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Subcellular localization of phospholipase C? in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

ABSTRACT: We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLC?), the sperm factor thought to induce the Ca(2+) oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLC? in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLC? and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLC? is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca(2+) oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLC? in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLC? in globozoospermic sperm is thus consistent and reinforces the role of PLC? as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

SUBMITTER: Escoffier J

PROVIDER: S-EPMC4311148 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

Escoffier Jessica J Yassine Sandra S Lee Hoi Chang HC Martinez Guillaume G Delaroche Julie J Coutton Charles C Karaouzène Thomas T Zouari Raoudha R Metzler-Guillemain Catherine C Pernet-Gallay Karin K Hennebicq Sylviane S Ray Pierre F PF Fissore Rafael R Arnoult Christophe C

Molecular human reproduction 20141029 2

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca(2+) oscillations at fertilization, was a ...[more]

PMID: 25354701

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Project description:We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia may be associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in the companion article that oocyte activation (OA) with globozoospermic sperm is very poor and due to the absence of phospholipase Cζ; therefore artificial OA (AOA) was used to bypass defective OA. Herein, we evaluated the developmental potential of embryos generated by ICSI + AOA in mice. We demonstrate that although OA was fully rescued, preimplantation development was impaired when using globozoospermic sperm. In human, a small number of embryos could be generated with sperm from DPY19L2-deleted patients in the absence of AOA and these embryos also showed a poor developmental potential. In conclusion, we show that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Most of the DNA breaks were already present when the sperm reached the epididymis, indicating that they occurred inside the testis. This result thus suggests that testicular sperm extraction in Dpy19l2-dependent globozoospermia is not recommended. These defects may largely explain the poor embryonic development of most mouse and human embryos obtained with globozoospermic sperm.

Project description:During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1-/-) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1-/--sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1-/- sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1-/- sperm and Plcz1-/- and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1-/-eCS-/-), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1-/- sperm + PLCζ mRNA, Plcz1-/- sperm + SrCl2 and Plcz1-/-eCS-/- sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1-/- sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1-/- sperm + SrCl2:4.0 ± 4.3% and Plcz1-/-eCS-/- sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1-/- sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1-/- sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.

Dataset Information

Subcellular localization of phospholipase C? in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

Publications

Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

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