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Dependence of sperm structural and functional integrity on testicular calcineurin isoform PPP3R2 expression.

ABSTRACT: After leaving the testis, mammalian sperm undergo a sequential maturation process in the epididymis followed by capacitation during their movement through the female reproductive tract. These phenotypic changes are associated with modification of protein phosphorylation and membrane remodeling, which is requisite for sperm to acquire forward motility and induce fertilization. However, the molecular mechanisms underlying sperm maturation and capacitation are still not fully understood. Herein, we show that PPP3R2, a testis-specific regulatory subunit of protein phosphatase 3 (an isoform of calcineurin in the testis), is essential for sperm maturation and capacitation. Knockout of Ppp3r2 in mice leads to male sterility due to sperm motility impairment and morphological defects. One very noteworthy change includes increases in sperm membrane stiffness. Moreover, PPP3R2 regulates sperm maturation and capacitation via (i) modulation of membrane diffusion barrier function at the annulus and (ii) facilitation of cholesterol efflux during sperm capacitation. Taken together, PPP3R2 plays a critical role in modulating cholesterol efflux and mediating the dynamic control of membrane remodeling during sperm maturation and capacitation.

SUBMITTER: Liu Y

PROVIDER: S-EPMC7493031 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Dependence of sperm structural and functional integrity on testicular calcineurin isoform PPP3R2 expression.

Liu Yue Y Zhang Chujun C Wang Shiyao S Hu Yanqin Y Jing Jia J Ye Luyao L Jing Ran R Ding Zhide Z

Journal of molecular cell biology 20200701 7

After leaving the testis, mammalian sperm undergo a sequential maturation process in the epididymis followed by capacitation during their movement through the female reproductive tract. These phenotypic changes are associated with modification of protein phosphorylation and membrane remodeling, which is requisite for sperm to acquire forward motility and induce fertilization. However, the molecular mechanisms underlying sperm maturation and capacitation are still not fully understood. Herein, we ...[more]

PMID: 31900494

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Project description:A global top priority for ameliorating male factor infertility includes identification of environmental factors and mechanisms that impact sperm function. Detection of endocrine disruptors (EDs) in seminal plasma and within the female reproductive tract has created an urgent need to understand how environmental stressors alter postejaculatory sperm function. Tributyltin chloride (TBT) is a model organotin, ED and epigenetic modifier that causes reproductive disorders in testicular sperm; however, consequences of TBT exposure on postejaculatory sperm prior to fertilization remain unknown. Our previous data indicate that TBT-exposed sperm affects initial cleavage and lowers embryo development potential. However, identifying reasons for idiopathic male factor subfertility is challenged by lack of routine clinical assessment for subtle phenotypes that compromise sperm function and therefore remain undetected with current diagnostic tools. Results The present study was aimed at identifying structural, genomic, and epigenomic consequences of TBT exposure to postejaculatory sperm in a temporal manner reflective of sperm transport prior to fertilization within the female reproductive tract. Bovine sperm were exposed to TBT (0 ,1, 10, 100nM) for 24 h under non-capacitating conditions at 25°C followed by quantification of sperm kinematics at 37°C, DNA integrity, and methylation status. No differences in sperm kinematics or capacitation status were detected after 24 h. However, acrosome integrity was compromised at both 0 and 24 h (P ≤ 0.05). Sperm DNA integrity was also negatively affected after 24 h. The methylation levels of select loci were quantified by PacBio single-molecule real-time (SMRT) long-read high-fidelity sequencing. Whole genome methyl-seq revealed 750 differentially methylated regions (DMRs) associated with exposure to TBT for 24 h. IPA and GO analyses identified embryo development, cell signaling, and transcriptional regulation as the most relevant bio-functions of TBT altered DMRs. Conclusions Postejaculatory mammalian sperm exposure to TBT negatively affects parameters important for sperm function while altering DNA integrity and the methylation profile of promotor regions. Consequences of sperm exposure to TBT include cellular and molecular mechanisms that are important for sperm function but remain undetected by routine clinical analyses. These findings provided new insight towards environmental impacts on postejaculatory sperm function.

Project description:Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10-12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.

Project description:BackgroundTesticular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age.ResultsComparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N).ConclusionsThis study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility.

Dataset Information

Dependence of sperm structural and functional integrity on testicular calcineurin isoform PPP3R2 expression.

Publications

Dependence of sperm structural and functional integrity on testicular calcineurin isoform PPP3R2 expression.

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