3

Paternal Investment and lntracellular

Sperm-Egg Interactions during and Following
Fert iIi zation in Drosophda
Timothy L. Karr
Department of Organismal Biology and Anatomy
University of Chicago
Chicago, Illinois 60637

I. Introduction
II. Sperm Structure and Production in Drosophilu
111. Sperm Transfer, Storage, and Utilization
IV. Syngamy (Sperm Penetration), Pronuclear Maturation, Migration, and Karyogamy
A. Syngamy
B. Pronuclear Maturation and Migration
C. Karyogamy
V. Structural Analysis of a “Sperm-Derived Structure” in the Developing Zygote
A. The Sperm Forms a Stereotypical Structure in the Fertilized Egg
B. The Early Cleavage Divisions
C. Sperm Fate in Later Stages of Embryogenesis
V1. Genetics and Molecular Biology of Fertilization and Early Embryonic Development in
Drosophila
A . Maternal-Effect Mutations
B. Paternal-Effect Mutations
VII. Cytoplasmic Incompatibility
VIII. Speculative Models of Sperm Function in the Fertilized Egg
A. Model 1-Nutritive Protein Import (Fig. 7A)
B . Model 2-Specific Protein Importation (Fig. 7 8 )
C. Model 3-DiffusioniGradient Production (Fig. 7C)
D. Model 4-Structural Role (Fig. 7D)
IX. Conclusions and Perspectives
References

1. Introduction

The predominant mechanism for sexual reproduction among eukaryotic organ-

isms involves fertilization of one specialized cell type, the egg, by another
specialized cell type, the sperm. The evolutionary mechanisms that gave rise to
sexual reproduction based on two sexes have been studied and debated by scien-
tists for over a century (Parker, 1982). While the seminal evolutionary events that
led to the development of a two-sex-based system of reproduction are not known,
they ultimately gave rise to anisogamy, i.e., two highly disparate cell types,

89
90 Timothy L. Karr
sperm and egg (Parker, 1982). These two cell types bear little resemblance-egg
cells are usually large and spherically or elliptically shaped and contain large
quantities of stored products, while sperm cells are almost invariably elongated,
thin cells containing little cytoplasm and are specialized for motility. They appar-
ently share only one common theme: both carry the haploid DNA complement of
each parent.
Over the past 2 decades, significant strides have been made in understanding
some of the molecular mechanisms of fertilization. Particularly impressive has
been the discovery of specific receptors in mammals and echinoderms responsi-
ble for species recognition and specificity (Wasserman, 1987). Thus, at least in
those organisms for which such molecules have been identified, we can hope to
eventually begin to understand how these highly differentiated cell types: (1) find
each other, ( 2 ) interact and fuse at their surfaces, and (3) ultimately form a
diploid zygote capable of realizing the developmental program.
A more thorough understanding of fertilization would benefit greatly from
study of a variety of animal species. However, fertilization has historically been
studied in only a highly restricted set of animals-mainly, chordates and echi-
noderms. Ironically, insects, which arguably represent the most diverse group of
animals, have received very little attention from developmental biologists inter-
ested in fertilization. As pointed out by Sander (1983, this bias in the field is, for
the most part, a practical one: insects usually fertilize their eggs internally and
generally produce smaller numbers of egg and sperm, making laboratory studies
difficult, if not impossible. Nonetheless, the potential for studying fertilization in
insects is enormous, considering the rich genetic heritage of Drosophilu and the
recent advances made in understanding the cellular biology and developmental
genetics in this model system. Also, from an economic and health perspective,
knowledge of the molecular mechanisms of fertilization in insects could repre-
sent a powerful and effective point of attack for the biocontrol of insects.
The potential involvement of the sperm and/or sperm-derived products in the
egg during and following fertilization was implied from our laboratory’s cyto-
logical and biochemical studies of Drosophila (Karr, 1991; Graner et al., 1994).
Our interest stems from the general observation by numerous investigators over
the years that sperm “gigantism” is a common feature in insects (Counce, 1963;
Hildreth and Lucchesi, 1963; Warn et al., 1984; Karr, 1991). For example,
D. melanogaster sperm, measuring 1.8 mm, are approximately the same length
as the adult males. The recent demonstration that these very large sperm are
completely engulfed into the egg, persist intact during and following fertiliza-
tion, and coil into a stereotypical structure may reflect a previously unappreciated
role(s) of the sperm in fertilization (Karr, 1991). While this claim remains to be
proven, it would, if true, significantly change our view of the role of the sperm
following egg penetration and provide new insights into the evolution of sperm
gigantism. An even more controverial idea, that extragenic paternal investments
participate in the development of the early embryo, will be discussed later.
3. Sperm-Egg Interactions in Drosophila 91
From a cell biological viewpoint, the real importance of these results is the
suggestion that intercellular sperm-egg interactions following sperm penetration
are central to fertilization, particularly in those insects where sperm gigantism
has evolved. Recent work by Schatten and colleagues (Simerly et al., 1993) has
also shown that the entire mouse sperm enters the egg and also persists for some
time after fertilization. More recent work has shown sperm tail entrance and
persistence to be a common feature in a number of related mammals (Schatten,
personal communication). The challenge now will be to integrate these new and
seemingly general findings into the overall picture of fertilization.
Our laboratory is engaged in the biochemical and cellular analysis of some of
the proteins associated with fertilization in Drosophila. The approach has been to
characterize sperm-associated proteins identified using monoclonal antibodies.
These antibodies have identified a large family of proteins, many of which are
specific to testes (Graner et al., 1994), related by their antibody reactivity.
Monoclonal antibodies have also allowed us to study sperm structure and fate in
the egg following fertilization. The extraordinary size of the sperm in D .
melanogaster aided in this description and has revealed previously unrecognized
aspects of sperm behavior and fate. The evolutionary and developmental conse-
quences of sperm structure in the egg, and the potential importance of sperm-
egg interactions during and following fertilization, will be discussed. In this
context, I will also discuss recent advances that have led to a deeper understand-
ing of early development, particularly the isolation of maternal-effect mutations
affecting fertilization and/or the very earliest stages immediately following fertil-
ization.
We are also currently studying a biological phenomenon related to fertilization
and early embryonic development known as cytoplasmic incompatibility (CI;
Karr, 1994). The phenomenon is characterized by blockage of the normal process
of fertilization in particular crosses of strains within the same insect species
(Jost, 1970; Werren et al., 1987; O’Neill and Karr, 1990). CI is closely associ-
ated with the presence of a bacterial endosymbiont, Wolbachia pipientis, found
in a wide variety of insect species (Breeuwer et al., 1992; O’Neill et al., 1992;
Boyle et al., 1993). interestingly, CI only occurs when infected males are mated
to uninfected females. Therefore, C1 can be viewed as a unique form of male
sterility similar to known patemal-effect lethal mutations in Drosophila.
This review relies heavily on previous excellent reviews of Sander (Sander,
1985, 1990), to which the reader is referred for a more comprehensive and
general assessment of insect fertilization. This review will focus on new results
and information since that time, particularly as they relate to paternal contribu-
tions, including extragenic contributions, to fertilization. Accordingly, this re-
view will only briefly describe the fundamentals of insect fertilization, focus on
fertilization and early embryonic development in the dipteran D . melanogaster,
and, where appropriate, refer to related insect species.
Another important purpose of this review is to provide a forum for speculation
92 Timothy L. Karr
about the significance and purpose of the evolution of sperm gigantism in insects.
In this context, four speculative models that may be relevant to this unique and
intriguing biological conundrum will be presented.

I I . Sperm Structure and Production in Drosophila

Although outwardly different in size and shape from their better-known mam-
malian, amphibian, and echinoderm counterparts, insect sperm and eggs contain
essentially the same components necessary for embryonic development. Insect
eggs are usually ellipsoidal and large relative to their body size. For example, the
mature Drumphila egg measures approximately 0.5 mm in length (approx-
imately the length of the adult abdomen) and 0.2 mm in width. The egg is
invested with the same cellular components as those found in all other eggs,
including large stores of mRNA, lipids, and proteins. Insect sperm, in addition to
the highly condensed chromatin in the head, also contain a flagellar axoneme
composed of a 9 +
2 structure and an acrosome (or a rudimentary acrosome;
Lindsley, 1980; Sander, 1985). However, unlike that found in most other animal
groups, insect sperm length can in some cases reach monumental proportions.
For example, D . melanugaster males produce sperm that are 1.8 mm in length or
about the length of the entire adult fly. This is hardly the record-sperm in excess
of 20 mm have been recorded in D . hydei, and the record now stands at at least
50 mm for D . bijiurcu (Pitnick et d., 1995).
During spermatogenesis, stem cell divisions occur at the proximal end of
paired testes, and fully differentiated sperm appear at the distal end, as shown in
Fig. 1 (Gonczy et al., 1992). Due to the highly regular and stereotypical develop-

~~ ~

Fig. 1 (A) Phase contrast view of an adult testis. The apical end (api) is left in all figures unless
otherwise noted: ter, terminal end. Bar = 50 Fm. ( 9 ) Schematic representation of five stages of
spermatogenesis. Arrows pointing to part (A) indicate where approximately in the testis each stage
begins; cells are displaced in an apical-to-terminal direction as they mature within each stage. Germ
line stem cells and somatic cyst progenitor cells are anchored around a hub of somatic cells (hub) at
the apical tip of the testis. Only one germ line stem cell (ste) and two cyst progenitor cells (cyp) are
represented for clarity. asy. asymetric divisions of a germ line stem cell and two neighboring cyst
progenitor cells give rise to one primary gonial cell (spg) and two cyst cells (cyc), respectively. mit,
the spermatogonial cell undergoes four mitotic divisions, while the cyst cells no longer divide. gro,
the resulting 16 spermatocytes (spe) grow dramatically. mei, the two meiotic divisions occur. mor,
the 64 haploid spermatids (spt) undergo dramatic morphological changes. Only 6 elongating sper-
matids are shown for clarity. Because of the length of the sperm tail, fully elongated spermatids have
their nucleus at the terminal end of the testis, while the tail extends almost to the apical end. During
this last stage, the two cyst cells become structurally distinct, the head-cyst cell (cyh) being associ-
ated with the sperm heads and the tail-cyst cell (cyt) elongating with the growing sperm tails. The
head-cyst cell then becomes entrapped by a specialized epithelial cell (tec) located in the terminal part
of the testis. Coiling of the sperm bundle ensues, followed by release of motile spermatozoa (spz)
into the seminal vesicle. Only one spermatozoon is shown for clarity. See text for additional informa-
tion. Reprinted, with permission, from Giinczv et al. (1992)
 Y
m
a
4
94 Timothy L. Karr
mental pathway of gametogenesis in Drosophilu and other insects, sper-
matogenesis (and oogenesis) has been a favorite subject of structural biologists
over the years. Gametogenesis has been elegantly described in great detail at the
light and electron microscopic levels; the reader is referred to reviews of this
subject (Lindsley, 1980; Mahowald and Kambysellis, 1980; Henning and Krem-
er, 1990). Recent excellent reviews of oogenesis (Spradling, 1993), sper-
matogenesis (Fuller, 1993), and embryogenesis (Foe e f al., 1993) have appeared
in the literature, to which the reader is referred for additional information.

111. Sperm Transfer, Storage, and Utilization

Since sperm of D . melunogaster are 1.8 mm in length, it seems unlikely that
sperm actively swim through the duct to the female. Instead, as discussed by
Sander (1990), some as yet unexplained force propels sperm into the female
genital tract. It has been estimated that D. melanoguster females can store, on
average, about 700 sperm (Lefevre and Jonsson, 1962; Fowler, 1973; Gilbert,
1981). This is in stark contrast to the number of sperm transferred and stored by
other animals and other species of insects. For example, a queen bee can store an
estimated 4 to 6 million sperm. Honeybee sperm are, of course, much shorter in
length. The extreme variation in sperm numbers is undoubtedly due, at least in
part, to the evolution of sperm gigantism in Drosophilu (Pitnick and Markow,
1994b). This extraordinary variation in sperm size and numbers raises many
intriguing questions about how and why sperm gigantism evolved and how this is
beneficial to those animals where it has occurred.
Once sperm transfer is completed, sperm move from the uterus into the sperm
storage organs of the female. Following sperm transfer and storage, the female
controls the patterns of sperm utilization, as documented in a variety of inspect
species (Sander, 1990). The efficiency of sperm utilization in insects is remark-
able. For example, the Drosophilu female lays about the same number of fertil-
ized eggs in her lifetime as sperm stored (Gilbert, 198I), indicating that virtually
every sperm stored is utilized. Obviously, adaptation of such efficient utilization
of gametic resources is a survival strategy employed by many species of insects.
The gametic strategies that have evolved in insects are, of course, quite different
from the reproductive strategies used by many animal groups utilizing both
internal (e.g., mammals) and external (e.g., echinoderms) strategies, where
enormous numbers of sperm are produced, but only a minute fraction are utilized
(Parker, 1982).

IV. Syngamy (Sperm Penetration), Pronuclear Maturation,

Migration, and Karyogamy
At the completion of oogenesis, the egg arrests in meiotic metaphase I (Huettner,
1924; Sonnenblick, 1950; Davring and Sunner, 1973). In response to either
3. Sperm-Egg Interactions in Drosophila 95
sperm penetration or egg hydration at the time of ovulation, or both, the egg is
activated, protein synthesis begins, and meiosis I1 is completed (Doane, 1960;
Mahowald et al., 1983). The time from ovulation to the first mitotic division has
been estimated at about 20 min in D . melanogaster (Rabinowitz, 1941). It has
not been possible to observe the earliest events of fertilization, particularly sperm
entry, as they occur inside the female. Therefore, only those events present after
eggs are laid are detectable, and the percentage of eggs in these very early stages
of fertilization represents a small percentage of the total (females tend to “hold’
their eggs for some time following fertilization). Although incomplete, some of
the basic events during this stage have been documented as described below.

A. Syngamy

Syngamy usually refers to the fusion of sperm and egg membranes that initiates
the subsequent events leading to karyogamy. However, very little is known about
syngamy in insects. The limited data available on this subject indicate that
syngamy in insects occurs by very different mechanisms than those employed by
other animals. The elegant electron microscopic study of Perotti (1975) has
shown that sperm penetration in D . melanogaster does not involve sperm-egg
membrane fusion, in direct contrast to what is known to occur in other animal
groups. Thus, at least in Drosophila, other mechanisms for sperm entry have
evolved that d o not include sperm-egg membrane fusion, and, technically, syn-
gamy does not occur (at least not at the cell surfaces). The electron microscopic
evidence indicates that the sperm enters by puncturing a hole in the egg oolemma
(Perotti, 1975). This opening is soon closed, apparently by a “curing” of the
membrane (Perotti, 1975). This raises intriguing and important questions about
the precise mechanism of sperm entry and the fate of the sperm membrane
following sperm entrance.

B. Pronuclear Maturation and Migration

Migration of the female pronucleus would appear to rely on an extensive array of

microtubles nucleated by the sperm aster (W. Theurkauf, persona1 communica-
tion). Some of the events of pronuclear maturation and migration are shown in
Fig. 2. The fertilized eggs shown in Fig. 2 were fixed and stained to reveal both
the maturing nuclei and sperm tail. Shortly after sperm entry, meiosis 1 and 11 are
completed, and, as shown in Fig. 2A, the four haploid products of meiosis are
aligned normally to the egg surface in the anterior-dorsal region of the egg.
Following sperm entry, maturation of the sperm nucleus and female pronucleus
commences (Figs. 2B and 2C). The female pronucleus migrates to the center part
of the egg near the anterior end at approximately 75% egg length (the posterior
end of the egg is considered to be 0% egg length). The rates at which the two
3 . Sperm-Egg Interactions in Drosophilrr 97
pronuclei mature are apparently different, as shown by a comparison of Figs. 28,
and 2C. Both the shape and extent of DNA decondensation are different in the
two pronuclei. The female pronucleus (top arrow in Fig. 2A) appears more
expanded and decondensed than the male pronucleus (bottom arrow in Fig. 2A).
The differences in the kinetics of maturation probably reflect the very different
nuclear structures involved. The sperm nucleus must first decondense from its
very compacted state and then import and/or exchange proteins. Presumably, by
the end of this process, both nuclei are identical with respect to their protein
compositions, both are invested with nuclear membranes, and both begin DNA
synthesis.

C. Karyogamy

In order to form the diploid zygote, the nuclear membranes surrounding the two
pronuclei must fuse (this fusion is known as karyogamy). During the entire
period of pronuclear decondensation and migration, DNA replication occurs and
presumably is completed by, or shortly after, the time the two nuclei complete
migration (Shamanski and Om-Weaver, 199 1). Following maturation and migra-
tion, the two pronuclei lie next to each other in the interior of the egg at
approximately 75% E.L., as shown in Fig. 2D. At this stage, nuclear membranes
have formed or are in the process of forming around each individual pronucleus
as they each condense following replication (Fig. 2E). The first mitosis ensues
(Fig. 2F) resulting in two diploid zygote nuclei. (Lin and Wolfner, 1991; Lopez
et d.,1994). The exact nature of the ensuing events of mitosis is only poorly
understood. These events have been recorded at the light microscopic level in
great detail using conventional sections and stains (Huettner, 1924; Rabinowitz,
1941; Sonnenblick, 1950) and, more recently, through confocal microscopy and
indirect immunofluorescence antibody staining (Karr, 1991; Lopez et al., 1994).
An excellent review of the current state of our understanding of these crucial
early events has recently been published (Foe et al., 1993). Other than these
classical descriptive studies, we know very little about the molecules mediating
these events. However, as discussed below, new insights are being provided by
genetic and biochemical studies of fertilization.

<
Fig. 2 Pronuclear maturation, migration, and fusion in Drosophila melunogaster. Young fertilized
eggs were fixed and stained with a DNA-specific fluorochrome. (A) Five products of meiosis, three
polar bodies (bracket) and two pronuclei (arrows) are observed in the anterior region. ( B and C) High
magnification views of female (B) and male (C) pronuclei showing the initial stages of pronuclear
decondensation. (D-F) Formation of the zygote nuclei. Fully decondensed and replicated nucici
apposed and touching (D); fully condensed nuclei lying immediately next to each other (E): anaphase
of the first mitosis (F).
98 Timothy L. Karr

V. Structural Analysis of a “Sperm-Derived Structure”

in the Developing Zygote

As previously shown (Karr, 1991), the sperm enters the egg intact and localizes
within the anterior region of the egg. This structure has some interesting features
that may provide clues to its function in the egg. It is important to keep in mind
that, because the sperm persists in the egg throughout early embryogenesis, the
sperm structure observed in the egg is more accurately referred to as a sperm-
derived structure. Although we know very little about the biochemical changes
occurring in, on, and around the sperm, it is safe to assume that proteins in the
sperm are degraded, modified, or bound by specific components in the egg.
Presented below are some of the data, accumulated in the laboratory over the past
5 years, that are relevant to the behavior and interaction of the sperm during and
following fertilization.

A. The Sperm Forms a Stereotypical Structure in the Fertilized Egg

Figure 3A shows a three-dimensional reconstruction of the sperm tail. The image

was produced from confocal optical sections of the sperm using the DROP-I . I
antibody (Karr, 1991; Graner et a / . , 1994). A unique feature of this structure is
the highly stereotypical folding and coiling of the sperm in the anterior end of the
egg. Observation of numerous fertilized eggs confirms the regularity of this
structure, suggesting that sperm-egg interactions are necessary for the observed
folding and coiling. Further indirect evidence of sperm-egg interactions comes
from numerous structural changes observed during and immediately following
sperm entrance (Karr, 1991). Presumably, sperm receptors and/or other interact-
ing molecules are present in this region. As discussed further below, some
maternal-effect mutations in D . melanogaster disrupt this structure, suggesting
that the proteins affected by these mutations interact with the sperm. The length
of the sperm tail in the egg was directly measured from three-dimensional recon-
structions like the one shown in Fig. 3A. These measurements confirm that the
entire sperm enters the egg. Similar results have now been found in 10 other
species of Drosophila (Karr and Pitnick, 1996).

Fig. 3 Localization of sperm tail during and following fertilization in Drosophila simulans. Sperm
in fertilized eggs were visualized using a mouse polyclonal antisera and a Auorescently labeled goat
anti-mouse antibody (A) or using an HRP-based detection system (B,C). Anterior is to the left.
(A) The entire sperm tail was computer reconstructed from confocal optical sections (A) and is seen
as a thin long string at one end of the egg (the image was contrast-enhanced to accentuate the faint
outline of the egg). (B and C) Arrows point to the close association of the end of the sperm tail to one
nucleus in the developing zygote at nuclear cycle 4 (B) and nuclear cycle 6 (C). Note that the sperm is
always found in the anterior end of the egg and that the sperm tailinucleus association is at or near the
anterior boundary of the dividing nuclei.
3. Sperm-Egg lnteractions in Dro.rophi/cr 99
100 Timothy L. Karr
B. The Early Cleavage Divisions

An even more striking (and perplexing) aspect of sperm persistence in the devel-
oping egg was discovered using polyclonal antibodies that stain the entire length
of the sperm tail, including the midpiece. Examination of embryos at various
stages postfertilization revealed that the sperm tail remains associated with a
single zygotic nucleus (Figs. 3B and 3C). During each nuclear division, the
sperm migrates and remains closely associated with the centrosome. Nothing
presently is known about how this attachment site is formed or how or why it
persists during embryonic development. This structure has no known correlates
in other animals, and it remains to be seen if similar behavior can be found in
other animal groups. However, one conclusion is inescapable: a paternally de-
rived structure persists in the developing zygote long after fertilization. Some
possible roles for the unique sperm-nucleus association are discussed below.

C. Sperm Fate in later Stages of Embryogenesis

The entire sperm structure appears to remain intact throughout much of embry-
ogenesis (Karr, 1991; Graner et al., 1994). During cellularization of the blast-
oderm, the sperm tail is sequestered in the yolk, excluded from the forming cells
(Karr, 199 1). Much later in embryogenesis, the sperm tail fragments and eventu-
ally disappears (unpublished observations).

VI. Genetics and Molecular Biology of Fertilization and

Early Embryonic Development in Drosophila
Over the past few years, the identification of maternal gene products essential for
embryonic viability by classical genetic and more recent enhancer-trap meth-
odologies has provided valuable new information about the genetic systems
controlling early development (Nusslein-Volhard and Wieschaus, 1980; Driever,
1993; Johnston, 1993). Analysis of mutations and the proteins encoded by them
forms the foundation of our understanding of the molecular mechanisms of
pattern formation in the embryo.
As discussed below, mutations that affect very early events, including the most
proximal events following sperm entry, have been identified.

A. Maternal-Effect Mutations

1. Young Arrest (fs(1)Ya)

Recently, the fs(1)Ya gene product has been shown to be necessary for pronuclear
fusion and possibly for the early embryonic mitoses (Lin and Wolfner, 1989). The
3. Sperm-Egg Interactions in Drosuphila 101
fs(Z)Ya protein accumulates in the sperm nucleus and female pronucleus prior to
pronuclear fusion, suggesting a role at this early stage of zygote formation (Lin
and Wolfner, 1991). Thefs(l)Ya gene product is localized to the forming nuclear
lamina and is speculated to be involved in the signal processes that regulate entry
into S-phase (Lopez et al., 1994).

2. Deadhead (dhd)
Another maternal-effect gene product thought to act early is deadhead (dhd).
Fertilized dhd eggs almost never initiate nuclear divisions (Salz et al., 1994).
The predominant phenotypes observed are anaphase-like mitotic figures associ-
ated with meiosis I, suggesting that dhd function is involved in the completion of
meiosis. In these eggs, the sperm nucleus does not undergo nuclear decondensa-
tion (H. K. Salz and T. L. Karr, unpublished communication), suggesting that
dhd is involved in some aspect of pronuclear maturation prior to DNA synthesis.
The cellular function of dhd is currently unknown. However, the predicted
amino acid sequence of dhd has extensive homology with thioredoxin, a multi-
functional protein implicated in a variety of cellular processes (Holmgren, 1989),
including the regulation of the rate of DNA synthesis (Muller, 1991) and micro-
tubule assembly (Khan and Ludena, 199 1 ). Two intriguing phenotypes observed
in the small percentage of dhd embryos that develop are: (1) defects in nuclear
migration in the anterior end of the egg and ( 2 ) defects in some of the segmental
structures of the head (Salz et al., 1994). Perhaps the two events are related,
suggesting that dhd either directly or indirectly acts specifically in the anterior
region of the egg.

3. Plutonium (plu) and Pan Gnu (png)

The plutonium (plu) and p a n gnu (png) genes are involved in the regulation of
DNA synthesis in the fertilized (or activated) egg (Shamanski and Orr-Weaver,
1991). plu and png have nearly identical phenotypes to a previously identified
maternal gene giant nudeus (gnu) also thought to regulate DNA synthesis (Free-
man and Glover, 1987). In all cases, mutant eggs indiscriminately synthesize
DNA without accompanying mitoses, resulting in giant, endoreplicated nuclei.
To date, little is known about the proteins encoded by these genes.

4. Maternal Haploid (mh)

The rnh mutation was originally recovered in genetic screens designed to detect
maternal-effect mutations (Gans et al., 1975). The mh mutation results in abor-
tive embryonic development, and the large majority of eggs die prior to blast-
oderm formation; the few eggs that make it past this stage die soon afterward.
Sperm enter mhlmh eggs, but the sperm does not form the typical folded and
coiled structure seen in wild-type eggs (T. L. Karr, unpublished observations).
102 Timothy L. Karr
The mh gene has not been cloned, but could be an excellent candidate for an egg
product that interacts with the sperm.

B. Paternal-Effect Mutations

The existence of a major structural entity, derived from the father, in the fertil-
ized egg suggests that mutations affecting this structure will influence the course
of fertilization. Additional factors brought in by the sperm also represent poten-
tial paternal elements that may be involved in development of the zygote. As
argued below, the purpose for this structure, if any, may be revealed by the study
of sperm-egg interactions.
To date, only two mutations have been characterized that affect the paternal
genome, paternal loss (pal [Baker, 19751 and ms(3)KBl (Fuyama, 1984,
1986a,b). The low number of paternal-effect genes isolated so far is not surpris-
ing, since no systematic genetic search has yet been accomplished. However,
large-scale genetic screens are being pursued that are designed to identify genes
involved in fertilization (B. Wakimoto, personal communication). With use of
the DROP-1 antibody to assess the state of fertilization, paternal genes affecting
early development can be screened. To date, no new paternal genes have been
identified, but it will be interesting to see the nature and number of mutations
isolated by such screens in the future.

1. Paternal Loss (pal)

Homozygous pal males produce progeny that, in a small percentage of cases,
lack the X, Y, or fourth chromosome (Baker, 1975). Also, high levels of embry-
onic lethality were observed, presumably due to paternal chromosome loss dur-
ing embryogenesis. The mutation is a strict paternal-effect, with no known
effects on the female. Thus, a gene product, not necessary for sperm develop-
ment but necessary for chromosome maintenance in the embryo, is defined by
this mutation. The eventual molecular cloning should yield interesting new data
concerning this gene and its product.

2. ms(3)K81
A strict paternal-effect mutation resulting in almost 100% embryonic lethality
was isolated and described by Fuyama ( 1986a,b). Homozygous ms(3)K81 males
produce motile sperm fully capable of fertilization (T. L. Karr, unpublished
observations). However, shortly after fertilization, a variety of developmental
defects are found. These range from defects in chromatin structure to mitotic
spindle structure and sperm structure, as shown in Fig. 4. The nature and timing
of the defects indicate a role in very early stages of zygote formation and
3. Sperm-Egg Interactions in Drosuphilo 103

Fig. 4 Cytological defects associated with the male-sterile mutation, ms(3)K81. Shown is a high
magnification view of a fertilized egg arising from a cross between a wild-type female and a male
homozygous for the ms(3)K81 mutation. The sperm tail has been visualized using an antisperm
antisera and counterstained with a DNA-fluorescent dye to reveal chromosome structure. The arrow-
head shows the location of the end of the sperm tail and the arrows point out various types of
chromatin defects. The majority of fenilized eggs show similar aberrant chromatin structures that
almost always lead to early embryonic death.

maintenance. We are currently comparing and contrasting the similarities of

defects induced by ms(3)K81 mutations to those seen in a related male-sterile
phenomenon, cytoplasmic incompatibility (see below).
The application of classical genetics and newer molecular genetic techniques
to the study of fertilization promises to yield valuable new information about
sperm entry, syngamy, and karyogamy. The available data already strongly sug-
gest that paternal products play a central role in fertilization and, perhaps, post-
fertilization processes in insects. As more attention is drawn to this area, other
genes associated with fertilization will certainly be discovered. Given the size of
the sperm and the interactions observed between sperm and egg (Karr, 1991;
Graner et a l . , 1994), we can expect genetic screens to identify mutations affect-
ing these interactions. In the future we can hope to see this area continue to
blossom and Drosophila become an integral member of the family of organisms
used as model systems to study fertilization.

VI I. Cytoplasmic lncompatibility

Cytoplasmic incompatibility is a phenomenon that disrupts fertilization in partic-

ular crosses of strains within the same insect species. CI occurs in at least five
orders of insects (Saul, 1961; Ryan and Saul, 1968; Yen and Barr, 1973; Wade
104 Timothy L. Karr
and Stevens, 1985; Hoffman et al., 1986; O’Neill and Karr, 1990). Interestingly,
cytoplasmic incompatibility is intimately associated with an endocellular
symbiotic bacterium. Wulbachia pipientis (Saul, 1961; Yen and Barr, 1973;
Breeuwer er al., 1992; O’Neill et al., 1992). This association was revealed by
use of antibiotics, which remove the bacteria and the incompatibility simul-
taneously. CI has been discussed as a possible mechanism of speciation (Laven,
1959) and as a tool for the biocontrol of insect pests (Laven, 1967; Karr, 1994).
As shown in Fig. 5 , cytoplasmic incompatibility is expressed in an asymmetri-
cal manner-only infected males mated to uninfected females are incompatible.
The reciprocal cross of infected females mated to uninfected males yields normal
progeny counts, as do crosses between either infected males and females or
uninfected males and females. In an incompatible cross, the sperm enters the
egg, and karyogamy and zygote formation does not occur (Ryan and Saul, 1968;
Jost, 1970; Breeuwer and Werren, 1990; O’Neill and Karr, 1990). The physical
entry of the sperm into the egg cytoplasm is sufficient to trigger the early devel-

r
Uninfectedegg ,

II 1I II
’
Fertilized by spenn from infected Wale ‘

Fig. 5 Cytoplasmic incompatibility. An uninfected egg fertilized by sperm from an infected male
fails to complete karyogamy and/or the first few cleavage divisions. By contrast, an egg from an
infected female fertilized by sperm from an infected male completes karyogamy and develops
normally. Reprinted with permission.
3, Sperm-Egg lnteractions in Drosophikr 105
opmental stages of cell division, but then most haploid embryos die at an early
stage. lncompatible crosses therefore result in very few, if any, viable adults. An
incompatible cross is formally equivalent to the paternal-effect mutation
ms(3)K81 in D . melanogaster discussed previously. It is also important to note
that mature sperm are devoid of detectable Wolbachia, implying that the bacte-
rium exerted its effect during spermatogenesis and that the effect was transmitted
in or on the sperm (again, formally analogous to a genetic lesion).
The molecular mechanism(s) of incompatibility is (are) currently unknown,
but the phenomenon raises intriguing questions about the role of the sperm in
fertilization and early embryonic development. Cytoplasmic incompatibility also
raises important questions about when, how, and why this form of symbiosis
arose initially. Most important for the present discussion, cytoplasmic incom-
patibility is an extragenic, paternally transmitted form of sterility that has pro-
found effects on the reproductive success of its host. In this respect, cytoplasmic
incompatibility is consistent with the idea that a sperm-derived or sperm-deliv-
ered product(s) can provide important factors to the egg during and/or following
fertilization in insects.
Cytoplasmic incompatibility also has important practical implications for strat-
egies of biological insect pest management (Karr, 1994). The rationale for using
C1 comes from the unique male-sterile effect described in Fig. 5-only infected
males mated to uninfected females result in inviable embryos. Therefore, release
of infected males into an indigenous uninfected population should rapidly reduce
the number of offspring in the next generation. Of course, care must be taken not
to release infected females, which would result in the spread of infected progeny,
rendering CI ineffective. A number of laboratory experiments with the tropical
warehouse moth Ephestia cuutella, an agricultural pest of stored grain, have
shown that cytoplasmic incompatibility can be successfully used as a means of
control of this insect (Brower, 1979, 1980).
Our laboratory has recently become interested in the cell biology of this
intriguing phenomenon. We are using as a model system Drosophila simulans, a
sibling species of D . melanogaster. Cytoplasmic incompatibility in D . simulans
was first discovered in crosses between strains of D . simulans from southern and
northern regions of California (Hoffmann et al., 1986).
By applying immunocytochemical techniques originally developed for obser-
vation of cellular substructure in the D. melanogaster embryo (Foe and Alberts,
1983; Mitchison and Sedat, 1983; Warn et al., 1984; Karr and Alberts, 1986;
Karr, 1991), we are examining the cellular defects associated with cytoplasmic
incompatibility. As shown in Fig. 6, our preliminary results suggest that cyto-
plasmic incompatibility disrupts the normal behavior of chromosomes during the
mitotic cycle. One rarely observes normal chromatin figures-instead, only
fragmented and aberrant chromosomes are observed. This has lead us to specu-
late that cytoplasmic incompatibility disrupts the normal process of protein incor-
poration into the sperm head during maturation (Lassy and Karr, 1996). The
fig. 6 Early embryological defects associated with cytoplasmic incompatibility. Eggs fertilized by
sperm from (a) tetracycline-treated parents (normal development) or (b and c) untreated males.
Shown in b is an aberrant cytoplasmic bridge formed during the first cleavage division (gonomeric
division). Parts of two embryos in c show (left) highly fragmented DNA and (right) two condensed
DNA bodies that are probably the result of failed karyogamy. Reprinted with permission from
O’Neill and Karr (1990).
3. Sperm-Egg Interactions in Drmophilo 107
disruption of the normal system of condensation and decondensation related to
the presence of a prokaryotic organism could arise by two (not necessarily
mutually exclusive) mechanisms: ( 1 ) incorporation of a bacterial protein or pro-
teins into the sperm during spermatogenesis, and (2) modification of specific
sperm proteins. Modification of sperm proteins has been recently observed by
high-resolution, two-dimensional gel electrophoresis, suggesting candidate pro-
teins for further study (W. Chang and T. L. Karr, unpublished observations). In
addition to providing molecular clues to the mechanism of cytoplasmic incom-
patibility, the eventual elucidation of the exact molecules involved in the expres-
sion of cytoplasmic incompatibility promises to yield new information about
general mechanisms of fertilization in insects.

VIII. Speculative Models of Sperm Function

in the Fertilized Egg
For the purposes of illustration, and to provide a forum for speculation and
discussion, four possible models suggesting roles of the sperm and/or sperm-
derived structure in early development are shown in Fig. 7. As in all disclaimers
that appear with speculative models, each model is not necessarily exclusive, and
the “real” model could be an amalgation of some, all, or none of the models
presented. With this proviso in mind, each will be discussed separately.

A. Model 1-Nutritive Protein Import (Fig. 7A)

One obvious consequence of the entrance into the egg of sperm of such extraordi-
nary length is the importation of a fairly significant amount of paternally derived
proteins. For example, it has been estimated that the total tubulin delivered to the
egg in the sperm is at least 0.1-0.5% of the total tubulin in the egg (Karr, 1991).
Other proteins, unique to the sperm, would introduce a new set of proteins into
the egg, and as such would represent an infinite change in protein concentration
from the egg’s perspective. It is logical to assume that molecular mechanisms in
the egg have evolved to utilize, alter, or degrade these paternal contributions. For
example, the disposition, fate, and possible function (if any) of sperm proteins in
the egg might be highly regulated and essential elements of early development.
In support of this idea, using a library of monoclonal antibodies, we have
observed differential patterns of antigen loss from the sperm following sperm
entry (T. L. Karr, unpublished observations). The eventual identification of these
proteins and their ultimate fate may provide important clues about their function
in the egg.
The evolution of sperm gigantism, in the context of male provisioning and
reproductive mating strategies in dipterans, has been examined extensively by
Markow and colleagues (Markow and Ankney, 1984, 1988; Pitnick e t a l . , 1991;
108 Timothy L.

1 1

1 1
D
Fig. 7 Four speculative models of postfertilization sperm function in Drosophila. A-D show pos-
sible roles for, and consequences of, sperm persistence during early embryonic development. The
first two can be broadly classified as provisioning models: (A) general and (B) specific provisioning.
The last two can be broadly categorized as structural models: (C)sperm structure participates in
gradient formation and (D) sperm structure interacts with anterior migrating nuclei. Also note that
inherent in all four scenarios is a possible fifth functional aspect-marking of an anterior boundary of
the early cleavage nuclei by the sperminucleus structure. See text for further details.

Pitnick and Markow, 1994a,b). In certain Drosophilidae, large amounts of acces-

sory gland proteins are taken up through the female reproductive tract and uti-
lized by females in somatic tissue maintenance and oogenesis. However, because
of multiple female matings, males run the risk of investing in progeny they do
not actually sire (Markow, 1988). One mechanism by which males may provision
eggs but remain assured of their paternity is to provision directly through their
3. Sperm-Egg Interactions in Drusophila 109
gametes in the form of long, protein-rich sperm (Pitnick and Markow, 1994b).
Collaborative experiments, designed to follow the fate of sperm proteins in the
developing egg using a library of monoclonal antibodies against sperm, are
planned. Proteins with interesting or novel patterns of utilization or localization
will be candidates for further study. Once identified, mutations of genes encoding
such proteins may provide insights into their functional significance vis a vis
provisioning.

B. Model 2-Specific Protein Importation (Fig. 7B)

Sperm may deliver to the egg specific molecules essential for either fertilization
or early zygote viability. Although many possibilities exist, three examples are
mentioned: (i) proteins involved in the generation of a functional centrosome
(i.e., the sperm basal body), (ii) enzymes necessary for the initiation or mainte-
nance of DNA synthesis, or (iii) proteins that either regulate or directly partici-
pate in cell cycle regulation (i.e., cyclins, protein kinases). These could include
as yet unknown proteins in addition to the known regulators of the cell cycle, that
are unique to the initiation of the first zygote division. Although Fig. 7B shows a
hypothetical factor surrounding the early cleavage nuclei, this factor (or factors)
may work at any stage during postfertilization development of the egg. Further
studies of the specific fate(s) of sperm proteins in the developing egg may
identify candidate proteins.

C. Model 3-Diffusion/Gradient Production (Fig. 7C)

Diffusion of a soluble factor has long fascinated biologists as a mechanism for

the generation of a gradient of morphological information during development
(Wolpert, 197 1). Over the years, models invoking diffusion-induced gradients
have become increasingly sophisticated. However, with one prominent exception
in insects, discussed below, very little substantive data on the molecular mecha-
nisms involved have been forthcoming. Nonetheless, diffusion models continue
to dominate the theoretical landscape of developmental biology, and, in lieu
of equally intellectually attractive and intuitive alternatives, they will continue
to do so.
The current leading candidate for such a molecule is the morphogen bicoid
(Driever and Nusslein-Volhard, 1988a,b; Driever, 1991). Bicoid RNA is lo-
calized to the anterior tip of the egg, where it is transcribed. The bicoid protein
produced at the anterior end then diffuses throughout the anterior half of the egg
where it later becomes incorporated into embryonic nuclei at the time of blast-
oderm formation (Driever and Nusslein-Volhard, 1988a,b). In the nucleus, the
bicoid protein acts as a regulatory molecule directing the transcription of other
110 Timothy L. Karr
regulatory genes (Driever and Nusslein-Volhard, 1989; Driever, 1993). There-
fore, the available data strongly suggest that the bicoid protein gradient in the egg
is generated by simple diffusion and that this gradient is intimately involved in
directing the overall segmented body plan of the embryo.
The sperm tail in the D . melanagaster developing egg forms a “natural”
gradient by virtue of its structure in the anterior end (Fig. 3). In terms of sperm
volume occupied per unit volume of egg, inspection of the distribution of the
sperm in the anterior end clearly indicates a “gradient” of sperm tail. It will be
interesting to see if experimental manipulation of the position of the sperm in the
egg (e.g., via magnetic microbeads attached to the sperm by anti-sperm anti-
bodies) may affect some aspects of early development.

D. Model 4-Structural Role (Fig. 7D)

There are two general ways that the sperm tail could provide an essential struc-
tural element to the developing egg. The first is more general and relates to the
concentration of the sperm tail in the anterior end of the egg. Its mere existence
suggests that the egg cytoplasm is organized differently in the anterior region of
the egg. This is consistent with the biochemical differences that exist in the egg
(see discussion of the bicoid protein above). For example, the sperm tail could
bind and organize specific egg proteins in the anterior region of the egg either
during or shortly following sperm penetration. This binding could in principle
organize other components in the anterior end. In doing so, this reorganization
would result in a gradient of proteins similar in shape to that of the sperm tail, as
alluded to in Model 3 (Fig. 7C).
Another structural role may involve the regulation of nuclear migration into
the anterior region of the egg. The pattern of nuclear movement to the egg
periphery on first inspection appears synchronous and uniform (Zalokar and Erk,
1976; Foe and Alberts, 1983; Karr and Alberts, 1986). However, upon closer
inspection, nuclei are slightly delayed in arriving in the anterior end of the egg,
as depicted in Fig. 7D (T. L. Karr, unpublished communication). Because nuclei
must pass by, over, and around the sperm tail in route to the egg surface, they
may be expected to interact either physically and/or biochemically. Preliminary
data suggest that microtubules associated with migrating nuclei and the sperm
tail interact. Double-label immunofluorescence using anti-sperm and anti-tubulin
antibodies demonstrates that microtubules and the sperm tail come into ex-
tremely close contact (physically touching at the resolution of the light micro-
scope), suggesting that this may be the mechanism of the delayed nuclear move-
ments. Therefore, the sperm may serve to physically impede the free movement
of nuclei into this region.
Another consequence of these interactions would be the incorporation of either
sperm proteins or egg proteins bound to the sperm into the advancing nuclei or
3. Sperm-Egg Interactions in Drosophila 111
into the domains of organized cytoplasm that surround them (Foe and Alberts,
1983; Karr and Alberts, 1986).

IX. Conclusions and Perspectives

Essentially, nothing is known about the evolution of sperm length variation.
However, several recent comparative studies examining the adaptive significance
of sperm length in a variety of taxa (Gomendio and Roldan, 1991; Roldan et al.,
1992; Briskie and Montgomerie, 1992, 1993; Gomendio and Roldan, 1993)
including Drosophila (Pitnick and Markow, 1994b; Karr and Pitnick, 1996),
have led to the following conclusion. Relatively long sperm either provide an
advantage in sperm competition, which is postcopulatory male-male competi-
tion for fertilization of the eggs of a specific female during a single fertile period
(Parker, 1970), and/or they represent the provisioning of sperm as a form of
paternal investment (Sivinski, 1984; Pitnick and Markow, 1994b). At present, no
hypotheses consistent with sperm competition theory to explain sperm length
evolution in Drosophila have been supported (Pitnick and Markow, 1994b).
Although the idea that sperm provide a functional role following sperm entrance
into the egg, particularly a role after formation of the zygote, is controversial, if
true it has intriguing and important implications for not only our understanding of
fertilization and development in insects, but for theories of the evolution of
sperm gigantism.
Taken together, the following facts support the notion of a functional role for
the sperm in the egg in Drosophila: (1) the sperm is always found asymmetrically
localized to the anterior region of the egg; (2) it persists intact during embryonic
development; (3) the sperm remains associated with a single zygotic nucleus; and
(4) the position of this unique sperm tail/nucleus structure within the egg marks
the anterior-most boundary of the dividing nuclei during the early cleavage
stages. A complete understanding of fertilization in Drosophila awaits the expla-
nation and integration of these observations.
Preliminary data suggest that gigantic sperm other than those of D .
melanogaster fully enter the egg. For example, in collaboration with Scott Pit-
nick and Therese Markow at Arizona State University, we have visualized the
17 min long sperm of D.pachea in a fertilized egg (Karr and Pitnick, 1996). We
are currently using confocal microscopy and indirect immunofluorescence to
create three-dimensional reconstructions of this enormous sperm. The entrance
and persistence of a sperm 10 times the length of the D . melanogaster sperm in
an egg that is essentially the same size (both eggs are approximately 0.5 mm in
length) strongly suggest that specific mechanisms coevolved in the egg to ac-
comodate such gigantic sperm. Given the considerable investment in energy and
resources needed to construct a sperm of this length (Pitnick and Markow,
1994a), it is unlikely that this occurred without some sort of adaptive and func-
112 Timothy L. Karr
tional significance. The models provided (Fig. 7) point out some of the possible
functions for this structure and will hopefully spark renewed interest in fertiliza-
tion in insects in general and in this paternal structure in particular.
Our understanding of factors responsible for the maintenance of anisogamy
suggests that sperm gigantism should not exist (Parker, 1982), and conventional
wisdom based on sexual selection theory fails to explain the occurrence of sperm
gigantism. Moreover, no theory of fertilization would predict the presence or
persistence of such a structure in the fertilized egg. Assuming an adaptive nature
of design, discerning the functional significance of sperm gigantism will change
our concept of direct paternal investment during reproduction in insects. The
challenge to biologists is to provide an explanation for the design and functional
significance of such adaptations,

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