Proc Natl Acad Sci U S A
September 1, 1985;
Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line.
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
Proc Natl Acad Sci U S A
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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.
Isolation of nuclear pore complexes in association with a lamina.