Marco Preventivo

The animale used in th is study were of the species Notoneota glauoa glauoa (Linn) and were c o lle c te d from ponds in Tentsm uir F o re s t, F if e , S cotland.

F or exaiiination by lig h t microscopy fre s h ly excised ov aries were fix e d f o r 2 h in 3*1 eth an ol a c e tic a c id (Clarke 1851), dehydrated, embedded in wax, and sectio ned a t 5/U. The se c tio n s were sta in e d w ith gallocyanine (p e r Sw ift 1955) washed, dehydrated and mounted in balsam. O ther sec tio n s were tre a te d p rio r to sta in in g w ith a s o lu tio n of

ribo n u clease (0,1 mg p er ml ribonuolease À in 0.01 M ptosphate b u ffe r 6 .0 ) fo r 2 h a t 37*0#

For ob serv ation s in p o la ris e d lig h t whole ovari#lQ8 were w unted in in se o t rin g e r emd exaadned w ith a C arl g e is s photomicrosoope POL f i t t e d w ith planapochrofaatio o b je c tiv e s , a p o la riz e r and an a la ly z e r. M iorotubules are d iso rg an ized and th e ir b ire frin g en c e destroyed by

exposure to low tem perature (Inoué 1952a; T iln ey and P o rte r 1967) o r to oolohioine (inoué 1952b; T iln ey I968) . vife have th sv efo re oom- pared the b ire frin g en c e in fre s h ly ex cised o v a rio le s w ith th a t in o v a rio le s previously su b jected to low tem perature and w ith th a t in oveurioles p rev io u sly tre a te d w itn c o lc h ic in e . F or low tem perature treatm ent liv e anim als were kept in a r e f r ig e r a to r a t 2"C fo r 12 hours. T h eir o v aries were removed and teased in to o v a rio le s under cold co n d itio n s. Single o v a rio le s were then mounted on s lid e s in in se c t rin g e r clo se

alongside o v a rio le s from u n tre a ted an im als, and the b ire frin g en c e in both types of o v ario le was cooqpared. F or co lch icin e treatm ent both o varies

were removed from an anim al. One was p laced in a s o lu tio n of 0.5# co lch icin e in in s e c t rin g e r. The o th e r was kept in in u ect rin g e r w ithout o o lsh ic in s.

Eaoh ovazy was g en tly teased In to i t s In d iv id u al o v a rio le s. At

15 minute in te z v a ls a f t e r e x c isio n of the o v aries p a irs of o v a rio le s , one from the oolohioine s o lu tio n and one from the

p la in rin g e r, were mounted on s lid e s and th e ir b ire frin g e n c e examined. Our au to rad io g rap h ic stu d ie s were c a rrie d out a s fo llo w s. Each of a number of anim als was in je c te d w ith 5^1 (5pC) of a s o lu tio n of u rid in e g e n e ra lly la b e lle d w ith tritiu m (1 .2 C /oii), and supplied by the Radiochemical C en tre, Amersham, England. The in je c tio n needle was in s e rte d through th e a rth ro d ia l membrane between the 2 most

p o s te rio r s te n a ite s . Animals were k ille d and th e ir o v aries fix e d 2 h, 8 h , 12 h , 24 h , and 3 weeks a f t e r in je c tio n . The o v aries were fix e d fo r 12 h in d a n fe lio e 's fix a tiv e (D arlington and La Cour 1942). They were subsequently washed in running w ater f o r 24 h , dehydrated in eth an o l and embedded in m ethacrylate. One-mioron se c tio n s of o v a rio le s were c u t w ith g la ss knives on a Porter-dlum ultram iorotom s, and mounted on s lid e s . M ethacrylate was removed from the se c tio n s w ith aspyl a c e ta te . S ectio n s were th en re hydrated, tre a te d w ith 5^ tric h lo ro a c e tic a cid a t 5*0 f o r 5 m, dehydrated and a i r d rie d from aceto n e. Mounted se c tio n s were co ated w ith Kodak NTB 2 liq u id emulsion d ilu te d 1:1 w ith w ater. They were then placed in lig n t- tig h t boxes and l e f t to expose f o r p eriod s of 20 to 40 days a t 18*0. P rep aratio n s were developed in Kodak D 19 b developer f o r 3 min, washed in w ater, fix e d in Kodak Acid F ix e r f o r 5 min, washed again in w a te r, and a i r d rie d . S ections were then s ta in e d w ith 0 .5 ^ methylene blue on 1# sodium te tra b o ra te (Borax) f o r 1 to 2 mins. A ll p re p a ra tio n s were dehydrated in eth an o l, mounted in balsam , and examined w ith a C arl E e iss photomioroscope f i t t e d w ith planapochroaatio o b je c tiv e s.

• 7 •*

F or o leo tro n adoroaoopy sin g le o v a rio le s were fre s h ly excised in to a % s o lu tio n of g lu tarald eh y d e in 0.06 M phosphate b u ffe r a t pH 7*3 and 18*0» A fte r 15 m inutes in glutaraldehyde the o v a rio le s were washed b r ie f ly in phosphate b u ffe r and tra n s fe rre d to 10 osmium

te tro x id e b u ffered to pH 7*3 w ith veronal a o e ta te (Palade 1952)»

The o v a rio le s were then dehydrated in aoetone and embedded in Vest opal W» S ilv e r to grey seo tio n s were out w ith g la ss knives on a Cambridge Ultramiorotome (A .F. Huxley p a tte r n ) , mounted on Athene 463 g rid s w ithout supporting film s and doubly s ta in e d w ith urapyl a c e ta te fo r 5 minutes and lead c it r a t e f o r 2 m inutes (Reynolds I963)# A ll s ta in in g so lu tio n s were f i l t e r e d through a M iU ipore f i l t e r (0#45y pore s iz e ) im m ediately befo re use. S ectio n s were examined w ith a Siemens Elmiskop I (80 kv) a t negative m agnifications of between 5,000 and

4 0,000.

QBSiiiRVATICNS

In our d e so rip tio n e of the o v a rio le s o f Notoneota we have sub­ s titu te d th e word "tube" f o r th e term " n u tritiv e cord" sin ce we do not thin k th a t the word "oord" s u its the s itu a tio n which we have found.

In an o v ario le of Notoneota the tro p h ic oore branches a t i t s p o s te r io r end in to about 20 tu b es. These tubes pass backwards and spread outwards in the p r e f o llio u la r regio n to become arranged around the periphezy of th e o v a rio le s, th u s e n o asii^ the more a n te rio r

f o ll io le s lik e the bones of a c o rs e t (F ig . 1 , 2 , 4)* As a r u le , eaoh oooyte i s served by one tu b e , and accordingly the number of tubes d ecreases from fro n t to re a r along the o v a rio le . Tubes extend back-

8 •

wards from the tro p h io oore to a l l f o ll io le s which co n tain eggs w ith­ out cap su les. The connections between tro ph io tubes and oocytes break down before a capsule form s. There a re a t le a s t as many tubes evident in tran sv erse se c tio n s through the region o f the f i r s t f o l l i c l e as th e re a re f o ll ic le s in the o v ario le ( F ig . 9 ). The tubes appear oval in cro ss s e c tio n . The w idth of the tubes in an o v arlo le v a rie s from lO ^ to The w idth o f a sin g le tube i s roughly co n stan t along i t s le n g th . The tubes are u su a lly bounded by f o l l i c l e o e lls b u t in some places an oocyte may form the in n e r boundazy of a tube. The tro p h ic oore and a l l tubes have a stre ak y o r fib ro u s appearance when seen in lo n g itu d in a l se c tio n (F ig . 2 ). In tra n sv e rse se c tio n however, they appear uniform ly g ra n u la r.

Five micron se c tio n s sta in e d w ith gallocyanine and w ithout p r io r d ig e stio n in ribonuolease served to show the distz*ibution of RNA in o v a rio le s. A ll cytoplasm in the tro p h io re g io n , in clu d in g the tro p h ic c o re , a l l tu b e s, and the cytoplasm of a U oooytes sta in e d in te n s e ly (F ig s. 1 , 2 ). Most of the n u c le i in the tro p h io region and a l l f o l l i c l e o e ll n u c le i had la rg e n u c le o li which sta in e d stro n g ly . In a l l our s ta in e d p re p a ratio n s the n u c lei of oocytes occupying the f i r s t 2 o r 3 f o llio le s appeared empty a p a rt from th in stra n d s of

chromosomal m a te ria l. N uclei of tne more p o s te rio r oocytes contained 3 to 5 sm all n u c le o li. In p re p a ratio n s p re -tre s te d w ith ribonuolease only nucleaur chrom atin was s ta in e d .

When a sin g le o v ario le i s mounted in in s e c t rin g e r and examined between cro ssed p o laro id s in a p o la ris in g microscope a l l tubes show stro n g p o s itiv e form b ire frin g e n c e w ith zwspect to t h e i r len g th

- 9 -

b iré fr in g e n t, but towards the edges of the oore the b iré frin g e n t m a te ria l bcoomes more d iffu se and f a in t (F ig . 3 ). There a re a s e r le e of narrow lin e s of b irefrin g en o e ra d ia tin g la te r a lly in to the tro p h io tis s u e from the trophio oore, Most tubes appear evenly b iré frin g e n t throughout th e ir le n g th s, although a g entle tw istin g of the b ir é f r in ­ gent m a te ria l in the tubes i s sometimes ev id en t. In a few in sta n ce s we have seen o v a rio le s b u rs t under p ressu re between s lid e and ooverw g la s s . When th is happens some of the tubes may become p a r tia lly is o la te d from the r e s t of th e tis s u e . These is o la te d tubes do not b reak , nor do they d is in te g ra te ; they remain compact and b rig h tly b iré frin g e n t.

The tro p h ic core and tubes in o v a rio le s su b jected to c o ld

treatm ent show weaker b ire frin g en c e than those in u n tre a ted o v ario les (P ig . 5 ).

O varioles tre a te d w ith oolohioine f o r 6 h show no b ir e f r in ­ gence so th a t n e ith e r tro p h io core n o r tub es are d is c e rn ib le in p o la ris a d lig h t (F ig . 6 ). O ther e f f e c ts of colchicine a re v is ib le however. The more obvious of these are sw elling of the neck of the tro p h io reg io n , and d iso rg a n isa tio n of the trophic reg io n so th a t tro p h io n u clei invade the tro p h io c o re . The o rd erly arrangement of tro p h o cy tes, o o cy tes, and tu b es in the p re -f o llio u la r reg io n d isap p e a rs, the o e lls in te rm in g le , and everything tends to flow back­ wards to f i l l the spaces o rig in a lly occupied by the tro p h ic tu b es.

In our autoradiographs of se c tio n s of o v ario les fix e d 2 h a f te r in je c tio n of % -u rid ln e only th e n u c le o li of n u c lei in the tro p h ic

reg ion were la b e lle d . In autoradiographs of seo tio n s from 3 h fix a tio n s n u c le i and cytoplasm in the trophio re g io n , inclu d in g the trophio core

- 10 -

were labelled* Tubes were lig h tly la b e lle d towards th e ir a n te r io r ends, but the la b e llin g of the tubes alongside the more p o s te rio r f o l l i o l e s was so aroely above background* Both n u c le i and cytoplasm of oocytes in the f o l l ic u la r reg io n were unlabelled* In autoradiographs made 12 and 24 hours a f te r in je c tio n s the trophio reg io n was heavily la b e lle d , a l l tubes were la b e lle d , and the cytoplasm o f a l l oooytes was la b e lle d above background (F ig s. 7# 8 , 9)# In autoradiographs of tra n sv erse se c tio n s through o v a rio le s which nad been exposed to % -u rid in e f o r 12 h o r more a few of the tro p h io tubes were q u ite u n la b elled (P ig. 9 ).

We employed the technique of autoradiography because we wished to see i f RM sy n th esised in the tro p h io region moved backwards along the tubes and accumulated in th e oocytes* oytoplaiUB. We p re d ic te d th a t i f th is were the case th en soon a f t e r in je c tio n of iso to p e in to the in s e c t the tro p h io reg io n should be la b e lle d , th e oocytes u n la b e lle d ,