Bioscience Reports 2, 959-966 (1982) 959 Printed in Great Britain

'De novo' centr ioles o r i g i na te at s i tes assoc ia ted w i t h

annu la te l ame l lae in sea-u rch in eggs

Rein 3. KALLENBACH

Department of Zoology, University of California, Berkeley, California 94720, U.S.A.

(Received 30 September 1982)

Hyper ton i c s t ress s t i m u l a t e s the formation of new centrioles in sea-urchin eggs. Those centrioles which a p p e a r away from the nuclear su r face o r ig ina te exclusiveJy at sites associated with annulate lamellae. Al though apparen t when nascen t centrioles become visible, the annulate lamellar association is gradually lost as nascent forms mature into centrioles.

Mature sea-urch in eggs no longer contain any centrioles; upon f e r t i l i z a t i o n , eggs reacqu i re cen t r io l e s from inseminating sperm (2 ,17 ,30) . Yet , u n f e r t i l i z e d eggs can readily produce numerous ma te rna l cen t r io les when over ly s t imula ted with parthenogenetic treatments (1t422). Some of these de novo centrioles originate at the nucleus (27), and a constant association with the nuclear envelope is observed dur ing the i r deve lopment in to mature centrioles (13). Addit ional centrioles appear in the cytoplasm away from the nucleus, forming the centers of cytasters (12). These supernumerary 'cyto- plasmic' centrioles by no means appear randomly in the cytoplasm, and it is their unique origin within eggs that is examined here. The findings explain how latent centrioles, which have been speculated to exist in mature eggs (13), may become dispersed in the cytoplasm during oogenesis. Also, various considerations suggest that the oocyte c e n t r i o l e s , before d isappear ing , may have produced these latent 'cytoplasmic' centrioles. If this is confirmed, then the concept of certain centrioles forming entirely de novo would become untenable; rather, the concept that a l l centrioles are generated only through self-replication would be more valid.

Mate r i a l s and Methods

Hypertonic seawater is an effect ive parthenogenetic agent that has classically been used to induce cytasters and centrioles (5 , t6) . This agent was used here, and details on experimental procedure and electron microscopy have already been published (12,13). Basically, u n f e r t i l i z e d eggs of the sea -urch in species Strongylocentrotus purpuratus were subjected to a continuous hypertonic seawater t reatment for near 8 h. During this t ime the eggs turned on, entered the cell cycle, formed numerous centrioles and cytasters, and pro- ceeded to early prophase (12,13). The procedure allowed a more detailed examination of centr iolar events than is possible with other parthenogenetic methods.

01982 The Biochemical Society

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R e s u l t s

'Cy top lasmic ' centrioles originate exclusively at sites associated with annulate lamellae (Fig. i ) . They may appear anywhere in the cytoplasm where annulate lamellae are located. Annutate lamellae (AL) , both in the form of single sheets of lamellae and the enveloping lamellae of heavy bodies, seem to be randomty distributed in S. purpuratus eggs, as seen in other sea-urchin eggs (25,28). Invariably, the total number of AL is always much greater than the number of centrioles which may appear. Osmiophilic material accrues onto early nascent bodies, forming relatively large aggregates. During this phase, the centriolar aggregates are usually associated with disrupting AL (Fig. 2). As nascent bodies begin to acquire recognizable centrioJe- like forms, close associations with any AS are frequently absent, though AL may be situated nearby (Fig. 3). Later, when fully mature centrioles are observed, no associations with AL can be detected (Fig. #).

These centrioles generally follow an identical 'maturat ion pat tern to that of centrioles originating in association with the nucleus. There is one primary difference: centrioles maturing at the nuclear envelope (NE) retain their association with this membrane, since the envelope r ema ins i n t a c t during i n t e r p h a s e (13) ; ' c y t o p l a s m i c ' cen t r io l e s originating with AL lose their association with these membranes. This loss involves either a partial or a complete disruption of the asso- ciated lamellae, resulting in the vesiculation of the membranes.

D i s c u s s i o n

The origin of newly induced centrioles at loci associated with both AL and the NE is not total ly unexpected. AL and the NE are considered to be homologous structures, since all AL are derived from the nuclear membrane during oogenesis (1,15,21). Centrioles do not reproduce by divis ion; instead, a 'generative' plan involving the 'seeding' of new centrioles by mature ones is hypothesized (19). It has been suggested that eggs contain many centriolar seeds or latent centrioles, some of which are associated with the NE, while others are dispersed in the cytoplasm; also, that the oocyte centrioles, before their disappearance, may have seeded these latent centrioles ( I3 ) . Conceivably, as the nuclear surface continued to enlarge during oogenesis, there might also have been repeated rounds of centriolar seed production by the oocyte centrioles. These 'seeds' may then have been left at the large germinal-vesicle envelope before its disruption. Since AL, including heavy bodies, do not appear in eggs until during and after germinal-vesicle breakdown (3,8), they may be involved in latent centriole dispersal. Possibly, as AL form, latent seeds may become associated w i t h them and together they disperse into the cytoplasm. Other latent seeds may become associated with the NE, following meiosis. The distribution oi latent centrioles throughout the cytoplasm may be in anticipation of some future development event, such as the formation of cilia in embryos.

The finding that centrioles contain RNA (#,i0,2#), required for both centriolar replication and aster formation (11,20,3t), implies that new centrioles and cytasters could not have been constructed unless

CENTRIOLAR ORIGIN AND ANNULATE LAMELLAE 961

Fig. i. Many 'cytoplasmic' centrioles appear at approx, the same time as egg activation (between 45 and 60 min). Before this time 9 no nascent centri- oles are detected 9 and all annulate lamellae (AL) appear structurally intact. Centriolar origin is a rapid event, during which those nascent forms (arrowheads) 9 which are the first to appear, are in full contact with lamellae. These contact points are presumably the loci where latent centrioles might have been located. Soon after their initial appearance 9 the centriolar precursor bodies (PB) lose their direct contact and become more loosely associated with lamellae; at this time~ lamellae of both AL and heavy bodies (HB) are already beginning to break up (arrow). a, x 52 000; b~ x 45 000

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Fig. 2. Nascent centrioles (PB) gradually enlarge through an accumulation of dense-staining material~ until relatively large granular precursor bodies form. It is within these aggregates that centriolar substructure develops. These large bodies are still loosely associated with either relatively intact segments or disrupted portions of AL and HB~ however~ the lamellae of both are now more fre- quently broken up at various points, revealing distended lamellae, loose vesicles 9 and lost pore complexes, a~ x 50 000; b 9 x 65 000.

CENTRIOLAR ORIGIN AND ANNULATE LAMELLAE 963

Fig. 3. As centriolar substructure develops and becomes more apparent~ the amount of osmlophilic

material diminishes. Procentriole-like bodies (PC) are often located near annulate lamellae (AL) 9 but close associations are not usually observed. Procentrioles are often surrounded by many micro- tubules and they usually lie within central astral regions which are relatively free of any organelles. a~ x 56 000; b x 76 000.

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Fig. 4. When mature centrioles are observed, they are seen within fairly large, developing, cytastral areas. Specifically, they are typically situated centrally within asters, and these central areas are generally organelle-free. Annulate lamellae are seen among the radially arrayed astral microtubules, but not near the centrioles. Not all de novo centrioles originate at the same time; they continue to appear from the time of egg activation up to 2�89 h later; however, the number of those forms which appear first constantly decreases, x 32 000.

nascent cen t r io les possess funct ional RNA. Moreover , consider ing the previous discussion on cen t r io la r seeding, it would not be unlikely tha t l a t en t cen t r io les also contain RNA, but in a dormant s ta te . It has a l r e a d y been s u g g e s t e d , in light of the finding tha t RNA seems r e q u i r e d for normal cen t r io le reproduct ion , tha t a def in i te genet ic con t inu i ty be tween cen t r io les exists over subsequent cen t r io le gener- at ions (7) .

T h e a l t e r e d in t r ace l lu l a r envi ronment in the hyper ton ic - s t r e s sed eggs may a c t i v a t e the l a ten t cen t r io la r forms by par t ia l ly de taching t h e m f r o m t h e i r associa ted lamel lar loci, since nascent cen t r io les typ ica l ly develop some dis tance away from thei r associa ted lamel lae . This ac t iva t ion , as well as the membrane disruption of the lamel lae , may involve an increase in in t race l lu lar Ca 2+ (12,32) , and may be a derepress ion reac t ion s t imulat ing RNA funct ion. This would allow the 'seeds ' to serve as d i rec t ing and organizing ent i t ies for the comple t e c o n s t r u c t i o n of new cent r io les . Other authors have made similar p r o p o s a l s with regard to RNA serving as nucleat ion si tes for the organiza t ion of prote in molecules for the fo rmat ion of basal bodies (9) , as well as r ibosomes (23) . (There is ev idence tha t the induced c e n t r i o l e s a r e a c t u a l l y p r e m a t u r e l y a c t i v a t e d basal bodies in

CENTRIOLAR ORIGIN AND ANNULATE LAMELLAE 965

preparation.) Mazia ( lg ) speculated some years ago that RNA could carry information for the recognition of proteins as well as for their synthesis.

Newly induced centrioles, then, are not in a true sense de novo, since information-bearing seeds for their complete formation probably r e s ide in the egg. Centrioles, with their unique morphology, are generally stable and permanent organelles in most cells; additionally, their role in mitosis and ciliogenesis makes them crit ically important for both deve lop ing and adult organisms. Also, newly appearing centrioles are capable of forming fairly rapidly, within minutes in some cases (6,26,29). In considering these facts, it would seem rather un l ike ly tha t such complex structures form fortuitously, involving randomly dispersed ions and molecules and chance reactions; rather, the existence of a detailed blueprint for complete centr iolar formation would be more likely.

One can maintain the view that centrioles (except basal bodies) universally are associated with nuclear membranes. A cell 's normal c o m p l e m e n t of c e n t r i o l e s is always typically associated with the nuc l e a r enve lope ; additional centrioles, which exist only as latent forms in unferti l ized eggs, are also associated with nuclear membranes (e.g. NE and AL). This association becomes apparent only af ter the la tent centrioles have begun to mature. 'Cytoplasmic' centrioles then, in a sense, are not really cytoplasmic, they only seem to be because, basically, pieces of nuclear surface are separated from the nucleus during the maturation of the egg and are dispersed throughout the cytoplasm. Since normal asters usually form around centrioles, this expiains why eggs, among cells, can make cytas ters when a cor rec t stimulus is introduced.

Acknowledgem ents

I thank Dr. D. Mazia for sharing with me his understanding, insights, and ideas; and E. Dmitrova-Kallenbach for her crit ical reading and editing of the manuscript.

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