Remodelling the paternal chromatin at fertilization in mammals

Abstract

At fertilization, the highly condensed and transcriptionally inert chromatin of the spermatozoa becomes remodelled into the decondensed and transcriptionally competent chromatin of the male pronucleus. The chromatin initially becomes dispersed and then transiently recondenses into a small mass upon entry into the ooplasm. This morphological change is coincident with and likely dependent on the replacement of the sperm-specific protamines by oocyte-supplied histones and the organization of the chromatin into nucleosomes. The chromatin then extensively decondenses within the male pronucleus and acquires many of the proteins that are associated with the maternal chromatin. Nonetheless, the paternal chromatin manifests distinct characteristics, including transient hyperacetylation of histone H4, increased transcription of endogenous and microinjected genes, and replication-independent demethylation of DNA. Sperm chromatin remodelling is controlled by an oocyte activity that appears during meiotic maturation and disappears approximately 3 h after activation (release from metaphase II arrest), and which requires factors associated with the germinal vesicle of the oocyte. The molecular components of this activity remain largely unknown. In frogs, nucleoplasmin is required to assemble histones H2A and H2B onto the paternal chromatin. Evidence is presented that related proteins may perform similar functions in mammals. Identifying the mechanisms that underlie sperm chromatin remodelling at fertilization may be relevant for understanding reprogramming of somatic cell nuclei after transfer into oocytes.

Fig. 1

Summary of sperm chromatin remodelling in mammals. During spermatogenesis, sperm histones become acetylated and are replaced by transition proteins, which are then replaced by protamines. After fertilization, the protamines are replaced by oocyte-supplied histones. This replacement occurs as the oocyte completes the second meiotic division and coincides with a transient recondensation of the paternal chromatin. After the protamine–histone exchange, 5-methylcytosine becomes undetectable on the paternal DNA. The paternal and maternal chromatin then decondense extensively within separate pronuclei, and the histones on the paternal chromatin become deacetylated. Other oocyte proteins (centromeric proteins CENP-A/B, and transcription factors TBP and Sp1) imported into the pronuclei further modify the chromatin and DNA replication begins.

Fig. 2

Expression of mRNA encoding nucleoplasmin 3 (Npm3) and nucleosome-assembly protein 1 (NAP-1) in mammalian oocytes at different stages of development. RT–PCR was performed using cDNA prepared from 30 oocytes. No RT: reverse transcriptase was omitted from the sample; M: 100-nt molecular size markers; GI: growing, incompetent; GC: growing, competent; FG: fully grown; MII: metaphase II.

Fig. 3

Effect of microinjection of antisense oligonucleotides on sperm chromatin morphology and histone assembly after fertilization in mammals. Oocytes arrested at prophase I were injected with oligonucleotides targeting nucleosome-assembly protein 1 (NAP-1) or nucleoplasmin 3 (Npm3), allowed to mature to metaphase II, and then fertilized. The zygotes were fixed and stained 4 h later using 4,6-diamino-2-phenylindole (DAPI) to assess maternal and paternal chromatin morphology (left panels) and an anti-histone antibody to assess histone content (right panels). Top: control uninjected oocyte. Two pronuclei (pn) containing immunodetectable histones are visible. Middle: oocyte injected with NAP-1 antisense oligonucleotides. Two pronuclei containing immunodetectable histone are visible. Bottom: oocyte injected with NAP-1 and Npm3 antisense oligonucleotides. Female pronucleus (inset) is fully developed and contains histones. Paternal chromatin has not progressed beyond the dispersed (ds) state and does not contain detectable histones.

Fig. 4

A model for the development during oogenesis of the activity that assembles histones on to paternal chromatin at fertilization in mammals. During growth, oocytes accumulate mRNAs encoding proteins that mediate DNA replication-independent assembly of histones on to chromatin. These mRNAs become translatable or reach a threshold level at the time that oocytes acquire meiotic competence, and through a process promoted by Ca²⁺ oscillations. During meiotic maturation the mRNAs are translated to generate histone-assembly activity. Although the activity is functional in maturing oocytes, it normally is not manifested until fertilization activates fully mature metaphase II oocytes.