Opinion

Sperm storage: distinguishing

selective processes and evaluating
criteria
Teri J. Orr and Patricia L.R. Brennan
Departments of Psychology and Biology, and Organismic and Evolutionary Biology Graduate Program, University of
Massachusetts, Amherst, MA 01003, USA

Sperm storage, the extended maintenance of viable

sperm, probably occurs in most internally fertilizing
animals. Because it temporally separates mating from
conception, sperm storage can be adaptive in ecologically diverse habitats and affect life histories, mating
systems, cryptic female choice, sperm competition, and
sexual conflict. Sperm storage can result from different
selective forces acting on females and/or males, sometimes resulting in coevolution. The various criteria often
used to determine the presence of sperm storage in any
given taxon can result from the action of any one or all of
these selective forces. Here we discuss the criteria used
to study sperm storage and how we can use these to
better understand the evolution of diversity in spermstorage adaptations.
A nearly ubiquitous and cryptic process: sperm storage
Internal fertilization results in a delay between mating
and fertilization and a need for sperm to remain alive long
enough to fertilize eggs. Some features that allowed sperm
to fertilize eggs in a liquid environment during external
fertilization are likely to have remained useful within the
female environment. However, selection on both sexes to
ensure viable sperm are available for fertilization has
resulted in many novel adaptations to store sperm.
Spermatozoa of animals with internal fertilization survive and are stored for variable periods of time before mating
inside the male or after mating inside the female, affecting
life histories, mating system evolution, and sexual selection
including cryptic female choice, sperm competition, and
sexual conflict [14]. Sperm storage can both drive and
respond to postcopulatory sexual selection [14]. Because
sperm storage increases the window between sperm production, mating, and fertilization, it provides increased
opportunity for postcopulatory sexual selection [15]. For
example, bat testis size is positively correlated with duration of female sperm storage (FSS), suggesting that longer
storage enhances sperm competition, and males of spermstoring species seem to produce more sperm than species
Corresponding author: Orr, T.J. (tjorr@cns.umass.edu).
Keywords: delayed fertilization; postcopulatory sexual selection; reproductive delays;
sperm competition; sperm longevity; sperm storage; sexual conflict.
0169-5347/
2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tree.2015.03.006

that do not store sperm [3]. However, sperm storage can

also be modified in response to selection. For example,
details of how sexual conflict plays out during sperm storage
in Drosophila presents evidence of antagonistic coevolution
at the molecular level (Box 1).
These examples would seem to suggest that sperm
storage is highly specialized and unique. However, sperm
storage is commonplace in internally fertilizing animals
and extremely diverse (Boxes 24 and Table 1). This
variation in sperm storage results from different evolutionary forces acting independently on males and females
or simultaneously in both. We should distinguish sperm
storage that results from selection on females and occurs
inside the female (FSS) from that resulting from selection
on males, either after manufacture but pre-copulation
[male sperm storage (MSS)] (see Glossary) or postcopulation occurring inside the female but driven by selection on
males, which can potentially result in sexual conflict.
The advantages to females of storing sperm are readily
apparent. Females are able to decrease the costs of mating,
ensure fertilization, potentially increase their choice of
sire, and time reproductive demands with periods of food

Glossary
Delayed fertilization
Due to delayed ovulation: copulation and insemination occur long before
ovulation. The opposite of this trait is induced ovulation, where the release
of one or more mature eggs occurs soon after mating.
Due to sperm storage: copulation and insemination are followed by a period
over which sperm are maintained in the females reproductive tract for an
extended period, after which the egg is fertilized by these stored sperm.
Delayed implantation: fertilization occurs shortly after mating [5]. Cell division
and development occur to the blastocyst stage (200300 cells) but the
conceptus remains in the uterine lumen for an extended period of time instead
of immediately implanting into the uterine lining.
Female sperm storage (FSS): extended maintenance of viable sperm within the
females reproductive tract. FSS is a general phenomenon that encompasses
the terms delayed fertilization and delayed ovulation. FSS results primarily
from selection on females but can also be influenced by selection on males.
Male sperm storage (MSS): extended maintenance of mature sperm in the
epididymis before mating.
Sperm longevity: the reproductive life expectancy of sperm independent of any
female processes (i.e., without female aid but under the same general
conditions).
Sperm storage: extended maintenance of viable sperm within the reproductive
tract (male or female). Implicit and often untested is that the sperm must be
capable of fertilization at the end of storage.
Sperm viability: refers to the capacity of sperm to successfully fertilize eggs. A
viability of 50% or greater is considered to be a demonstration of fertility based
on the human infertility literature [39].
Trends in Ecology & Evolution, May 2015, Vol. 30, No. 5

261

Opinion
availability [1,5,6]. Similarly, males may benefit from manipulating sperm storage inside the female by biasing
paternity on their favor or by increasing their overall
paternity from any one mating (e.g., Drosophila; Box 1).
However, the benefits to males from storing sperm before
copulation have been discussed less, although they can be
just as important because they can pre-adapt sperm to
survive longer inside the female and sperm are unlikely to
be used immediately after spermatogenesis. Males can
benefit from storing sperm before mating if separating
sperm production from sperm delivery is advantageous.
Sperm-storing males can have sperm ready to use during
the reproductive season earlier than other males [58] or, if
males spread the costs of reproduction between gamete
production and obtaining a mate, this strategy is likely to
be adaptive [79].
At present, we lack a framework that unites the selective forces responsible for purported sperm-storage traits

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

with the criteria for defining sperm storage. Some features

of sperm storage, such as the presence of sperm-storage
organs, derive from selection on females, others, such as
sperm longevity, can stem from selection on males or
coevolutionary processes between the sexes, while others
still for example, the arrangement of sperm in the female
might simply be a byproduct of the architecture of the
female reproductive tract. However, some studies seem to
equate any sperm storage with FSS and report FSS in
species where selection on males, or no selection for storage
at all, could explain the same observations. For example,
FSS has been reported solely because sperm are found
inside the females reproductive tract at a time later than
expected (Table 1; mammals [9,10], rattlesnakes [11],
sharks [12]). However, in such cases matings might be
difficult to observe. Other studies, including some experimental approaches, claim sperm storage based on births
occurring later than expected (skinks [13]), a phenomenon

Box 1. Molecular basis of sperm storage

Detailed studies of the molecular pathways involved in sperm
storage, and their physiological consequences in Drosophila, are
elucidating the complex coevolution and the roles of males and
females in storing sperm.
Knockouts and mutants of Drosophila melanogaster have shown
how seminal proteins and peptide hormones play key roles in the
evolution of sperm storage [5659]. Given that many of the
documented compounds are conserved from insects to mammals,
this research represents a key step in understanding the molecular
and genetic basis of sperm storage across a range of taxa [5862].
Ejaculate proteins are key for sperm storage and include several
accessory gland proteins (Acps) such as the well-studied SP. SP
(Acp70A) is gradually released from storage sites for up to 5 days and
has many impacts on female physiology (e.g., egg production) and
behavior (remating) [63,64]. Several additional, short-lived Acps are
known and may have roles in earlier sperm dynamics (processes
preceding storage). Acps including CG1656, CG6289, CG8137,
CG9334, and CG14560 aid sperm entry to sperm-storage organs and
may be involved in sperm maintenance during storage [57
59]. Furthermore, several genes have been identified that encode
proteins upstream of SP although details of these are still being
investigated [57].
Females also produce compounds required for sperm storage
(Figure I) [59,60,62]. Furthermore, in Drosophila melanogaster three
female organs are key for FSS: the seminal receptacle, the spermathecae, and the female accessory glands (also called parovaria). The
spermathecal secretory cells release compounds that are known to be
important for maintaining sperm and for sperm and egg motility
[62]. These spermathecal substances exert their effects on sperm stored
in the seminal receptacle and are integral for successful ovulation
[40,65]. The less-understood parovaria are likely to secrete compounds
important for ovulation and sperm storage as well as other functions,
but the details of this organ remain to be fully established [62].
Here we illustrate the key steps and many compounds of the
Drosophila postmating sex response as relevant to sperm storage
[40,46,55,56,66,67]. Steps both known and implicated by current
studies are indicated in Figure I.
(1) Copulation.
(2) Ejaculation includes the transfer of both sperm and ejaculate to
the females vagina. The ejaculate contains proteins, sugars, small
molecules, and vesicles. Some compounds, like SP, may affect
female postmating behavior (not reviewed here). Others are key
for subsequent steps of reproduction including sperm storage and
fertilization and may bind to receptors in the female or on the
sperm themselves. Many male-derived compounds have been
identified thus far including: Acp29AB, Acp36DE, Acp70A (SP),
262

(3)

(4)

(5)

(6)

(7)

CG11864, CG1652, CG1656, CG17575, CG9997, CG14061,

CG30488, CG12558, and carboxylesterase. Some compounds
may be closely associated with the sperm themselves [60].
The ejaculated sperm mass (spermatozoa and aforementioned
compounds) is moved from the site of ejaculation in the vagina to
the uterus (bursa) quickly [56]. In turn, the uterus expands and
changes shape. Various male-derived compounds interact with
the females reproductive tract at this juncture to aid sperm
transfer and storage (e.g., Acp29AB, Acp36DE) [32,59]. The
females nervous system is likely to be responsible for contractions that enable sperm movement. Simultaneously, male compounds such as Acp36DE are likely to help stimulate contractions
that enable sperm movement to stores [59].
Sperm eventually reach the seminal receptacle and spermathecae.
As sperm reach these storage sites, they encounter spermathecal
substances (orange circles denoted SS in Figure I) that are
secreted by cells in the spermathecae and have far-reaching
effects. However, the role of these substances is largely unknown.
Unlike mammals, Drosophila sperm remain fairly active during
their stay in the storage organs [62].
Sperm are gradually released from storage sites, possibly enabled
by female spermathecal substances and male-produced carboxylesterase and CG12558 [56,57,62]. Stimulation of the females
nervous system results in ovulation [62]. Ovulation is not required
for sperm release (*) nor is the converse true [62].
Eggs move to the site of fertilization. Spermathecal secretory cells
may play a role in this process [68] and experimental females
lacking these cells ovulate fewer of their ova and occasionally
retain fertilized eggs in their reproductive tracts; these eggs may
then hatch internally [60]. There is also evidence that sperm from
the seminal receptacle are more likely to fertilize eggs relative to
sperm contained in the spermathecal secretory cell-rich spermathecae [60,61,68]. Another male protein ovulin enables egg
laying [59]. Sperm may be released from storage for an extended
period of time of up to several weeks [59]. However, a steep
decline in the number of sperm in storage is noted between 6 and
48 h post mating [63].
Recent findings in Hymenoptera are consistent with the documented dynamics of the D. melanogaster model system regarding
opportunities for coevolution between the sexes at the molecular
level in relation to sperm storage [69]. Given the successful
identification of the compounds discussed here and the availability of mutants, the development of the Drosophila model
system and its application to non-model taxa will expand even
further our understanding of the mechanics of FSS and coevolution between males and females.

Opinion

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

1.
Copulaon

2.
Ejaculaon

Sperm mass transfer

(sperm + ejaculate)

Sperm from

3.
vagina to uterus (bursa)
Movement
Uterus changes shape,
expands

4.
Storage
(FSS)

5.
Sperm
release &
ovulaon

Female substances (relevant to sperm storage):

Spermathecal secretory cells release
compounds referred to here as spermathecal
substances (SSs)
(sperm molity, storage, movement,
egg movement)
Glucose dehydrogenase (Gld)
CG32939 (fra mauro)
CG5630 (hadley)

Uterine shape changes.

Sperm to seminal receptacle or
spermathecae.

Sperm to storage
organs: receptacle and
spermatheca

Sperm released
from storage, eggs
ovulated*

6.
Sperm used to
Ferlizaon ferlize eggs

Sperm mass
(sperm +
ejaculate)

Transcriptomes dier between seminal

receptacle and sperm atathecae

Seminal
receptacle

Sperm from vagina to uterus.

Spermatatheca
SSs

Egg released upon

smulaon of nervous
system (via male and
female compounds).
SSs may be required
for proper ovulaon
(indirect evidence).

Male substances (relevant to sperm storage)

Ejaculate: Sugars and proteins
glucose dehydrogenase and esterase, ovulin. On
sperm themselves; Sdic and wasted.
Acps (sperm entry, maintenance and storage)
Acp29AB (retenon in stores)
Acp36DE (accumulaon, contracons)
Acp70A (Sex pepde)
Acp62F (Precidence of sperm)
Lecns:
CG1652
C type:
CG11864
CG1656
CG1757
Cysteine non-protein
Serine protease homolog CG9997
CG10586
Carboxylesterase (Est-6)
(sperm release from stores)
Upstream
CG14061, CG30488,
CG12558 (release from stores)

Sperm move to
storage structures
where they remain
mole.
Sperm movement
from stores.

Sperm in receptacle
more likely to ferlize
(uorescent studies).

Parovaria

TRENDS in Ecology & Evolution

Figure I. A schematic representation of the key steps and molecules involved in sperm storage in Drosophila melanogaster.

that could instead have been due to delayed implantation

or delayed development [1,14]. The mistaken assumption
that sperm storage was responsible for what is now known
to be parthenogenetic reproduction in vertebrates (e.g.,
Komodo dragons, hammerhead sharks, pythons)
[1,14,15] highlights the risk of using only one criterion
(in this case sperm longevity) to assign sperm storage.
To refine our thinking about sperm storage, we develop
a framework based on the most common criteria used
to study sperm storage. We organize criteria to highlight
increasing degrees of evidence for sperm-storage specializations typically associated with FSS, also identifying
those that can result from selection on males and those
that might not be under selection for storage. Developing
such a framework has the advantage of deepening our
understanding of sperm-storage evolution by fully considering existing phenotypic diversity. Comparative studies
on sperm storage have been limited to taxa with spermstorage organs [1618] or with extreme duration of sperm
storage (e.g., bats [19]). Our framework allows comparative
studies between taxa that might lack sperm-storage structures (Table 1) or have only a short duration of storage, to
better understand the evolution of sperm storage.
In this review we focus primarily on vertebrate examples, but examination of the literature reveals that the
issues we highlight here are also pervasive in the study of
sperm storage in invertebrates and therefore we believe
that our arguments and framework are broadly applicable.
Many exciting questions can be addressed with a more

refined framework regarding sperm storage in various

taxa. Is the phylogenetic distribution of this trait driven
by selection on males or females? Which criteria evolved
in different taxa under dissimilar ecological conditions
(Box 5)? A possible way to examine these and other questions is to quantify the existing evidence of sperm storage,
which we have attempted in the form of an index that
weighs and combines different lines of evidence to evaluate
FSS (Box 3). A previous attempt to quantify this trait in
squamate reptiles [7] has not been broadly applied, perhaps because it is too taxon specific. Analyses using our
suggested index can include a broad range of species for
which only partial data are available, potentially making
these species valuable in a comparative framework. Although preliminary, this approach will hopefully spark
discussion and further refinement of these ideas.
Below, we review criteria of sperm storage in order of
the strength of selection that might be operating on them
and whether they are likely to derive from selection on
males, females, or both. Listed from most passive to most
active (and possibly varying degrees of selection), these
criteria are all utilized in our proposed sperm storage index
(SSI). Six general criteria for sperm storage follow below
(Box 3).
The general criteria for sperm storage
Sperm arrangement
In vertebrates and possibly some invertebrates [20], sperm
typically line up with their heads facing the epithelial
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Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

Box 2. Sperm storage is widespread and nearly ubiquitous in internal fertilizers, as seen in this phylogeny
Colors indicate the number of species known to have sperm storage
in each branch. Fertilization mode is indicated as I (internal) or E
(external) or a combination, with the most common type listed first.

Duration of sperm storage is indicated in days as well as known

locations of sperm storage (Figure I).

Ferlizaon
Key:

Duraon Locaon

Most FSS
Many FSS
Some FSS
None FSS
Suspected FSS

Alligators
Birds
Turtles
Lizards
Snakes
Bats
Other placental mammals

Marsupials
Monotremes
Amphibians
Lungsh
Coelacanths
Bony sh
Sharks and rays
Urochordates
Hagsh and lamprey
Hemichordates
Echinoderms
Cnidarians
Ctenophorans
Arthropods
Nemotodes
Flatworms
Lophorates
Annelids
Mollusks
Sponges

I
I
I
I
I
I
I
I
I
I/E
E
I
I/E
I/E
E
E
E
E
E
E/I
I
I
I
E/I
I
E/I
E

7
7-117
90-1,460
14-540
90-2,555
16-300
0.5-30
0.5-16
unk.
120-900
90-240
30-730
14-4,000
5
55
180-365
70-1,460
-

Oviduct
SSTs in uterovaginal juncon
Albumin glands in oviduct
SSTs, sacs, crypts in oviduct, infundibulum, vagina, uterovaginal junct.

SSTs in uterine tube

Oviduct, utero-tubal juncon, uterus
Uterine glands, vagina, cervix
Furrows, crypts, reservoirs in oviduct
unk.
Spermathecal tubules, cloacal glands, spermathecae

Infundibumulm, ovarian follicles, ovary

Oviductal glands

Spermathecae, seminal recepcal

Spermathecae
Tubae organs near oviduct
Spermathecae
Spermathecae
TRENDS in Ecology & Evolution

Figure I. Schematic phylogeny of female sperm storage in major groups of animals.

lining of the females reproductive tract, which might or

might not include specialized sperm-storage organs. Some
authors have suggested that this arrangement indicates
that sperm are being stored by females in the location
where they are lined up [14,15,21]. However, sperm arrangement might also result from selection on males alone
to arrange their sperm in an advantageous position waiting to fertilize the females eggs [22] or from other evolutionary processes in the absence of direct selection.
Therefore, sperm arrangement is not a particularly informative criterion to study the mechanisms responsible for
the evolution of sperm storage and should not be used as
direct evidence of FSS.
Sperm behavior
In internally fertilizing species, sperm have to find storage
sites and eggs within an environment that can be closely
regulated by the female (e.g., hyperactivation of sperm
stored in the oviducts [23]). Sperm motility is under selection in males, as sperm that can more efficiently find their
target will result in higher male fitness. Active movement
in the form of rheotaxis (movement in response to a
current) has been demonstrated in several mammalian taxa
(e.g., mice, humans [24]), suggesting a plausible directed
movement of sperm to storage or fertilization sites. Selection
264

for efficient sperm movement within the female tract has

led to the evolution of sperm cooperation. For example, in
mice (Apodemus sylvaticus) sperm have evolved hooked
sperm heads to form sperm trains that move more efficiently
inside the viscous fluid of the female reproductive tract
[25,26]. Trains are formed preferentially among the sperm
of the same male, suggesting selection on males via sperm
competition. High sperm competition also correlates with
more hooked sperm heads among species [2527]. Masses
of sperm (conjugation) observed in many invertebrates may
serve a similar enhanced motility function [26].
When sperm storage is under selection in females, they
might actively move sperm to storage sites. Female marsupials appear to be especially good at sperm transport, with a
large percentage of sperm reaching storage or fertilization
sites (roughly one in every eight sperm) relative to many
eutherians (roughly one in every 10 000 sperm), perhaps
aided by cilia lining the reproductive tract [2629]. However,
even eutherians exhibit directed sperm movement. Sonography in humans reveals directed movement of fluids in
the female reproductive tract [30]. Female mammals can
use contractions, in conjunction with movement of cilia
lining the lumen of the oviducts, to move sperm as far as
the uterotubal junction [28]. In reptiles, sperm are moved
from the site of ejaculation to storage (Anolis, 624 h) also

Opinion

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

Box 3. The SSI and sperm storage ratio (SSR)

Based on several criteria including sperm behavior, sperm longevity,
and the presence of sperm-storage structures, we propose a
standardized index to evaluate the likelihood that a species has
evolved FSS, even when not all lines of evidence are available. An
additional method would evaluate the ratio of sperm longevity
relative to the duration of storage within the female.
To calculate the SSI, one would assign values to each criterion (1 for
conclusive evidence, 0.5 for some evidence, and 0 for no evidence) and
sum the cases providing any evidence relative to the criteria for which
data were available. This value would be standardized across taxa by
dividing the value by the total number of criteria evaluated (here, 12).
Because the SSI is relative to both the number of criteria examined and
the possible criteria, future studies could add additional criteria for FSS.
SSI = (sum of supported criteria  N criteria examined)/total criteria
Using the 12 criteria we ha;ve listed, the standardized index would
range from 0 to 12. A key point of the SSI is that species with varying
certainties of FSS assignment can still be compared with one another.
Life history

Anatomy

Physiology

Behavior

 Sperm live longer in the females than in similar

conditions not within the female
 Sperm viable after storage
 Storage structures (organs)
 Sperm stored in a particular regions
 Multiple (types) of sperm storage are evident
 Organized arrangement of sperm (e.g., heads
aligned with female anatomy)
 Female immune suppression (localized)
 Sperm receive nourishment
 Specialized sperm (ejaculate) biochemistry (e.g.,
Acps; Box 1)
 Ovulation only at the end of the sperm storage period
 Sperm activity diminished during storage but returns
at termination of storage (thus, obviously
synchronized with female reproductive patterns)
 Sperm moved in targeted manner to storage site

possibly due to female muscular contractions [31]. Female

contractions are likely to be important for sperm placement
in storage sites in insects. Reproductive tract contractions
might facilitate sperm storage in Drosophila, triggered by
Acps [32], but sperm can reach storage sites in some insects
even if muscles are disabled [14], suggesting that selection
on males favors sperm that can reach their target unaided.
Sperm viability
The presence of viable sperm inside the female tract is a
necessary but not sufficient criterion in support of FSS.
The presence of sperm could be due to selection on
females to actively maintain or at least not degrade
sperm. However, it could also arise from selection on
males to produce long-lived sperm independent of female
processes, including specialized biochemistry of the ejaculate (Box 1).
Fertilization occurs at varying intervals after copulation
(18 h in cows and up to 10 years in some social insects
[14,15,33,34]; Table 1); therefore, sperm can remain viable
inside the female for different periods of time. In species
with MSS, sperm are selected to be long lived, and this
adaptation could help them survive and remain viable
inside the female in the absence of any selection on
females. For example, in bats the duration of MSS appears
to be qualitatively similar in magnitude to the duration of
FSS [9].

12 criteria
SSI = (sum of supported criteria  N criteria
examined)/total criteria

A generic example is illustrated in Box 4.

Possible ways to refine the SSI include requiring studies investigating FSS to demonstrate two prerequisites:
(i) Sperm must live longer in the female than under similar
conditions outside her reproductive tract.
(ii) Sperm must be viable at the end of storage.
Strong evidence (i.e., of more active forms of FSS) could be given
extra weight in calculations of the SSI (e.g., structures, nourishment,
chemistry).
The SSR
An alternative way to examine FSS would be to use a ratio of sperm
longevity relative to duration of storage within the female reproductive tract. This ratio allows sperm storage to be examined as a
continuous variable. A limiting feature of the SSR is that it requires
knowledge of sperm longevity outside the female and such data are
rarely available. Interpretation of FSS would be tempered given the
lower level of certainty.
SSR = duration of FSS (days)/duration of sperm longevity without the
female (days)
Broad taxonomic differences in the SSRs of a few species demonstrate why this metric might be interesting. Human sperm appear to
be viable in the female reproductive tract for up to 5 days, while
sperm outside the female are viable for less than 1 day [40,70]
(SSR = 5). However, bee sperm can live for up to 2 years in the female
(730 days) but only 6 months (180 days) with reasonable viability
outside the female [71] (SSR = 4). Examination of more closely related
species may allow examination of this metric in a phylogenetic
context or in the context of total lifespan.

We can learn more about the selective processes acting

on sperm survival by examining sperm longevity inside
and outside the female. Currently, the only way to distinguish between sperm longevity and FSS is to compare
sperm with and without female assistance. If spermatozoa
in the absence of female assistance (but with, for example,
similar temperature and pH) live for a shorter period of
time, FSS might be occurring. If sperm live for the same or
a longer period of time outside the female, this would not
support FSS. This can occur if there is no selection on
females to maintain sperm or if females evolved to get rid of
unwanted ejaculates (e.g., squid [35], spiders [36], bats
[37], birds [38]). However, accurately mimicking the conditions inside the female would require a greater understanding of the female reproductive environment than we
have at present. Perhaps an easier first step may be to
gather information about the percentage of viable sperm
(sensu [39]) after storage.
Sperm and immunity
The immune response of females that exists to protect the
reproductive tract from potential infection can quickly
decimate inseminated sperm [40]. If selection operates on
females to store sperm, we would expect that sperm are
protected from this typical house cleaning. For example,
they could be stored in areas of low immune regulation
(e.g., oviduct [40]) or the immune response could be
265

Opinion

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

Box 4. The strength of selection on FSS can be visualized in various taxa using our suggested SSI
Note that values in parentheses have partial support (and thus are
weighed as 0.5 rather than 1 in SSI calculations). Here we also

Aributes

Variable

Annelid
Shark

Life
history

Mammal:
Marsupial

Amphibian

illustrate sperm-storage anatomy in various taxa (anatomical illustrations by T.J.O.) (Figure I).

Mammal:
Human

Sperm live longer in

female than w/o

Mammal:
Bat

Reple

Bird

Mollusk:
Snail

Insect:
Fruit y

Insect:
Honey bee

MSS*

Viable aer storage

Anatomy

Physiology

Structures (organs)
Region
Mulple types of
storage sites
Sperm in organized
arrangement
Female localized
immune suppression
Sperm receive
nourishment
Sperm have specialized
biochemistry
Ovulaon only at end of
SS

Behavior

Sperm acvity diminished

during storage, acve preferlizaon
Sperm move or moved to
storage site in targeted
manner

SSI

Sum of evidence
N lines of evidence
Index (SSI)

( )
( )

( )

( )

( )
( )
( )

4
5
1.7

5
5
2.1

4.5
6
2.3

6
7
3.5

5
9
3.8

5.5
9
4.1

6.5
8
4.3

8
9
6.0

9
9
6.8

8.5
11
7.8

12
12
12

Anatomy

TRENDS in Ecology & Evolution

Figure I. An illustration of how the SSI can be applied to taxa where some criteria for sperm storage are known. General anatomical features associated with sperm
storage in each group are also depicted.

downregulated where sperm are found. Females can even

go a step further and protect sperm from fungal or
bacterial attack [41,42]. Males too may protect their
sperm during storage; for example, by including antimicrobial peptides (AMPs) in the ejaculate.
Immune responses might occur systemically or locally
in areas of sperm storage. Maintaining a low systemic
immunological response could be costly if this makes
females more susceptible to infection. Systemic immune
costs of FSS have been demonstrated in insects (e.g.,
crickets [42], ants [43]) and are likely to be observed in
many sperm-storing taxa. Localized decreases in immunological response to sperm can be a means of mitigating
immunological costs and thus immune responses might be
lowered in storage sites only. These localized responses
are known primarily from vertebrate studies [40]. Therefore, taxon-specific differences in immune physiology require taxon-specific predictions and closer investigation of
localized immunity. Immunity regulation in response to
sperm storage may not allow straightforward predictions
because other factors can influence the females immune
response, including protection of her reproductive tract
and embryos.
Leukocytic attack of foreign cells, including spermatozoa, is the norm inside female vertebrates [28] and sperm
are often targeted for phagocytosis [14,28,40,41]. In
reptiles, degradation of sperm by leukocytes has been
266

interpreted as an absence of FSS [44]. In invertebrates,

the immune response involves hemocytes, which attack
foreign cells via an encapsulation response that seems to be
systemically reduced postmating in some but not all taxa
(e.g., damselflies, Scathophaga stercoraria) [45,46].
Downregulation of the female immune response could
also result from selection on males to manipulate FSS and
halt the destruction of their sperm, or from issues related
to other aspects of the females biology. Regulation of the
female immune response in humans is known to occur
through repeated exposure to the partners ejaculate, presumably due to multiple exposure and developed tolerance
of foreign cells [47]. Within Drosophila, males express and
transfer AMPs to females in their seminal fluid, while
females express AMPs in the oviduct and sperm-storage
organs [45].
Sperm maintenance and nourishment
During extended periods of storage, sperm might require
physiological support, which can include protection from
oxidative stress as well as nourishment. Some physiological support for sperm is provided by ejaculate components
[8], but some might be provided by the female if sperm
storage is to her advantage. Although female sperm nourishment would present strong evidence of FSS, its occurrence is debated [6,9,1315,34]. Verifying that sperm are
gaining nourishment from female-derived substances is

Opinion

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

Table 1. Example species illustrating the variety of methods used to assign the presence of sperm storagea
Invertebrates
Arthropoda
Crabs (various over
Decapoda
110 species) including
(Brachyura)
Pseudocarcinus gigas,
Chionoectes bairdi
Spiders (generalized)
Arachnida

Insecta

Gastropoda

Red flour beetle (Tribolium

castaneum)
Boll weevil (Anthonomus
grandis)
Bed bug (cimicids)

Duration (days)

Location

Evidence

Refs

14 years
including some
with trans-molt
retention
365

Thelycum, spermatheca,
seminal receptacle

Viable offspring without access to a

male; histology and macro dissections to
look at anatomy

[18]

Spermathecae (two, with

separate entrances)

Orb web spider (Argiope) morphology,

paternity analyses, spermatheca filling
patterns
Mating and viable offspring for this
period of time after mating
Looking for living sperm after different
periods of storage
Offspring after female sexual isolation
from males (mates)

[8,36]

140

Spermatheca

14 or longer

Spermatheca

3550 or longer

Seminal conceptacles
(sperm-storage
receptacles and transport
ducts in oviducts and
elsewhere modified from
immune cells)
Spermatheca and seminal
receptacles

[72]
[31]
[33,54]

Fruit fly (Drosophila

melanogaster)

14

Panamanian leaf-cutting
ant (Atta colombica)

>3650

Spermatheca

Odonata (dragonflies
various species)

Days to >2
weeks

Spermatheca

Snails: round rocksnail,

Leptoxis ampla), smooth
hornsnail, (Pleurocera
prasinata), skirted
hornsnail, (Pleurocera
pyrenella), silty hornsnail
(Pleurocera canaliculata)
Japanese pygmy squid
(Idiosepius paradoxus)

At least length
of reproductive
season

Spermatophore bursa (no

seminal receptacle)

Until
spawning

Seminal receptacle (near

buccal mass) not
connected to ovary

Achatinidae, Helicidae

>365

Subdivided spermatheca
or spermathecal tubule

Earthworm (Hormogaster
elisae)

820

Spermathecae

Virgin females mated with several males

in different orders and allowed to
produce cocoons

[48]

Nematode
(Caenorhabditis elegans)

Spermatheca formed from

a widening of oviduct

Looking for living sperm after different

periods of storage; of 164 sperm in an
ejaculate, 100 will reach organ that could
hold 350

[33]

Human (Homo sapiens)

Uterine glands

[40]

Little brown bat (Myotis

lucifugus)
Southern yellow bat
(Lasiurus ega)
Nectar-feeding bat
(Macroglossus minimus)

138

Uterotubal junction

Unknown

Unknown

Fertilization after timed copulation,

histology, etc.
Histology, uterine sperm relationship,
known insemination period
Interval between mating and ovulation

Unknown

Uterine cornua

[10]

0.523

Lower isthmus of oviduct

Orientation of sperm in uterus (histology

of samples from a single time period)
with no mature follicles in ovaries
Histology, experimental (controlled
mating), demonstration of sperm activity
from stores

Fertilization after copulation, microscopy

of storage organs, genetics of storage
including knockouts that no longer store
sperm; of 5000 sperm in ejaculate,
approximately 1200 reach spermatheca
Single mating event followed by life
cycle without males but maintained
fertility using sperm from mating
Matings of virgin females and continued
fertility of females; many detailed
morphological studies
Histology; some reports of orientated
sperm but not a rule

Histology, virgin squid experimentally

introduced showed differential
spermatangia removal from seminal
receptacles; fertilization is likely, external
details unclear

[33,56]

[43]

[55]

[20]

[35]

[73]

Annelida

Chromadorea

Mammals
Theria

Laboratory mouse (Mus

musculus)

[74]
[9]

[1,28]

267

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Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

Table 1 (Continued )
Duration (days)
0.6

Location
Lower isthmus of oviduct

0.9

Lower isthmus of oviduct

30

Uterus

Dog (Canis lupus

familiaris)
Horse (Equus caballus)
Domestic pig (Sus scrofa)
and sheep (Ovis aries)

Quoll (Dasyurus
viverrinus)
Opossum (Didelphis
virginiana)
Dunnart (Sminthopsis
macroura)
Fat-tailed dunnart
(Sminthopsis
crassicaudata)

Common rat (Rattus

norvegicus)
Guinea pig (Cavia
porcellus)
European hare (Lepus
europaeus)

Evidence

Refs
[1]
[1]

Uterine glands

Prepartum estrus, histology, timed

matings, lavaged vagina, and verified
matings
Electron microscopy, timed matings

[21]

6
2.0

Uterus
Uterus

Histology, timed matings

Histology, timed matings

[1]
[1]

14

Isthmic crypts of oviduct

Histology and experimental

[1,53]

0.5

Isthmic crypts of oviduct

[1,53]

2.5

Isthmic crypts of oviduct

[1,53]

2.5

Isthmic crypts of oviduct

Brown antechinus
(Antechinus stuarti)

16

Isthmic crypts of oviduct

Echidna (Tachyglossus
sp.)

Implied

Blue mouth rockfish

(Helicolenus dactylopterus
dactylopterus)
Trinidadian guppy
(Poecilia reticulata)

[1,67]

Marsupials

Histology and experimental,

demonstration of sperm activity and
directed movement from crypts to sites
of fertilization
Histology

[1,53]

Unknown

Timing of births and known mating

period

[51]

300

Ovary (epithelial crypts)

Histology

[15]

300

Ovary micropocket

Production of offspring in the laboratory

after removal of males, genotyping of
introduced population of females

[75]

365

Oviduct tubules

[66]

390

Oviduct gland spermstorage tubules (SSTs)

(terminal region)

Histology of wild-caught animals

compared with individuals reproductive
state
Microscopy of wild-caught sharks
illustrating sperm presence; presence of
secretory cells near sites of putative
sperm storage, presence of stored sperm
in pregnant animals, and a biannual
ovarian cycle

SST in ovisacs (oviduct)

Histology

[34]

1316

SST in uterovaginal
junction

Histology; controlled-mating
experiments, some demonstrating
motility

[1]

11117
8
1316

SST
SST
SST

120

Uterus or vagina

90

Vaginal furrows

[1]

Monotremes

Fish
Osteichthyes

Chondrichthyes
Blue shark (Prionaca
glauca)
Gummy shark (Mustelus
antarcticus)

[12]

Amphibia
Tailed frog (Ascaphus
truei)
Aves
Passeriformes

Galliformes
Columbiformes
Anseriformes
Reptilia (Squamata)
Snakes
Neotropical rattlesnake
(Crotalus durissus
terrificus)

Garter snake (Thamnophis

sirtalis)

268

[1,38,51]
[1]
[1]

Lavage of uterus and vagina to verify

sperm motility, histology, some allowed
to continue reproduction to verify
viability; evidence of nourishment from
female epithelium found via staining for
mucosal function
Histology, captive births

[11]

[1,31]

Opinion

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

Table 1 (Continued )
Duration (days)

Location

Evidence

Refs

Ground skink (Scincella

lateralis)

Unknown

Rugae and crypts of

oviduct

[13]

Fringe-fingered lizard
(Acanthodactylus
scutellatus)
Green anole (Anolis
carolinensis)
Tropical house gecko
(Hemidactylus mabouia)

120

Crypts of the infundibulum

Sampling across time, histology (light

microscopy and transmission electron
microscopy), secretory vesicles
Timed histology, pores and secretory
vesicles found

210

Sperm receptacles

Histology, breeding of captive animals

[1,30]

Unknown

Uterine tube (and not

vagina)

Secretory cells near putative sites of

sperm storage, phagocytosis outside
putative storage region

[44]

Painted turtle (Chrysemys

picta)
Desert tortoise (Gopherus
agassizii)
Soft-shelled turtle
(Lissemys punctata)

1095

Albumin-secreting glands
in oviducts
Albumin-secreting glands
in oviducts
SST, isthmus
(infundibulum
oviduct to uterus)

Paternity testing of offspring from

captive females as well as histology
Paternity testing of offspring from
captive females as well as histology
Timed dissection of wild-caught turtles,
estrogen treatment to examine sperm
movement

[76]

Lizards

[1,13]

Turtles

730
120

[76]
[52]

Sperm storage has been described in a many taxa using various criteria. Even within the same taxon, the criteria used to assign FSS differ between studies. Data include
duration, location (if known), and other supportive evidence.

challenging [28]. Female production of carbohydrates in

the reproductive tract of mammals might serve such a role
[28], as might secretions from specialized glands (e.g.,
annelids [48], spiders [49], crabs [18]). However, demonstrating that females provide special compounds just for
sperm, and not for their own physiological needs, is difficult. Furthermore, determining unequivocally that these
products are the result of selection on females versus males
is also a challenge, particularly because males may be able
to parasitize female somatic resources. The presence of
glands in areas where sperm are stored is known but does
not provide convincing support for sperm nourishment

without further knowledge of their function. However,

sperm not in association with the females reproductive
tract die or lose fertilization capabilities [28] and the
alignment of sperm heads in close association with the
females tissues could possibly allow nutrient uptake from
the female (bats [37], fowl [38], earthworms [48], marsupials [50], and possibly some gastropods [20]).
Sperm-storage organs
Most internally fertilizing animals possess sperm-storage
organs (Table 1 and Box 4). Specialized structures to store
sperm clearly derive from selection on females. The degree

Box 5. Future directions in sperm storage research

Many exciting questions regarding the causes and consequences of
sperm storage remain. Key aspects that deserve further inquiry
include the female and male roles in sperm storage, the evolutionary
steps in the origins of FSS, and the degree of sexual conflict in species
with varying levels of sperm storage.
Do we see phylogenetic signal or other evolutionary patterns in the
presence of sperm storage?
 Here we have proposed that sperm storage is nearly ubiquitous in
internally fertilizing animals. However, some groups are likely to
exhibit evidence of stronger selection on sperm storage relative
to others. Future studies could examine a broad range of taxa in a
comparative (phylogenetic) context to understand how sperm
storage may have evolved. By mapping the acquisition of features
like sperm-storage organs, nourishment of sperm, and active
transport of sperm onto a phylogeny, we may begin to grasp the
evolutionary history of sperm storage. Additionally, we can test
whether and how traits commonly associated with MSS are related
to the evolution of FSS.
 Furthermore, by including aspects of ecology, life histories, and
mating systems, such studies may begin to elucidate the driving
forces behind the evolution of sperm storage.
 Using the SSI we propose (Box 3) might allow comparisons of taxa
despite disparate evidence.
What is the evolutionary history of FSS?
 By mapping onto a phylogeny the losses and gains of sperm
storage criteria, we might begin to gain insight into what features

are key in initial transitions to or from FSS and determine

whether any of the criteria we discussed are prerequisites for its
evolution.
 Ancestral-state reconstructions could be used in well-studied clades
to elucidate additional aspects of the evolutionary history of this
trait. Once we have a better understanding of features relating to
FSS (primarily measured as aspects of the females biology), we
can begin to investigate coevolution between the sexes.
 A key next step would be to investigate whether one sex ever wins
with regard to driving the evolution of sperm storage.
Do any aspects of FSS such as storage organ morphology provide
more or less opportunity for cryptic female choice?
 Researchers have begun to investigate the consequences of FSS on
males (e.g., [14,52,75]). However, does the strength of selection
on FSS correlate with the level of female control of precopulatory
events? Does female precopulatory choice interact with more
complex forms of FSS so that species with less precopulatory
choice evolve more complex postcopulatory choice (e.g., birds [51]
versus Drosophila [61])?
 Studies investigating aspects of cryptic female choice in closely
related taxa with different SSIs might yield insight into the
opportunity for postcopulatory female choice.
 Future studies might benefit from utilizing proteomics and gene
expression (Box 1) associated with the proximate aspects of female
control to determine which key aspects of FSS have a greater
influence on a females ability to choose and how this may
influence evolutionary patterns.
269

Opinion
of sophistication in the morphology of sperm-storage structures can reflect the strength of selection and costs and
constraints in evolution. For example, sperm-storage
tubules in birds have a single opening and sperm are lined
up in the order in which they enter the tubule. As a result,
the last sperm to enter is the first to exit, leading to lastsperm precedence in birds [51]. In spiders, females place
sperm from separate matings into different storage organs
[49]. This might reflect a different degree of precopulatory
choice that selects for more refined sperm-storage organs
if females have less control over mating.
In species lacking specialized sperm-storage organs,
selection on females can still favor sperm storage, and
such storage might occur in structures of the female reproductive tract that originally evolved for other functions.
For example, the uterine glands of dogs [20], albuminsecreting glands in turtles [52], and the oviductal glands
of sharks [15] have been found to contain sperm and have
been suggested as storage sites. However, it is unknown
whether sperm simply arrives in these sites by chance
and are easily trapped because of their architecture and
location [28], so the mere presence of sperm within these
structures is not sufficient evidence of FSS. The best
support that these are sperm-storage organs would be
information on whether sperm from these structures are
used to fertilize eggs. In the absence of such data, examining multiple individuals would reveal at least whether
sperm reliably end up in the same location. Other anatomical lines of evidence could include examination of epithelial differences (ultrastructure) in regions of putative
storage. For example, the presence of glandular tissue
could be investigated for secretion of sperm-nourishing
materials (criterion number 4).
Further evidence that females store sperm in the absence of specialized storage organs could derive from the
examination of ontogenetic changes of the putative spermstoring regions. If sperm-storing features are apparent
only when a female reaches sexual maturity, this is supportive that such features play a role in FSS. However,
anatomical or physiological changes that occur in the
reproductive tract after mating could result from selection
on both males and females and coevolutionary processes.
Changes induced by copulation have been observed in
the epithelial folds of the brushtail possum (Trichosurus
vulpecula) [50], secretory cells in the soft-shelled turtle
(Lissemys punctata) [52], the grey short-tailed opossum
(Monodelphis domestica) [29], and the cervix of some mammals [40]. If the mechanism by which these structures
become modified to store sperm is derived from chemical
or mechanical changes that occur during copulation, males
have the potential to manipulate females to preferentially
store their sperm.
Wherever they are located, putative sperm-storage sites
must be conducive to fertilization either because of proximity to ovulated eggs or because sperm can be moved from
these stores to areas where unfertilized oocytes occur. The
fat-tailed dunnart (Sminthopsis crassicaudata) moves
sperm from crypts closer to the oviduct around ovulation
[53]. In cephalopods (e.g., Architeuthis) sperm are moved
from under the skin of their arms to fertilization sites
[14]. Another extreme example of sperm movement is seen
270

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

in the traumatically inseminated bed bug (Cimex lectularius), which has evolved specialized structures to direct
sperm to newly derived (from mesoderm versus ectoderm
as in other insects) storage organs [54]. Other females
might move sperm to particular areas [33]; for example,
to sites where males cannot remove them (Odonata [55]).
If sperm consistently position themselves or are positioned
by the female in putative storage sites, this could be taken
as support of FSS and the role of these structures in sperm
storage.
Strength of selection on sperm storage and weighing
the evidence for FSS
Sperm storage is widespread and diverse in species with
internal fertilization and we need a more refined way to use
data to understand patterns of sperm-storage evolution,
including the action of the various mechanisms of selection
that act on adaptations to store sperm. One way to achieve
this understanding is to use the criteria outlined above to
determine the relative support for different mechanisms of
selection acting on various species. We have developed a first
approximation in the form of a simple quantitative index
(Box 3) where individual criteria are weighed differently to
examine the evolution of FSS. This could also be done for
MSS, to examine selection on males once their sperm are
inside the female, and to study coevolutionary processes
after further refinement of the criteria we present. Further,
this approach will allow us to examine the phylogenetic
patterns of the various criteria for sperm storage and their
correlation with the ecologies of animals with differing
levels of support for sperm storage (Box 2). Our proposed
SSI might have utility for comparing taxa (such as those
outlined in Table 1) and could reveal surprising and previously unappreciated patterns in sperm storage.
Concluding remarks and future directions
We were prompted to write this article due to frustration
with the lack of clarity in studies of sperm storage, which
seem to assume that any sperm storage is equivalent to
FSS. We hope to have shown that this is not necessarily
the case and that different mechanisms of selection on
males and females can act on sperm storage. Further, we
also call attention to the fact that multiple selective mechanisms can affect sperm storage and that these sources
of selection should be distinguished in future studies. We
outline (Box 5) a few ideas for future research into sperm
storage. We view this synthesis as a call for greater
attention to this issue rather than as a definitive set of
conclusions and hope other biologists will use our ideas as
a starting point for studies of the prevalence and evolution
of sperm storage mediated by selection on males and
females.
Acknowledgments
The authors thank Jeff Podos, Thomas Eiting, Casey Gilman, Duncan
Irschick, Norman Johnson, Diane Kelly, the OEB graduate program, Paul
Craze, and two anonymous reviewers for useful comments on early drafts
of the manuscript. Dr Mariana Wolfner helped us to clarify the molecular
pathways in Drosophila shown in Box 1. The authors also thank Marlene
Zuk for insightful discussions. T.J.O. was supported by a National Science
Foundation Postdoctoral Research Fellowship in Biology under Grant
No DBI-297 1202871.

Opinion

Trends in Ecology & Evolution May 2015, Vol. 30, No. 5

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