Indi an Journal of Experimental Biology
Vol. 4 1, December 2003, pp. 1372- 1383
Review Article
Male accessory gland proteins in Drosophila: A multifacet field
K Ravi Ram" & S R Ramesh*
Drosophi la Stock Ce nter, Department of Stud ies in Zoology, Uni versi ty of Mysore, Manasagangot ri, Mysore 570 006 , Ind ia
aprese nt address: Depart mcnt of Molecul ar Biology and Genetics, Co rnell Uni versity , Ithaca, NY 14853, USA
Male accessory gla nd in Drosophila is a secretory tisslle of the reprod uctive system. The proteins sylll hes ized in the
accessory gla nd are tiss ue specific, stage specific-seen only during the ad ult stage and sex specific in the se nse of ma le
limited ex press io n. These secretions th at form a component of th e semi nal flu id are transferred to the fe male at the time of
copu lation and play an important ro le in reproduction. In conj uncti on with sperm , these secretory protei ns ass ure
reprod ucti ve success by reducing the fe male's recepti vity to mating and escalating the rate of egg laying. Some of these
proteins are antibac teri al in na ture wi th a likely funct ion of protecti ng the fema le's ge ni ta l tract agai nst microbial infec ti on
during/after mat ing. Most of th e ge nes involved in the sy nth esis of acccsso ry gland proteins are autosomal but a few are still
X-linked. Their male specific ex press ion is achi eved at the time of sex determin ation. The level of ex pression of these ge nes
is dose dependent and th ey fo llow Mc nde li an pattern of segregation. Further, majority of these protei ns are rapidly evolving
with high rates of non-synonymo us subs titutions. In thi s rev iew, by considering the work carried ou t in differe nt fields , we
have tried to generate a co mprehensive pictu re abou t the ma le accessory g land and the ro le of its proteins in the reproduction
of Drosophila.
K ey words: Drosophila, Evo lution , Fert il ity, Gene expression, Ma le accessory
"Love is li ke mag ic and it always wi ll be, for love still
remains li fe's sweet mystery". For a biologist, love is
no longer a mystery but it's indeed chemistry, which
is always fue lled by chemi cals. Then co mes another
point - Is love gettin g or giving? In terms of bi ology,
it's a benefi cial relati onship that provides an ample
opportunity both for the co ntributor and the recipient
to perpetuate their gene pool. Maki ng love is not
unique to hi ghly evolved animals bu t is see n even in
insect world. In the insects, most of the reactions of
this complex love chemi stry are catalyzed by th e
proteins sy nthes ized by th e male accessory gland.
The accessory glands are two lobes that branch off
the ejaculatory duc t l . They develop from a spec ial set
of cells in th e ge ni ta l imagi nal disk upon instructi ons
by genes that determ ine th e sexual phenotype of the
animaI2-s. They sy nthes ize a complex mixture of
proteins, carbohydrates, lipids as we ll as amino acids
th at form co mponents of th e seminal fl uid and are
transferred to the female du ring mating6.7. Several
studies have been made with a focus main ly on
biochemistry and ge neti cs of the secretory proteins of
these glands. The ultrastructure of cpithelia l cell s and
the general reprod uctive ro les of the secretory
*Correspondi ng author
E-mail: srramesh2000 @yahoo.com
Fax: 9 1-821-242 1263
Pho ne: 91-821-2472218 & 5550218 (Res)
g l and~
M ating behav iour, Reprod uctio n
products, i.e., the repression of the female recepti vity
and the stimulation of oviposi ti on are well
documented. Ex periments dealing wi th the isolation
as well as structural analysis of specific peptides, th e
genes codin g for them and th eir functions have been
reviewed peri odi call/· 8• 12 • However, only in recent
years, signifi cant progress has been made to specify
the function of indi vidual components, mechanism of
act ion and their evolutionary status. He nce, in thi s
rev iew, we will be dealing with all the major aspects
of accessory gland protei ns and the functions of
specific peptides, the mechani sm of th eir act ion as
well as evol uti onary perspecti ves in greater detail.
Existi ng wealth of informatio n on male accessory
glan d pro teins in Drosophila is the outco me of
extensive work on D. melanogaste r. However,
investigations on other species of Drosophila such as
D. jill1 ebris, D. simulans, D. sechellia, D. mauritiana,
D. suzukii, D. subobscura and di ffe rent me mbers of
D. nCisula subgrou p have enabled LIS to obtain a
comparative picture among various species/subspecies
of thi s ge nus .
Structure and synthesis
The accessory glan d (Fi g. 1) is a bilobed stru cture,
each of which opens into prox ima l part of vas
deferens th at holds mature sperms. T hese lobes are
composed of a single layer of cells su rroundi ng a
RAYI RAM & RAMESH: MALE ACCESSORY GLAND PROTEINS IN DROSOPHILA
lumen that are in turn surrou nded by a muscle sheath.
There are two morphologically distinct secretory cell
types namely main cells and secondary cell s l. The
main cells th at acco unt for 96% of the secretory cells
are pri sm shaped and binucleate, wh ile remaining 4%
are the pear shaped binucleate secondary cells (Fig. 2),
which that are interspersed amo ng the main cells at
the distal tip of th e gland l3 . In D. melanogaster, each
lobe consists of abo ut 1084 main ce ll s and 24
secondary cells, each cell type producing a
characteristi c set of proteins, beginning at about the
time of eclosionI 3- ' 5 . In different members of D.
nasuta subgroup we l6 have show n th at th e number of
cells are higher and ranged from 1600 (in D. kohkoa)
to 2900 (in D. s. neonosuta) . Our studies have
Testis
Accessory gland
Ejaculatory duct
Ejaculatory bulb
Fig. I - Drosophila ma le reproducti ve systelll sho wi ng th e
position and propo rt ion of accessory gla nds ( from Flybase hllp://fl ybase.org.)
1373
revealed the absence of correlation between the
number of cells/gland and the qu antity of secretions
but shown a positive correlation for gland size and the
quantity of secretions synthesized that fa lls in line
with the findings of Cunnigham et al. 17 who have
demo nstrated that th e increase in the size of the gland
is associated with increased secreti ons. Further, al l
these parameters let it be th e quantities of secreti ons,
number of cells/gland or size of the gland were fo un d
to increase in their value as the di stance fro m the
ancestor increases though the genetic basis of each of
these parameters is different from another ' 6 (see Fig. 3) .
Stromnaeus and Kvelland 18 reported th at the new ly
emerged males need 12 hr to sy nthesize th e glandular
secretion . On the contrary , Federer and Chen 19
reported that secretory prod ucts are fo und in the gland
lumen in the pupae prior to eclosion. Ho weve r, in all
the cases unmated males were fo und to possess
max imal amount of secreti ons only at about 5-7 days
fo llowing eclosion and maintained at a constant level
for at least about 20 day s.5· '4.2o. In the unmated males
7-10 days post eclosion, the secretory proteins that get
acc umul ated in the gland lumen acco unt fo r about
80% of the total soluble proteins 6 Even in th e
D. iJl7l11igrans group, th ere is a progress ive increase
up to day 7 and from day 8 onwards the increase is
. l"'
?? QuaI'Itatlve
. I y tIlere .IS no cIlange
on I y margllla
- '--.
fro m th e day of eclosion and the onl y exception being
a 70 kD protein frac ti on whi ch increases in quantity
Fig. 2 - Phase contrast 'images of A-Portion of accessory g land wa ll main ce ll ; B-Seco ndary cell interspe rscd among main cells
N-n ucleus, G-goigi complex; {b-fil aillentou s bodies; M-Illusc le fibres; m-mitochondria; yc-large vacuo le in the secondary cell. The
numericals I &2 in subscript represen t main cell and secondary cell respec ti vely (fro m Bairati I).
INDIAN J EXP BIOL, DECEMBER 2003
1374
the X-chromosome, 20-60 in the second chromosome,
60-100 in the 3rd chromosome and 101 -102 in the 4th
chromosome), 'A' indicates first division of 26 and
'a' denotes subdivision within ' A'. The gene and the
transcript names are italicized and the name in nonitalics indicates protein. i.e., Acp26Aa is the gene
while Acp26Aa is its protein.
In D. melanogaster, Acps have been mapped onto
2 nd and 3rd chromosomes using different techniques
such as electrophoresis 27 , screening for male specific
.
~6 ~8 29
· Situ
. lb
. )9 30 . Some 0 f
transcnpts.-. an d In
ly n·d·lzatlon-··
these genes are either tightly linked (as in case of
Acp26Aa and Acp26Ab that are apart by just 20 base
pairs)14.31 or clustered (as in case of Acp57Da,
Acp57Db and Acp57Dc according to their localizati on
in polytene band 57D, i.e,. 2ml chromoso me)32. Very
rece11lly, an EST screen by Swanson el a(\3 coupled
with the el ucidation of D. lIIelan ogaster genome
helped to iden tify most of the candidate Acps
accounting to nearly 90% of the male accesso ry gland
genes. A striking feature is that all these genes are
autoso mal. Wolfner el al?9 have opined that the
absence of Acp genes ill the X-chromosome mi ght
relate to their ma le limited expression and even
suggested that the autosomal pl acement of Acp genes
was advantageous because the genes could be
up to day 7 followed by its gradual disappearance23 .
Following mating, these secreti ons are replenished 15
in order to avoid the fertility problems as multiple
matings in rapid succession depl ete accessory gla nd
contents24 .
Genetics of male accessory gland proteins
Genetics of these proteins has been extensively
worked out. At present 50-70% of the ge nes coding
for these ti ss ue specific proteins have been
r
localized-J • The DNA/RNA sequences have been
analyzed for many genes ancl the current path Iies in
elucidation of the regu latory path ways of their
expression. To beg in with they we re termed as mst
(male lipecific lran scripts)26 and later named as Acp
(Accessory gland proteins) II . The nomenclature is as
follows: Acp is followed by a number and then an
alphabet in uppercase whi ch is in so me cases suffixed
by lower case alphabet. The number indicates the
chromosome locati on; upper case alphabet indicates
the division (if the gene stretches into another divi sion
th en two uppercase alphabets are used) and the lower
case alphabet indicate subdi vision. For· example, in
Acp26Aa - Acp stands for accessory gland protein ,
'26' indicates location in chromosome 2 (as per the
genetic nomenclature of D. JIlelanogasl er , 1-20 are in
A;
2905; B: 0.257 ;C: 15.20
A: 2214; B: 0.268; C: 13.00
D. sulfurigaster
sulfurigaster
A: 1544; B: 0.215; C: 9.50
I
1
D. kohkoa
A: 1902 ; B: 0.273; C: 13 .00
1
I~
A: 2219; B: 0.224 ; C: 10.50
~I
D. II. kepulauana
I
I D. lias uta nasuta I
A: 1942; B: 0.301; C: 20.00
D. II . albomicans
Fig. 3 - Nctwork of D. rla s Ul CI subgroup sho wing thc trends of dirfe rcnt paramctcrs of accessory gland. (from Ravi Ram and Ramcsh
A: Number of ccll s/gland; B: Sizc of the gland (cm"); C : Quantity of secrelio ns (/l g)
I6
).
RA VI RAM & RAMESH: MALE ACCESSORY GLAND PROTEINS IN DROSOPHILA
expressed at high levels without also needing to
acquire dosage compensation. However, recent
studies in our lab on accessory gland secretions
among a few members of D. nasuta subgroup have
shown that some protein fractions follow X-linked
pattern of inheritance, which is a first report of its
kind in Drosophila 22 ,34 . By analyzing accessory gland
protein samples from F}, F2 and backcross progeny,
we have found that 40 kD fraction in D. n. nasu/a, 55,
40, 25 kD fractions in D. albomicans and a 24 kD
fraction in D. n. kepulauana follow X-linked pattern
of inheritance. Thus our studies have shown that
though the accessory gland proteins are male limited,
some fractions show X-linkage which attracts the
attention of researchers working on gene regulation.
Recently, Lopez et al. 35 have shown that X-linked
locus male-female steril e in region 6E [Ir~fs( 1 )6E] is
required for the production of normal seminal t1uid.
This probably has regulatory function as its mutants
fail to reduce the receptivity and increased egg laying
which are normally seen in mated females. The
involvement of Y -chromosome in the synthesis of
these proteins has been rul ed out long ago 36 . The
mRNAs of Acps that have been studied in detail so fa r
and the number of amino acids in their pro-proteins
are listed in Table 1.
Regulation and expression of Acp genes
Genes expressed specifically in the accessory gland
are subject to several le ve ls of regulation. These genes
Table I-Somc of Acp genes, size of their mRNA and the
number of amino acids in thcir proteins
Gcne
Acp26Aa
Acp26Ab
Acp29AB
AcpJ/F
AcpJ2CD
Acp3JA
Acp36DE
Acp5JEa
Acp53Eb
Acp57Da
ACJ!57Db
Acp57Dc
Acp62F
Acp63F
Acp70A
Acp76A
Acp95EF
Acp98AB
Size of mRNA
Pro Protcin Reference
(kb)
(aa)
0.9
0.5
0.95
3.5
0.95
282
III
234
OA5
2.7
0.6
241
115
716
120
OA
0.75
0.65
0.85
55
46
OA5
OA5
115
81
55
388
52
31
0.23
1.3
0.3
OJ
42
Monsilla and Wolf'ncr7
Monsma and Wolfner7
Wolfner et ai. 29
Wolfner el al. 29
Wolfncr el ai. 2<)
Wolfner el a1.2~
WolfnLr et al 29
Wol fncr ef al. 29
Wolfne r el al.29
Simmerl el al. 32
Simmerl el (t/J2
Simmer! el cli ..12
Wolfner el al 29
Wol fner el al. 29
Chen el al. "J
Wol fne r el al. 29
Di Bencdetto el al.l.l
Wolfner el al."
1375
are controlled indirectly by the loci controlling sex
determination, which specify during larval Iife, the
formation of the accessory g land at a later stage5.7.
Subsequent tissue specific and temporal tran scriptional controls result in the onset of tran scription of
5 14 1accessory gland genes late in the pupal stage' .). A
third level of control is translational, in that these
transcripts are not tran slated until after eclosion .
Further, mating has been found to induce a general
1.5 to 2-fold increase in translation in the accessory
gland, presum ably in order to replenish the sec retions
37
that are transferred to th e female during mating .
Therefore, the expression of these genes is subject to
deve lopmental and mating induced regulation. When
the quantities of the accessory gland proteins were
estimated from 7 days old unmated males , there were
l6
statistically significant differences between species .
As the study included the quantitative analysis in
unmated males , the observed differences can be
attributed to differential developmental regulation.
Hormonal role is another facet of Acp gene regul ation .
Application of juvenile hormone in vitro can mimic
mating stimulated increase in tran slation 38 ,39 . The
co nclusive evidence for the hormonal role was
40
provided by Cho et al . They found a high level of
Acp57Dc expression right after eclosion when th e titer
of juvenile hormone TTl (JH III) is at the peak.
The males of Drosophila posses, two major sets of
autosomes in homozygous condition while the sex
chromosomes name ly X-chromosome and Y -chromoso me always exist in hemizygous condition. The
interspecific hybrid males would have one set of
aurosomes and th e X-chromosome inherited from the
female parent and the other set of autosomes and
Y -chromoso me inherited from the male parent.
Hence, in the F I hybrids , th e genes of one species are
represented in only one dose. Studies on the Acp gene
expression have shown that the auto somal Acp genes
coding for proteins specific for one or the other parent
species are expressed normally in the hybrid cells
with the rate of synthesis and accumulation being half
when co mpared with the homozygous parent
41
species • However, the X-linked fractions reach th e
parental leve ls since there will not be any difference
in the gene dose as males of both the parent species
?74'
a nd the Ily bn'ds Ilave on I y one X -c hromosome--·-.
Figure 4 depicts the expression levels of X-linked and
autosomal fractions in the parent as well as the
hybrid. These studies havc thus shown that the
sy nthesis of specific accessory g land proteins
corresponds to the gene dose.
[N DIAN J EX P BIOL, DEC EMB ER 2003
1376
Biochemistry of secretions
The accessory gland secretions consist of proteins,
carbohydrates, lipids and amino acids 6. Electrophoretic
separation of the accessory gland proteins from male
D. melanogasler on 7.5% native gels yields about J 2
bands and on 10% SOS gels at least 40 fractions
co uld be noticed4J ,44 . Stumm-Zollinger and Chen 44
b
0
,j
u
)
,
.3
rig. 4- Express ion levels o r dirferent autosoma l and X- li nked
accesso ry gland pro tcins among pareills as we ll as hybrids.
DifTerc nces in O. D. va lu es o r X-linkcd fractions in the parent and
hybrid samples (4, 5 & 7) are statist ically insignifica nt 22 ,42
I) 26kD of D. II . II(/S llla
5) 55kD of D. II. a/bolllicans
(Autoso mal)
(X-linked )
2) 25 kD o f D. s. a/boslriga/(l
6) 40kD of D. II. a/boll1icalls
(Autosomal)
(X- linked)
3) 40kD of D. II. lI([sula
7) 24 kD of D. II. keplliaualla
(X-linked)
(X-linked)
4) 25kD of D. II. a/boll1iclillS
(X-l inked )
were able to localize at least 85 fractions by way of
2-D electrophoresis and these gels when subjected to
autorad iography36 yield about 1200 protei ns. Unli ke
in D. melanogaster the SOS-PAGE patterns in
different members of D. nasuta subg roup are very
simple with only 4-8 major fractio ns. They mi grate in
three groups with group I having hi gh molecular
weight fraction s and group III with low molecul ar
weight frac ti ons22 .34 . Biochemically, Acps general ly
fall into three categori es as represented in Fig. 5. The
number of amino acids va ry from 31 (as in
Acp98AB)29 to 7 16 (as in Acp360E)45 . The number
of amino ac ids in different Acps is li sted in Table 1.
In Drosophila, regu lated proteolysis seems to play
very important role in the accessory gland function as
Acp26Aa which is a pro hormone li ke molecul e l 4.3R is
subjected to rapid and specific cleavage within the
female genital tract 46 . Further, presence of proteases 33
?93147·
id
an d protease .In h'Ib'Itors-'
" 1I1 t h
e accessory
g an
secretions also support the involvement of reg ulated
proteo lytic cascades. Most of the protease inhi bitors
found in accessory gland secretions belong to selv in
super fam il y. Members of serpin super famil y are
extra cellular serin e protease inhibitors, helping to
control proteo lyti c events associated with a wide
varicty of regulated biochemical pathways48 . A
second possibl e ro le for a protease inhibitor is in the
coagu lat ion of semen after mating to form mating
pl ug as Acp76A whi ch is a serine protease inhibitor is
transferred to the female during mating and majority
Secretions
(m ixture of Proteins, carbohydrates and lipids)
Simple peptidesiProhomormone
Precursors IS.29.32.4?
Prominent Acps
that come under
respccti ve groups
Acp95EF
Acp70A
Acp26Aa
Aep57Da
Acp57Db
Acp57Dc
Acp98AB
Proteases
Acp36DE
Modifiers . .
Protease inh ibito rs
Acp76A
Acp62F
Fig . 5 - Catego ri cs of accesso ry gland proteins and differe nt Acps Ih al fall into each category
Lipases
RA VI RAM & RAMESH: MALE ACCESSORY GLAND PROTEINS IN DROSOPHILA
of the Acp76A is found in the mating plug 29 . Lipase
activity has been demonstrated in the secretions,
which has some similarities to vertebrate bile salt
stimulated lipase. Lipase activity is significantly
reduced in males and increased in females shortly
after mating; thereby suggesting that it is transferred
from male to the female, which may be important in
mating and reproduction in Drosophila49 . Posttranslational modifications of accessory gland proteins are
a common feature. Acp36DE is a large glycoprotein 29
while in D. nasuta subgroup, all the three groups of
fractions are glycosylated and group III fractions in
particular are highly glycosylated22 •34 . AcpS7DC
which is preferentially expressed but not restricted to
accessory glands is phosphorylated4o.5o . Further, a
peptide (Acp62F) having sequence similarity to
neurotoxin 39 and another peptide with antimicrobial
activity'l have been reported . These pep tides might
play an important role in the reproduction thereby
helping the species on the whole and in future might
have an applied value too.
Functions of accessory gland proteins
Male accessory gland proteins perform a variety of
functions. Table 2 embodies different Acps whose
runctions have been identified. In short, the important
ones include increasing the rate of oviposition,
decreasing the receptivity to mating, sperm storage,
sperm utili zation and sperm displacement. These
secretions have also been implicated in reducing the
life span of the mated female. Most of the postmating
responses caused by the Acps are only short term in
nature. Two Acps namely Acp70A and Acp26Aa
have been shown to modulate egg production.
Acp70A commonly called as sex peptide (SP) is
T ab le 2 -
Different accesso ry gland proteins and th eir fun ctio ns
Protei n
Function
Reference
Acp26Aa
Ovulation and Elevation
of egg laying
Signiticant assoc iat ion
wi th Sperm co mpetition
Significant association
with Sperm competition
Sperm sto rage a nd also
sperm competitions
Significant assoc iation
with Sperm competition
Toxicity
Stimulation of
oviposition and
Reduction of rec eptivity
Regulated protcolysis (?)
Antibacterial activ ity
Heifetz
Acp26Ab
Acp29AB
Acp36DE
Acp53Ea
Acp62F
Acp70A
Acp76A
28 kD protein
et
al. 65
Clark
el
al 6U
Cl a rk
el
al.
6O
Be rtram e l a/ 45 ,
Clark et a/. w
Clark e l al.60
Lung
Chcn
el
al. 52
el
al. 3O
Wolfner el al. 29
Lung el (11. 51
1377
invol ved in repressing the female receptlYlty and
stimulate oviposition 47. A short-term drop in mated
female's attractiveness is triggered independent of the
receipt of the sperm and Acps. However, maintenance
of the lowered attractiveness is dependent upon
sperm52. Acp26Aa plays a role in the elevation of egg
laying by the mated female 46 and its effect on the
elevation of egg laying is apparent only on the first
day after mating 38 . Acps and sperm are
complementary stimuli for inducing high rates of
oogenesis progression and rapid egg deposition.
While Acps alone can induce egg deposition and
oogenic progression, both Acps and sperm are
required for maximum stimulation of oogenic
progression and egg deposition immediately after
. 53
matll1g'
Accessory gland proteins also playa role in the fate
of sperm transferred to female during mating. They
are not essential for the transfer of sperm to the
female, but play an important role in the storage of
sperm. Tram and WolfI)er5~ by direct counts of stored
sperm have shown that the female stores only 10% of
normal levels whose mates transfer little or no Acps
along with sperm. The involvement of accessory
gland proteins in the sperm storage is a very important
feature as it profoundly influences the number, timing
and paternity of the female progeny. Acp36DE is
required for efficient sperm storage in the mated
females 55 . Further, when mated to males without
Acp36DE, females failed to maintain an elevated egglaying rate and decreased receptivity behaviour whose
persistence normally depends on the presence of
stored sperm. Mutations in genes that affect sperm
storage can drastically affect the out come of sperm
competition. Males lacking Acp36DE suffer in sperm
competition because fewer sperm are retained or
stored 56 . Using spermless mutant, Chapman et al. 56
have shown that significantly more second male
offspring were produced following the transfer of
Acp36DE by spermless first mating males . This
implies that the transfer of Acp36DE itself by the first
male faci litated the storage or use of the second
male's sperm and that co-transfer with sperm is not
necessary for Acp36DE effects on second male' s
sperm storage. The PS-l in D. fun eb ris is shown to
function as an acrosin inhibitor57 .
Females of most animal species are usually
inseminated by more than one male, which allows
sperm from different males to compete for
fertilization. However, to prevent invasion of sperm
from other males, Drosophila males elicit rejection
INDIAN J EXP BIOL, DECEMBER 2003
1378
behaviour in their mates after copulation. This
rejection behaviour is induced exclusively by the
secreted accessory gland products transferred to the
female during copulation and the activities of sperm
as well as accessory gland products are co mplementary and interdependent i.e. , both sperm fertility and
rej ection behaviour of females depend on accessory
gland products, the prolonged activities of which in
turn require the presence of sperm 58 . However, sperm
di splacement is an important component of male
reproducti ve success in cases where females remate
frequently relative than duratio n of sperm storage and
rate of sperm use. Prout and Clark59 have show n th at
male accesso ry gland fluid has a role in sperm
competition mediated by incapacitation or inefficient
60
have shown
use of resident sperm. Clark et al.
significant associations between particular Acp alleles
at four different loci (A cp26Aa/A b; Acp29AB;
Acp36DE and Acp53Ea) and the abi lity of males to
resist displacement by subsequent sperm.
61
Chapman et al. have demonstrated that Main ce ll
produ cts of the mal e accessory glands are involv ed in
elevating the rate of female egg-laying, in reducing
the female receptivity to further matings and in
removing or destroying sperm of previous mates .
They have also shown that seminal fluid products
from the main cells of th e male accessory gland are
respo nsible for the cost of mating in females with the
rel atio nship between the transfer of these products
and the lon gev ity of the mated female being inverse ly
proportional. Acp76A plays a rol e in the regulation of
Acp proteolysis/or in th e coagulation of sem in al fl uid
to form mating plu g that prevents remating and
Acp62F is supposed to be the factor contributing to
2
the cost of mati ng throu gh its toxicitl .
Further, Acps seem to have immunologi ca l
functions also; a 28 kD male accessory g land derived
protein is found to have antibactelial activit/I . This
could assis t in protecting the male 's reproductive tract
and after transfer to the female , the fema le
reproductive tract or eggs against bacterial infection.
As these proteins are tran sferred along with th e
sperm, they may also protect sperm from bacterial
infection.
How does Acps bring about these functions'?
Attempts to understa nd the mechanism through
which Acps bring about the function started with the
whole transplantation of accessory glands, or by
inj ecti ng specific peptides or by the use of transgenic
flies and the latest being the use of fluorescent
markers coupled to the specific Acp transcripts to
keep a track over the protein movement. Transplantation experiments in the unmated females have shown
increased oviposition and reduced receptivity in the
recipient female implying that the accessory gland is
the source for the factors that could elicit these
behaviors. Since these gla nds were implanted into the
abdominal cavity, they have to act via he molymph. At
least two Acps (Acp26Aa and Acp62F) have been
l4
shown to enter the hemolymph of the mated female .
The sperm storage protein Acp36DE does not enter
the female circulatory system and mu st therefore, act
within the reproductive tract45 .55 . Thus, there are two
kinds of Acps, (a) those which act within the genital
tract and (b) those which reach the target site through
hemolymph and elicit the behaviors.
i) Acps enter hemolymph through ventral posterior
vagina
Lung and Wolfner63 analyzed the entry of seminal
fluid proteins into hemolymph using two peptides that
enter the hemolymph (Acp26Aa and Acp62F) and the
one that does not (Acp360E). Th ey reported that
Acps e nter the female circulatory system from the
posterior vagina, which has hi gher permeability thus
affording access immed iately after inse mination. Th e
ab il ity of the Acps to enter the fem ale hemolymph
was correlated with their abil ity to cross the intim a
that lines the posterior vagina. The ventral vagina is
structurally different from other parts of the female
reproductive tract in that it lacks mu sc les64 . Acp26Aa
and Acp62F enter the hemolymph through the ventral
posterior vag ina by 5 min of initi ation of cop ulati on.
Acp360E is trapped within the gen ital trac t due to its
large s ize/or its tight associatio n w ith the other
components of the seminal fluid or the genit al tract.
About 10 min after the start of mating , co mponents of
the sem in al tluid coagulate into a solid mass (mating
plug) in the posterior part of the femal e reproductive
tract. As a resul t of this, whi le Acps are being
transferred to the female, entry into the hemolymph is
decreased or terminated.
ii) Acp36DE facilitates sperm storage by corra1linf:j
the sperm near sperm storage orgal/.s
In the mated female, Acp36DE associates with the
wall of the oviduct, just anterior to the openings of the
sperm storage organs. It also associates with the
leading edge of sperm mass. Acp36DE or a complex
containing it forms a barrier that "corrals" sperm near
the openings to the sperm storage organs and this
RAYI RAM & RAMESH: MALE ACCESSORY GLAND PROTElNS IN DROSOPHILA
45
process facilitates the sperm storage . However,
55
Neubaum and Wolfner have shown that Acp36DE is
not required for barrier formation but both Acp36DE
and batTier are required for maximal sperm storage.
iii) Acps affect the rate of egg laying through
mechanical and sensory stimuli
Egg laying is a multi ste p process. It begins with
oocyte release by the ovaries, fo llowed by eggmovement down the oviducts and its deposition on a
substratu m. Acp26Aa elicits this response by
stimul ating the first step in egg lay ing -release of
oocyte by the ovary. Durin g mating, Acp26Aa begins
to accu mulate at the base of ovaries, a pos ition
consistent ,vith the actio n on the ovari an mu sc ulature
to mediate oocyte release and cause the clearin g out
of ex isting mature eggs, thereby indirec tl y allowin g
oogenes is to be initiated immedi ately after mating 65 .
Sex peptide (SP) stimulates egg production,
oviposition and also reduces receptivity in the mated
fe male. Studi es have show n th at a memb rane bound
fo rm of SP induced typi cal post mating behavio ur,
indicating that SP mu st be outside the ce ll in order to
66
exert its biologica l effects . Peripheral nerves,
co nnective,
suboesophageal gangli on, cervical
discrete parts of the thoracic gan g li on and the gen ital
tract are the SP binding sites in th e mated female 67 .
Prominen t binding of th e peptides to afferent nerves
suggests modification of sensory ou tput. Sex peptide
activates the juvenile hormo ne III-sepox ide (JHB3)
synthesis in the corpus all atum 68 th at in turn acts by
stimul ating
vitell ogenic
oocy te
progression 69 .
However, juvenile hormon e analogue does not eli cit
increased oviposition and red uced receptivit/ 6 and
hence sex peptide mu st have an additional separate
effect on these two post matin g responses . Sau dan et
70
al . have found that an ejaculatory duct peptide
(DUP99B ) elicits the sa me post mating responses as
sex-peptide. These two peptides also bind to sperm
and thei r gradu al release from the sperm in the mated
female might be a reason for the pers istence of
reduced receptivity. Fi g. 6 ill ustrates the localization
of so me of the Acps in different parts of the mated
female reproducti ve trac t.
Evolutionary perspectives
Analysis of Acp genes has contributed enormou sly
to the field of evo luti onary biology. Starting from
simple protein pattern comparison to the present day ' s
Expressed Sequence T ag (EST) approach has helped
us to understand the intricacies involved in th e rapid
1379
27
evolution of proteins . Whal en and Wilson detected
considerable polymorphism for nearly one half of the
major protein bands, including null alleles for three
bands. Further, th e gland specific proteins were found
to be more polymorphic than testes specific proteins,
as well as those found in both testes and glands 71 .
Later on, Thomas and Singh77 have shown that the
reproducti ve tissue (testis and accessory gland)
showed more divergence than did the nonreproducti ve ti ssue. Exuberant SDS-PAGE pattern
polymorphism was observed in the natural
populations of both D. n. nasuta and D. s. neonasuta
collected from various altitudes and in both, the low
molecular weight fractions have been found to
mediate the introduction of vari ability (Rav i Ram and
Ramesh, communicated).
Mutations are the raw materials fo r evolution and
therefore it's quite natural for the substituti ons to
occ ur in any given gene. Th ese substitutions might be
sy nonymous or non- sy non ymous. Since most nonsynonymous changes are deleterious, for a given
protein, the ratio rate of non-synonymous substitutions
(Ka)/rate of synonymous substitutions (Ks) should be
less than unity (1) and this demonstrates purifying
selection. However, in positive selection, the ratio of
Ka/Ks exceeds unity ( 1) indicating that more nonsynonymous changes are occurring than synonymous
changes thereby leading to rapid evoluti on of certai n
F - - - O vary
~(,fi,~G~~---- Lateral oviduct
- -- - -- - - Oviduct
f t - . , ; - - - -- - - -
Spennathecum
~~6i:'~------ Scminal
receptacle
>""f71rr"'J..------ Accessory gland
- - - - - - - Uterus
Fig. 6 - lll ustration showing local ization of so mc Acps in
different parts of fema le reproducti ve sys tem (the positions of
alphabet are purely arbi trary and j usl for ex planatory purpose).
The image of fe male reprod ucti ve sys tem is adap ted from Fl ybase
(http://fl ybase.org) . A- Acp26Aa: B-Acp36DE, C-Acp76A, DAcp70A, E-Acp62F
1380
INDIAN J EXP BlOL, DECEMB ER 2003
proteins wherever favorable mutations occur. Many
genes pertain ing to male reproduc tion are show n to
evolve rapidl / 3 among species and the evidences
increasingly suggest the influence of pos itive
selectio n. Sequence comparison of ge nomes or
Expressed Sequence Tags (ESTs) from related
organisms provides an insight into functional
co nservation and diversifi cation. EST approac h has
shown that genes, which encode putative accessory
gland specific seminal fluid proteins have a
sign ificantly elevated level of nonsynonymous
substitu tio n when compared to non accesso ry gland
specific ge nes suggesting that their divergence has
been accelerated by positive Darwinian selecti on and
at least 11 % of predicted Acps are evo lving rap idli 3 .
Prominent Acps that are shown to be under positive
selection include Acp26Aa74 .77 , Acp70A 7H and
79
Acp29AB . Working with the duplicated sex-peptide
gene (Acp70A) region in D. slibobscura, Cirera and
Aguade 80 have opined that positive selec ti on caused
the initial differentiation of the N-terminal ends of the
gene product, whi le pu rifyi ng selection cou ld be
respo nsible for hi gh conservation of th e C-terminal
ends. To further analyze the patterns of polymorphism
and divergence in genes encoding accessory gland
proteins, Begun et al. 25 conducted a sequencing
survey of 10 Acp ge nes in sampl es of D.
melallogasfer as well as D. simulans (Acp29AB,
Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F,
Acp63F, Acp76A, Acp95EF alld Acp98A B) and have
shown that the elevated divergence of Acp ge nes is
accompanied by elevated intraspec ific polymorphism.
ln addition to th e already repo rted departu res of
Acp26Aa, Acp29AB and Acp70A fro m ne utral ity, the
data of Begun e f aZ. 2S reject neutrality at Acp29AB and
Acp36DE.
One possible reaso n for thi s rapid evol ution may be
due to the fact that adap tations in one sex may impai r
fitness in the opposite sex. As accesso ry gland
proteins tri gge r several changes in the female
including the increased female death rate/redu ced
longevity, there is a hypothesis of a tradeoff between
defensive sperm-competiti ve abi lity of males and life
history parameters of mated female whi ch has bee n
supported by th e study of Civetta and Clark 81•
Therefore, this kind of tradeoff always keeps genes
coding for these proteins in a state of flux leading to
higher rates of evoluti on. Tsaur ef al .82 reported that
th e level of amino acid poly morp hi sm in the Nterminal quarter of Acp26Aa in D. mauriliana is
hi gher than in its sibling spec ies, D. lIIeLanogaster and
based on th e above hypothesis, they postul ated that D.
mauritiana may have been under much more intense
sex ual selection than other species .
Civetta and Singh 83 opined that trai ts in volved in
reproduction are better predictors of species
distinctness as th ey diverge much faster than the ones
in vo lved in so mati c function. In their · study, the
sex ual trai ts did not manifest lu xuriance in the
interspecific hybrids and testes showed an average
additive effect with a trend towards paternal
dominance. However, our stud y involvi ng the hybrids
of D. n. nasllta and D. n. alboll1icalls has show n th at
as far as quantity of accesso ry gland secretory
proteins are concerned, such a trend (paternal
dominance) was not observed; instead th ere was a
trend towards D. n. nasuta in the progeny of different
crosses. Further, the accessory gland proteins neither
manifest th e phenomena of luxuriance nor breakdown
in the interspecific hybrids 84 . Hihara"4 has show n th at
in D. l1Ielanogaster th e number of eggs laid is closely
associated with the quantity of accessory gla nd
secretions in th e adult male (about 70% of whi ch are
transrerred to the fe ma le during mating). However,
our study involving D. n. Jlasura , D. n. albomicans
and their hybrids has revealed that the FI fem ales
produced sign ifi cantly less nu mber of eggs when
mated to FI males, leading to F2 breakdown though
the quantity of accessory gland secretory protein in FI
males was similar to that of D. II. nasuta. These
findings suggest th at the ge netic consti tution of an
individual has a bear ing on the fec undity of the
fema le rather than the qua nti ty of accessory gland
secret ions in the adu lt male that are transferred to the
. R4
·
remae
I d un ng mat ll1 g .
Research on mal e accesso ry gla nd protein s in
Drosophila has led to th e contribution of info rmation
to different field s such as mo lec ular biology,
bio logy,
developmental
bio logy,
evo lut ionary
behavioral biology, endocrinology and even to
neurobiology. There is no do ubt that wilh more and
mo re peptides in volved in vita l fun ctions are being
identified alo ng with the mechanism of action, the
po tential of accessory gland proteins is increasing as
prospective substances for biological co ntrol thereby
helpi ng the hum an ca use. In add ition to this, the
identification of X- linked fract ions22 is another
important contribution that would require furth er
stu dies to unders tand th e dynamics of regul ation and
subsequent expression of the X-linked gene that is
male s£ecific. Further, the EST screen by Swanson
el al. 3 has paved the way for large scale
RA VI RAM & RAMESH: MALE ACCESSORY GLAND PROTE[NS IN DROSOPHILA
investigations on these proteins and the future lies in
the identification of receptor molecules for th ese
proteins which would provide an insight into the
function as well as mechanism of action o( th ese
proteins.
IS
Acknowledgement
17
The authors are grateful to DST and CSIR, New
Delhi for financial support. Thanks are also due to
Prof. H . A. Ranganath, Chairman of our department
for facilities and encouragement.
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