Benavente R and KrohneG null (1985), Change of karyoskeleton during spermatogenesis ...

XB-ART-29079

Proc Natl Acad Sci U S A 1985 Sep 01;8218:6176-80.

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Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line.

Benavente R , KrohneG .

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Lamins are the major constituent proteins of the nuclear lamina. In the frog, Xenopus laevis, they are the products of a multigene family whose expression can be correlated to certain routes of cell differentiation. For example, lamins LI (Mr, 72,000) and LII (Mr, 68,000) is expressed, together with LI/LII, in certain highly differentiated cell types such as neurons and muscle cells and is the only lamin present in diplotene oocytes. Here we report the identification by means of two monoclonal antibodies of a fourth lamin (LIV) of Mr 75,000, which is expressed specifically during the later stages of spermatogenesis. In the seminiferous tubules, Sertoli cells contain LI/LII and LIII whereas, among the spermatogenic cells, spermatogonia contain only LI and LII. In contrast, in spermatids and sperm cells these lamins are completely replaced by lamin LIV. Primary spermatocytes are negative with both antibodies, indicating that a switch in the expression of lamins occurs early in spermatogenesis. Lamin LIV is distributed in patches along the nuclear envelopes of elongated spermatids and sperm cells rather than in the characteristic continuous lamina pattern found in most other cells. We hypothesize that the specific expression of lamin LIV is related to the conspicuous changes of nuclear architecture and chronmatin composition that are known to take place during the late stages of sperm development.

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Species referenced: Xenopus laevis
Genes referenced: adm alb lmnb2
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	FIG. 1. Immunofluorescence microscopy on frozen sections through Xenopus testes with monoclonal lamin antibodies [a'-c'; the same sections were stained with 4',6-diamidino-2-phenylindole for visualization of nuclei (a-c)]. (a') Antibody PKB8 reacts with the nuclear periphery of Sertoli cells (arrows), spermatogonia (some are denoted by G), and connective tissue cells (C). (b') Antibody L046F7 stains the nuclear periphery of Sertoli cells (arrows), spermatids (S), and sperms (Sp). (c') Testis of an immature animal after incubation with antibody L046F7. Positive reaction is found only in the Sertoli cells. Primary spermatocytes (SI) are negative with both antibodies (a'-c'). (Bars = 20 um.)
	FIG. 2. Electron microscopic immunolocalization using antibody L046F7 on frozen sections through testis, showing positive reaction in the nuclear (N) periphery of a round spermatid (a) and sperms (c and d). Second antibodies coupled to 5-nm colloidal gold particles (a and c) or to peroxidase (d) were used. (a) Round spermatids show gold label over most of the nucleoplasmic surface of the nuclear envelope (NE). (b) Control section similar to a after incubation with the gold-coupled secondary antibodies only. Gold particles are extremely sparse and randomly distributed in such preparations. (c and d) In sperms, the positive reaction at the nucleoplasmic surface of the inner nuclear membrane is more disperse and usually appears in the form of small clusters of gold particles (arrows in c). A corresponding patchy pattern is seen after immunoperoxidase reaction (arrows in d). (Bars = 0.2 Am.)
	FIG. 3. Identification of lamins present in testis of Xenopus by immunoblotting of polypeptides separated on NaDodSO4/polyacrylamide gel (12% acrylamide; a, b, c', and d, autoradiographs; c, Coomassie blue staining). (a and b) Nuclear lamina-enriched fraction of testes obtained from adult animals (lane 2 in a and b) is shown in comparison with the related fraction of diplotene oocytes (lane 1 in a and b). Two polypeptides ofMr 75,000 and Mr 68,000 corresponding to lamins LIV (arrowheads) and LI,, (arrows) react specifically with antibody L046F7 (lane 2 in a) and with the guinea pig antibodies (lane 2 in b). (c and c') Nuclear lamina-enriched fraction of purified sperms (lane 2), in comparison with the related fraction of oocytes (lane 1). Reference proteins (R) are, from top to bottom, bovine serum albumin albumin and actin. (c') Immunoblot of a parallel gel after incubation with antibody L046F7. Only lamin L1v is detected in sperms (lane 2, arrowhead). (d) Immunoblot of nuclear laminaenriched fraction from testes of immature animals (lane 2; lane 1 shows lamina fraction ofoocytes). L,,, (arrow) is the prominent lamin but small amounts of Lv (arrowhead) are already detectable. The positive lower molecular weight band represents a degradation product of LI,, (cf. ref. 21).
	FIG. 4. Autoradiography showing polypeptides of nuclear lamina- enriched fractions obtained from testes (a) and purified sperms (b) as revealed after two-dimensional gel electrophoresis and immunoblotting with monoclonal antibody L046F7 (a) and guinea pig antibodies against lamins (b). The position of coelectrophoresed bovine serum albumin is denoted by an asterisk. NEPHGE, non-equilibrium pH gradient gel electrophoresis in the first dimension; SD, NaDodSO4/PAGE (12% acrylamide) in the second dimension.
	FIG. 5. Maps of l25l-labeled tryptic peptides of lamins Liv (a) and LI,, (b). Prominent peptide spots are not coincident. E, first dimension by electrophoresis; C, second dimension by chromatography.

References [+] :

Aaronson, Isolation of nuclear pore complexes in association with a lamina. 1975, Pubmed