A detailed cyto lo gica l study has been conducted fo r R. prolixus ovarioles by Heubner and Anderson, (1972 a, b and c). The account which follows deals mainly w ith structures th a t are apparently concerned with f o l l i c l e shaping. Most o f these structures were not examined in d e ta il by Heubner and Anderson.
(a) The tunica propria
During e a rly p re v ite llo g e n ic growth, the tunica propria is about 320 nm th ic k . By la te previtellog e n e sis the tunica is reduced in th ic k ness to about 255 nm, and during vite llo g e n e s is is fu rth e r reduced to 95 nm (Figs. 29, 30 & 31). Throughout growth o f the tunica p ro pria , i t s composition remains f a i r l y constant. Fibrous elements ramify between the amorphous material o f the tunica. The fib re s are concentrated in to a layer up to 128 nm th ic k on the side o f the tunica nearest the
f o l l i c u l a r epithelium . Fibres are often grouped in to bundles th a t are 25 - 30 nm in diameter. The fib re s are mainly oriented c irc u m fe re n tia lly in d ire ctio n s at r ig h t angles to the polar axes o f oocytes and p a ra lle l to the outer surface o f the f o l l i c u l a r epithelium (Figs. 32 & 33).
(b) The f o l l i c l e c e lls
During p re v ite llo g e n ic growth, bundles o f m icrofilm ants th a t are about 300 nm in diameter are apparent at the apex o f each c e ll d ir e c tly beneath f o l l i c l e c e ll outer surfaces (Fig. 29). Bundles are oriented c irc u m fe re n tia lly at r ig h t angles to oocytes* polar axes and p a ra lle l to the outer surfaces o f f o l l i c l e c e lls . Microtubules with the same
o rie n ta tio n occasionally in te r d ig ita te between the m icrofilam ent bundles (Fig. 32). Microtubules also run p a ra lle l to the long axes o f c e lls (Fig. 34). These may help to maintain the columnar shape o f f o l l i c l e c e lls (Heubner & Anderson, 1970; 1972 a).
34 The outer surfaces and apical la te ra l membranes o f f o l l i c l e c e lls are highly irre g u la r in shape. Filopodial c e ll extensions th a t are about 190 nm in diameter p ro je ct from f o l l i c l e c e lls . M icrofilam ent bundles extend along the in te r io r s o f these extensions (Fig. 35).
At the bases o f f o l l i c l e c e lls , the plasma membranes o f adjacent c e lls are connected by desmosome complexes. These include septate desmosomes, gap junctions and desmosomes o f the zonula adhaerens v a rie ty
(Fig. 36). At the level o f these basal zonula adhaerentes, and closely associated with these desmosomes, m icrofilam ent bundles (about 300 nm th ic k ) extend across each c e ll (Fig. 37). Nearly a ll o f the bundles are c irc u m fe re n tia lly arranged. The microfilaments run p a ra lle l to the outer surfaces o f f o l l i c l e c e lls and a t r ig h t angles to the polar axes o f oocytes. The same arrangement o f desmosomes and m icrofilam ent bundles occurs throughout previtellog e n e sis although during la te previtellogenesis
(when f o l li c l e s are about 600 pm long) the f o l l i c l e ce ll/o o c yte boundary becomes 'peaked' (Fig. 38). The 'peaks' occur at the point where ad jacent f o l l i c l e c e lls meet each other. In these instances, the micro filam ent bundles extend from 'peak' to 'peak*. Basal m icrofilam ent bundles are only found in elongating f o l li c l e s . Very early p r e v it e llo genic f o l li c l e s are spherical. Their f o l l i c l e c e lls do not have basally positioned m icrofilam ent bundles.
V ite llo g e n ic oocytes (about 1100 pm - 2000 pm in length) are d is tinguished by the presence o f channels between f o l l i c l e c e lls (Fig. 39). These channels are e x tra c e llu la r spaces th a t are widest (about 3 - 5 pm) at the extreme outer and inner surfaces o f the epithelium (Figs. 40 & 41). Channels only form between f o l l i c l e c e lls along the sides o f oocytes
while those c e lls which form the cap or apical region o f a f o l l i c l e are longer and closely apposed to one another. Thin bundles o f microfilaments
(about 120 - 130 nm th ic k ) fo llo w the margins o f the developing
v ite llo g e n ic channels along c e ll membranes at the sides o f f o l l i c l e c e lls (Figs. 42 & 43).
Along the sides o f f o l l i c l e s , c e lls re ta in th e ir association with each other, despite the presence o f channels, via c e ll surface pro je ctio n s. Bundles o f microfilaments extend along the in te rio rs o f these p rojections. Projections from the outer surfaces o f f o l l i c l e c e lls approach the tunica propria as is the case during e a r lie r stages o f oogenesis (Fig. 44) while those from the inner surfaces o f f o l l i c l e c e lls approach the oolemma (Fig. 45).
The large apical and basal m icrofilam ent bundles observed in f o l l i c l e c e lls o f p re v ite llo g e n ic oocytes are absent during v ite llo g e n e s is .
DISCUSSION
The re su lts presented above show th a t oocytes and th e ir f o l li c l e s grow anisom etrically as they pass p o s te rio rly down the o va rio le.
Furthermore, there are changes in the cytoskeletal organisation o f f o l l i c l e c e lls during the tra n s itio n from p re v ite llo g e n ic to v ite llo g e n ic growth o f oocytes (Fig. 46). This may be an in d ic a tio n th a t the f o l l i c l e c e lls are closely involved in f o l l i c l e shaping. There are no ind ica tion s th a t the oocytes and the ovariole sheaths play a major role in f o l l i c l e shaping. For example, the oocyte contains no well oriented cytoskeletal arrays. The two ovariole sheaths are c o n tra c tile (Heubner & Anderson, 1972 a). However, each sheath is a r e tic u la r network o f c e lls separated from one another and from the f o l li c l e s by haemolymph. Any tension transm itted from m yoepithelial c e lls to f o l li c l e s yi_a the haemolymph could provide 'corset-1 ike^ support to f o l li c l e s but i t is doubtful whether they play a major ro le in f o l l i c l e shaping.
36 la rg e ly responsible fo r defining the elongate form o f R. prolixus
oocytes. Most oocyte elongation occurs during the period preceding vite llo g e n e s is (de Wilde & Loof, 1974). During p re vite llo g e n e s is, a system o f c irc u m fe re n tia lly oriented microfilaments and specialised
junctions is situated a t both the outer and the inner surfaces o f f o l l i c l e c e lls . The arrangement o f m icrofilam ents is such th a t they may be
responsible fo r the active production o f te n s ile forces between and w ith in f o l l i c l e c e lls . There is evidence th a t cytoplasmic c o n tra c tile pro teins may function as a cytoskeletal system in the cytoplasmic m atrix
(P o lla rd , 1976). Microfilaments found in Drosophila melanogaster f o l l i c l e c e lls have been found to bind to heavy meromyosin (Weilings, J.V., Personal Communication), This reveals th a t the microfilaments are a ctin filam ents and therefore probably act in conjunction w ith myosin to produce a c o n tra c tile e ffe c t. I t is highly lik e l y th a t the microfilaments observed in R. p ro lixu s f o l l i c l e c e lls are s im ila r ly composed although fu rth e r experimentation is necessary to substantiate th is . The 'peaked' arrangement o f f o l l i c l e c e ll bases o f la te
p re v ite llo g e n ic f o l li c l e s may be an in d ic a tio n o f tension production. The circum ferential tension could be d is trib u te d throughout the
f o l l i c u l a r epithelium (the cytoplasmic projections may play a support ing role in th is respect) thereby r e s tr ic tin g circum ferential expansion o f the f o l l i c l e . I f th is is so, f o l li c l e s would become more elongate in d ire ctio n s which p a ra lle l the polar axis o f the oocyte.
The re su lts show th a t the growth o f f o l li c l e s during v ite llo g e n e s is is v ir t u a lly isom etric. The v ite llo g e n ic channels would obviously im p a ir the effectiveness o f the p re v ite llo g e n ic m ic r o fi1 ament/junction system, so during v ite llo g e n e s is , circum ferential resistance to f o l l i c l e growth is apparently provided by the tunica propria alone. This would account fo r the less marked resistance to circum ferential growth observed
during vite llo g en e sis as compared with p re vitellog e n e sis. I t is not known to what extent organised, oriented e x tra c e llu la r s tru ctu re s, such as the fibrous tunica p ro pria , are responsible fo r the form and in te g r ity o f the c e llu la r systems they c le a rly support. However, i t is generally supposed th a t e x tra c e llu la r fib re s confer r i g i d i t y in maintaining shape perm itting the range o f c e ll action to extend in space fa r beyond th a t o f a single c e l l .
Circumferential systems have been found in a ll f o l li c l e s th a t have been selected w ith a view to examining examples o f each o f the three ovariole types as shown in Table 1. Therefore, presumably most insect f o l li c l e s have such a system. I t is in te re s tin g th a t a circum ferential system o f both microtubules and microfilaments appear to be u tiliz e d during P. americana f o l l i c l e elongation, whereas a circum ferential system consisting predominantly o f microfilaments (circum ferential microtubules are ra re ly encountered) is apparently u tiliz e d during R. prolixus f o l l i c l e elongation. Microtubules do occur in te lo tro p h ic ovarioles where they act in other developmental capacities. For
example, microtubules apparently f a c i l i t a t e ribosomal flow in n u tr itiv e tubes o f R. prolixus (McGregor & Stebbings, 1970; Stebbings & Bennett, 1975).
A ll th is fin e stru c tu ra l evidence fo r f o l li c u l a r control o f oocyte shaping and elongation in P. americana and R, prolixus is circum stan tia l. However, experimental support has been provided by Went, (1978) fo r
Heteropeza pygmaea. Oocytes grow is o m e tric a lly and f a i l to elongate when they develop in v itr o a fte r treatments which prevent the enclosure o f oocytes by a f o l l i c u l a r epithelium .
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CHAPTER 3
THE MECHANISM OF OVIPOSITION AND CORPUS LUTEUM FORMATION IN THE PANOISTIC OVARIOLES OF P. AMERICANA
AND THE TELOTROPHIC OVARIOLES OF R. PROLIXUS INTRODUCTION
The passage o f the oocyte from the ovariole in to the oviduct is one o f the main ovulatory events in insects. I t involves the escape of an oocyte from the f o l l i c u l a r epithelium and the breakdown o f the e p ith e lia l plug at the entrance to the oviducal pedicel. V ir tu a lly nothing is known about the means by which these events are effected (Davey, 1965). Various theories have been proposed to account fo r the posteroid move ment o f oocytes in the ova rio le and th e ir subsequent release. These theories include 'crowding' (Raven, 1961), muscular p e ris ta ls is of myoepithelial c e lls o f the ovariole sheaths (King & Aggarwal, 1965;
Chapman, 1972), and e la s tic action on the part o f the tunica propria (Singh, 1958; Bonhag & Arnold, 1961; Chapman, 1972). However, i t seems lik e ly from my studies th a t no one o f the factors described above is e n tir e ly responsible and th a t, in a d d itio n , cytoskeletal elements and e x tra c e llu la r fib re s may also be involved. I t seems th a t f o l l i c l e c e ll cytoskeletons may be concerned with propulsion o f oocytes in to oviducts (a fte r helping to define oocyte shaping in ce rta in insects) and th a t they adopt new configurations to accomplish th is task as they enter a post v ite llo g e n ic phase o f mechanochemical a c tiv it y .
RESULTS