role of rRNA epigenetic modifications during spermatogenesis and provides novel theoretical explanations for the m6A modifications. Trial registration number Not applicable

Telomere Distribution in Human Sperm Heads and Its Relation to Sperm Nuclear Morphology: A New Marker for Male Factor Infertility?

Infertility is a problem affecting an increasing number of couples worldwide. Currently, marker tests for male factor infertility are complex, highly technical and relatively subjective. Up to 40% of cases of male factor infertility are currently diagnosed as idiopathic therefore, there is a clear need for further research into better ways of diagnosing it. Changes in sperm telomere length have been associated with infertility and closely linked to DNA damage and fragmentation, which are also known to be related to infertility. However, telomere distribution is a parameter thus far underexplored as an infertility marker. Here, we assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts. In addition, we used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups. Our findings indicate that the infertile cohort sperm nuclei were, on average, 2.9% larger in area and showed subtle differences in sperm head height and width. Telomeres were mainly distributed towards the periphery of the nuclei in the control cohort, with diminishing telomere signals towards the center of the nuclei. Sperm nuclei of infertile males, however, had more telomere signals towards the center of the nuclei, a finding supported by 3D imaging. We conclude that, with further development, both morphology and telomere distribution may prove useful investigative tools in the fertility clinic.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization.

Flowering plants have a unique sexual reproduction system called 'double fertilization',in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes' nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.

Morphological nuclear integrity of sperm cells is associated with preimplantation genetic aneuploidy screening cycle outcomes

To examine the effect of sperm morphology on embryo development at the chromosomal level. Prospective study. Assisted fertilization center. Couples who underwent IVF-PGS cycle, as a result of advanced maternal age, were randomly allocated into two groups: intracytoplasmic sperm injection (ICSI; n = 60) or intracytoplasmic morphologically selected sperm injection (IMSI; n = 60). IVF in conjunction with preimplantation genetic screening (PGS). Sperm nuclear morphology at high-magnification ICSI and incidence of aneuploidy in derived embryo. There was a significantly increased incidence for sex chromosome aneuploidy in ICSI embryos when compared with IMSI embryos (23.5% vs. 15.0%, respectively). High-magnification sperm selection was associated with a significantly lower risk of sex chromosome abnormalities (odds ratio [OR], 0.57; confidence interval [CI], 0.37-0.90). The incidence of chaotic embryos was also significantly higher with the ICSI procedure (27.5% vs. 18.8%), while the IMSI procedure was associated with a significantly lower risk of chaotic embryos (OR, 0.64; CI, 0.43-0.96). Moreover, the cycle cancellation rate was significantly higher in ICSI cycles (11.8% vs. 2.5%). High-magnification sperm selection was a significant predictor of the likelihood of cycle cancellation (OR, 0.26; CI, 0.11-0.62). Spermatozoa free of nuclear morphological malformations were found to be significantly associated with the lower incidence of aneuploidy in derived embryos, resulting in lower rates of cycle cancellation.

Presence and Release of Bovine Sperm Histone H1 During Chromatin Decondensation by Heparin-Glutathione

During spermatogenesis, changes in sperm nuclear morphology are associated with the replacement of core somatic histones by protamines. Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones. Histone H1, along with protamines, mediates chromatin condensation into an insoluble complex that is transcriptionally inactive. In vitro, heparin-reduced glutathione causes sperm decondensation, and the structures formed are morphologically similar to the in vivo male pronucleus. To study the participation of histone H1 in bovine sperm chromatin remodelling, we measured the presence and release of histone H1 by immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis, and immunoblotting. Nuclear decondensation was induced by 80 μM heparin and 15.0 mM reduced glutathione (GSH) for 7, 14, and 21 h at 37°C. Additionally, nucleons, composed of nuclei isolated from the sperm, were decondensed with 20.0 μM heparin and 5.0 mM GSH for 4.0 h at 37°C. Controls were incubated in buffer for similar periods of time. Immunofluorescent localization of histone H1 was carried out with mouse monoclonal antibody, and DNA localization was visualized by 0.001% quinacrine staining. Chromatin decondensation was accompanied by increased sperm nuclei and nucleon surface area. We observed that histone H1 was localized exclusively in the nuclei of intact sperm and nucleons. Histone H1 immunofluorescent intensity did not change in control samples but decreased over time in samples treated with heparin-GSH. There was a negative correlation between the surface area of sperm nuclei and immunohistochemical intensity of histone H1 (P < 0.05). Nucleon decondensation showed a similar relationship. By electrophoresis and immunoblotting, we verified the loss of histone H1 from the sperm and nucleons and its release into the incubation media. Based on these results, we propose that histone H1 is present in the bovine sperm nuclei. H1 depletion may participate in chromatin decondensation and nuclear swelling induced by heparin-GSH.

Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo ( Bubalus bubalis) semen quality

The objective of this study was to evaluate sperm functional attributes in relation to in vitro sperm-zona binding ability and cleavage rate in assessing frozen thawed buffalo ( Bubalus bubalis) semen quality. Frozen-thawed forty-eight ejaculates from eight Surti buffalo bulls (six ejaculates/bull) obtained by artificial vagina were used. Frozen semen from each bull was thawed, pooled, and subjected for sperm functional (six replicates) and in vitro fertilization (four replicates) tests. The progressive forward motility, plasmalemma functional integrity assessed by fluorogenic [6-carboxyfluorescein diacetate (CFDA), and propidium iodide (PI)], hypoosmotic swelling (HOS), and hypoosmotic swelling-Giemsa (HOS-G) test, mitochondrial membrane potential, sperm nuclear morphology, the number of sperm bound to zona and cleavage rate differed significantly ( P < 0.05) between bulls. When the animals were grouped based on cleavage rate (group I, >40% cleavage rate, n = 5, and group II, <40% cleavage rate, n = 3), in vitro fertility parameters and all the sperm functional attributes except sperm nuclear morphology differed significantly ( P < 0.05). The proportions of sperm with functional plasmalemma in the tail and intact acrosome assessed by HOS-G test (25.33, range: 17.48–40.27) were significantly ( P < 0.001) lower than the functional plasmalemma in the tail assessed by HOS test (39.80, range: 27.85–54.67). The number of sperm bound to zona had significant correlations with the mitochondrial membrane potential ( r = 0.90, P < 0.01) and plasmalemma integrity (fluorogenic, r = 0.74 and HOS, r = 0.79, P < 0.05) and HOS-G, r = 0.87, P < 0.01). The cleavage rate had significant ( P < 0.05) correlations with the mitochondrial membrane potential ( r = 0.70) and plasmalemma integrity measured by HOS-G test ( r = 0.68). The present study indicates that these attributes could represent important determinants of buffalo sperm quality influencing cleavage rate.

EVALUATION OF SPERM NUCLEAR MORPHOLOGY IN SPECIMENS WITH ABNORMAL SPERM CHROMATIN STRUCTURAL ASSAYS

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000x. The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9+/-16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.

Reconstitution of sperm nuclei of zebrafish (Danio rerio) in Xenopus egg extracts.

Cell-free extracts of Xenopus eggs cause cyclic change in permeabilized sperm nucleus, nuclear envelope breakdown, chromosome condensation, and reformation of nuclei. In this study, the ability of cell-free extracts to cause similar changes in zebrafish sperm was examined. When lysolecithin-treated sperm from zebrafish were incubated in Xenopus egg extracts, a series of changes in sperm nuclear morphology were observed periodically. These changes correlated with maturation-promoting factor (MPF) activity. Furthermore, sperm nuclei of zebrafish replicated DNA during reconstitution in Xenopus egg extracts. These results showed that cell-free extracts of Xenopus egg possess the ability to cause cell-cycle-dependent changes in zebrafish sperm, implying the possibility of generating transgenic zebrafish in a similar way to transgenic Xenopus.

Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects.

An important goal of modern analyses of semen is to elucidate the molecular traits of mammalian sperm chromatin structural abnormalities, defined here as 'uncompensable', that lead to abnormalities in fertility, pronuclear formation, early embryo quality and pregnancy outcome. Sperm with uncompensable nuclear abnormalities are able to fertilize oocytes both in vivo and in vitro; however, due to the uncompensable trait(s), the embryo development may be abnormal. Uncompensable nuclear traits can be experimentally induced in bull sperm by a mild thermal insult to the testis. Sperm nuclear morphology abnormalities seen in ejaculates 11-days post stress are likely related to molecular changes in chromatin observed 3-days post stress by the flow cytometric sperm chromatin structure assay (SCSA). The SCSA measures the susceptibility of sperm nuclear DNA to denaturation in situ. This susceptibility has been correlated with the presence of DNA strand breaks that may be derived in part by oxidative stress and possibly by a unique, abortive apoptotic mechanism. The extent of DNA denaturation is not significantly related to the level of disulfide bonding between the chromatin protamines. The use of human sperm with uncompensable nuclear traits for artificial reproductive techniques is also discussed. The goal of this research is to remove from semen doses those sperm with uncompensable nuclear traits and thereby increase male fertility potential.