Seasonal patterns of sperm storage in the Yellow-headed Blackbird Xanthocephalus xanthocephalus

The seasonal pattern of sperm storage was examined in a wild population of Yellow-headed Blackbirds Xanthocephalus xanthocephalus by collecting females at eight different stages of their breeding cycle. Sperm storage tubules (SSTs) were present in simiktr numbers throughout all stages of the breeding season, from arrival on the breeding grounds to departure for winter. In contrast, the size of SSTs varied enormously over the season, reaching maximum size just prior to clutch initiation, when SSTs were more than three times the length of those found in post-breeding birds. The amount of sperm stored also varied over the season, in parallel with the growth and regression of SSTs. Sperm were first observed in SSTs approximately 2–5 days before laying and increased rapidly in abundance until clutch initiation. The amount of sperm stored then decreased gradually during the egg-laying period so that by clutch completion approximately 70% of stored sperm had disappeared from the SSTs. The rapid seasonal development and subsequent regression of SSTs in the Yellow-headed Blackbird contrast sharply with the pattern found in domestic poultry, where the imposition of continuous laying regimes has; led to the maintenance of SSTs at maximum size for extended periods of time.     

Apical blebs on sperm storage tubule epithelial cell microvilli: Their release and interaction with resident sperm in the turkey hen oviduct

Located at the anterior end of the turkey hen's vagina are numerous discrete tubular invaginations of the surface epithelium, collectively referred to as the sperm storage tubules (SSTs). After mating or artificial insemination, sperm ascend the vagina, enter the SSTs, and over the ensuing days and weeks, gradually exit the SSTs and are transported to the anterior end of the oviduct to fertilize a daily succession of ova. Little is known regarding the cellular and molecular mechanisms responsible for sperm subsistence in the lumen of the SST. In this study, the origin of microvillus blebs (MvBs) on the apical tips of SST epithelial cells was examined, and their possible role in sperm survival was discussed. Regardless, if sperm are present or not, transmission electron microscopy revealed two types of microvilli differentiated by the presence or absence of pleomorphic unilaminar MvBs localized to their apical tips. Although some MvBs appeared to be discharging their contents into the SST lumen, others appeared to have pinched off the microvillus stem. When SSTs contained clusters of densely packed sperm, the sperm heads of those sperm adjacent to the SST epithelial cell surface were surrounded by the microvilli. Associated with the plasmalemma of sperm throughout the SST lumina were membrane fragments and small vesicles (30–130 nm in diameter), some of which appeared to have fused with sperm. It is concluded that the MvBs are a form of shedding vesicle released from the SST epithelial cell microvilli by apocrine secretion. On the basis of observations described herein and those of other authors, it is suggested that the MvBs contribute to sustained sperm storage in the SSTs by (1) supplying metabolic substrates used by resident sperm, (2) serving as fusogenic vehicles providing exogenous macromolecules that reversibly suppress sperm functions associated with fertilization (decapacitation?) and stabilize the sperm plasmalemma, and (3) acting as transport vesicles actively transporting fluid from the SST epithelial cells to the SST lumen.     

THE EVOLUTION OF SPERM SIZE IN BIRDS

Sperm size varies enormously among species, but the reasons for this variation remain obscure. Since it has been suggested that swimming velocity increases with sperm length, earlier studies proposed longer (and therefore faster) sperm are advantageous under conditions of intense sperm competition. Nonetheless, previous work has been equivocal, perhaps because the intensity of sperm competition was measured indirectly. DNA profiling now provides a more direct measure of the number of offspring sired by extrapair males, and thus a more direct method of assessing the potential for sperm competition. Using a sample of 21 species of passerine birds for which DNA profiling data were available, we found a positive relation between sperm length and the degree of extrapair paternity. A path analysis, however, revealed that this relationship arises only indirectly through the positive relationship between the rate of extrapair paternity and length of sperm storage tubules (SSTs) in the female. As sperm length is correlated positively with SST length, an increase in the intensity of sperm competition leads to an increase in sperm length only through its effect on SST length. Why females vary SST length with the intensity of sperm competition is not clear, but one possibility is that it increases female control over how sperm are used in fertilization. Males, in turn, may respond on an evolutionary time scale to changes in SST size by increasing sperm length to prevent displacement from rival sperm. Previous theoretical analyses predicting that sperm size should decrease as sperm competition becomes more intense were not supported by our findings. We suggest that future models of sperm-size evolution consider not only the role of sperm competition, but also how female control and manipulation of ejaculates after insemination selects for different sperm morphologies.     

Expandable spermatheca influences sperm storage in the simultaneously hermaphroditic snail Arianta arbustorum

Summary

In many simultaneously hermaphroditic land snail species, the sperm storage organ (spermatheca) is highly structured, suggesting that the female function might be able to influence offspring paternity. Physical properties of the sperm storage organ, including its initial size and sperm storage capacity, may also affect fertilization patterns in multiply mated snails. We examined the structure, volume and tubule length of empty spermathecae in the land snail, Arianta arbustorum, and assessed differences in spermatheca size following a single copulation. The number of spermathecal tubules ranged from 2–7, but was not correlated with the volume of empty spermathecae. The volume of sperm stored in the spermatheca after a copulation was correlated with neither the number of spermathecal tubules nor copulation duration. Mean spermathecal volume more than doubled between two and thirty-six hours after sperm uptake, but the length of the spermathecal tubules did not change. Interestingly, the volume of sperm stored in the spermatheca seems not to be related to the size of the spermatophore and thus not to the number of sperm received (= allosperm). The amount of allosperm digested in the bursa copulatrix was highly variable and no significant relationship with the size of the spermatophore received was found. These findings suggest that numerical aspects of sperm transfer are less important in influencing fertilization success of sperm in A. arbustorum than properties of the female reproductive tract of the sperm receiver.     

Microscopic structure of the sperm storage tubules in the polygynandrous alpine accentor, Prunella collaris (Aves)

We describe the microscopic structure of the sperm storage tubules (SSTs) of the polygynandrous alpine accentor, Prunella collaris. The SSTs were found at the utero‐vaginal junction of the oviduct and were composed of a single layer of columnar epithelium. The cells of the tubule proper were non‐ciliated and had a round or oval nucleus in their basal portion. Their cytoplasm was finely or coarsely vacuolated due to lipid inclusions. Under the electron microscope, the epithelial cells exhibited a number of mitochondria, Golgi bodies, occasional lysosome‐like dense bodies, granular endoplasmic reticula, junctional complex, and tonofilaments. The apical margin of the cells was fringed with numerous microvilli. The epithelial lining of the SSTs was devoid of mucous cells, but showed occasional infiltration of lymphoid cells. No contractile elements were found in association with the SSTs, but a close apposition of unmyelinated nerve fibres to the basal part of the SST cells was recognized. Intraluminal sperm were arranged in bundles, and their heads were orientated towards the distal portion of the SSTs. Evidence for engulfment of sperm by the SST cells was obtained for the first time. A sign of atrophy or regression of the SSTs was found in one specimen.     

Dynamics of sperm storage in the grasshopper Eyprepocnemis plorans

Abstract. Sperm storage duration in the grasshopper Eyprepocnemis plorans Charpentier (Orthoptera, Acrididae) was found to be 58.5 days but with a wide between-female variation (from 26 to 113 days). One copulation was enough to fertilize two to eight egg-pods and to produce 130 eggs (28–313) and 119 embryos (28–290), on average. Sperm availability, however, decreased progressively with time, so that the majority of females spent a long final period of their lives without laying any pod. Finally, sperm storage duration was positively correlated with clutch size and total production of eggs and embryos.     

Development of sperm storage tubules in the quail during sexual maturation.

The development of the utero-vaginal sperm storage tubules (SST) in the quail during sexual maturation was studied using light microscopy and image analysis. SST development starts at around 28 days with low columnar cells, 10.9 +/- 0.7 microm high, found at the base of mucosal folds in the distal uterus. Seven days later, small bud-like invaginations consisting of columnar, 16.4 +/- 1.1 microm high cells with basal nuclei were found in this region. An extremely rapid growth of the oviduct occurred at approximately 40-42 days of age with considerable variation in oviductal length between birds, coefficient of variation (CV) 64.8% and 66.2%, respectively. Two of these birds had SST containing spermatozoa but were not laying. At 49 days, oviductal length was 24 +/- 0.5 cm (CV 2.0%), and all birds had functional SST with spermatozoa and had started to lay. Mature SST consist of columnar, nonciliated cells, 19.8 +/- 0.7 microm high. Although development of SST in the quail, to a large extent, coincides with the development of the rest of the oviduct, the present findings suggest that utero-vaginal sperm storage is possible before the complete maturation of the oviduct and subsequent onset of lay.     

Lack of sperm production and sperm storage by arctic-nesting shorebirds during spring migration

Birds nesting at high latitudes may copulate during migration to arrive on the nesting grounds ready to breed. We surveyed 12 species of shorebirds during spring migration to determine whether (1) males produced abundant sperm and (2) females harboured functional sperm storage tubules (SSTs). Sperm production by males on migration was rare. Only four of seven species (9.8% of 41 males) of long-distance migrants harboured sperm, whereas all four species (100% of eight males) of short-distance migrants held sperm. In females, no long-distance migrants held sperm in their SSTs ( n  = 28 females) and SSTs were small compared to long-distance migrants collected on their breeding grounds. Our results indicate that shorebirds nesting at high latitudes were generally not reproductively active during migration and that any sexual behaviour on migration is unlikely to lead directly to fertilizations.     

Multiple paternity in a natural population of a salamander with long-term sperm storage

Sperm competition appears to be an important aspect of any mating system in which individual female organisms mate with multiple males and store sperm. Post-copulatory sexual selection may be particularly important in species that store sperm throughout long breeding seasons, because the lengthy storage period may permit extensive interactions among rival sperm. Few studies have addressed the potential for sperm competition in species exhibiting prolonged sperm storage. We used microsatellite markers to examine offspring paternity in field-collected clutches of the Ocoee salamander (Desmognathus ocoee), a species in which female organisms store sperm for up to 9 months prior to fertilization. We found that 96% of clutches were sired by multiple males, but that the majority of females used sperm from only two or three males to fertilize their eggs. The high rate of multiple mating by females suggests that sperm competition is an important aspect of this mating system. Comparison of our data with those of other parentage studies in salamanders and newts reveals that multiple mating may be common in urodele amphibians. Nevertheless, the number of males siring offspring per clutch in D. ocoee did not differ appreciably from that in other species of urodeles with shorter storage periods.     

Specific features of in vivo and in vitro sperm storage in birds

This review focuses on some of the main features of sperm selection and storage in birds mainly on the basis of studies performed in poultry species, with emphasis on the initial selection of sperm at the female vagina level prior to migration towards the sperm storage tubules. Sperm originating from low-quality males or subjected to inappropriate in vitro storage conditions are rapidly discarded, resulting in impaired fertility in corresponding flocks. In the absence of accessible and appropriate technology for matching the 'storing' potential of sperm in the oviduct, conditions for prolonged sperm storage under a liquid (through the use of semen extenders) or a solid state (cryopreservation) have received only limited attention, despite their potential interest to facilitate male and female management in poultry flocks. Despite this, technology for short-term liquid storage is currently used in turkeys, guinea fowl and muscovy ducks and also in progress in chickens. In addition, technology for cryopreservation of avian semen has become available for some species (chicken, goose) to facilitate the management of genetic resources, including the preservation of rare and economically important breeds.     

Selection on sperm size in response to promiscuity and variation in female sperm storage organs

Sperm cells are exceptionally morphologically diverse across taxa. However, morphology can be quite uniform within species, particularly for species where females copulate with many males per reproductive bout. Strong sexual selection in these promiscuous species is widely hypothesized to reduce intraspecific sperm variation. Conversely, we hypothesize that intraspecific sperm size variation may be maintained by high among-female variation in the size of sperm storage organs, assuming that paternity success improves when sperm are compatible in size with the sperm storage organ. We use individual-based simulations and an analytical model to evaluate how selection on sperm size depends on promiscuity level and variation in sperm storage organ size (hereafter, female preference variation). Simulations of high promiscuity (10 mates per female) showed stabilizing selection on sperm when female preference variation was low, and disruptive selection when female preference variation was high, consistent with the analytical model results. With low promiscuity (2–3 mates per female), selection on sperm was stabilizing for all levels of female preference variation in the simulations, contrasting with the analytical model. Promiscuity level, or mate sampling, thus has a strong impact on the selection resulting from female preferences. Furthermore, when promiscuity is low, disruptive selection on male traits will occur under much more limited circumstances (i.e. only with higher among-female variation) than many previous models suggest. Variation in female sperm storage organs likely has strong implications for intraspecific sperm variation in highly promiscuous species, but likely does not explain differences in intraspecific sperm variation for less promiscuous taxa.     

Female sperm storage in amphibians.

The three orders of extant amphibians are Gymnophiona, Anura, and Urodela. Although all gymnophionans apparently have internal fertilization and many are viviparous, female sperm storage is unknown. Internal fertilization has convergently evolved in a few anurans, but females of just one species, Ascaphus truei, are known to possess oviductal sperm storage tubules (SSTs). The SSTs of A. truei are similar anatomically to such glands in squamate reptiles. This similarity is convergence due to similar functional adaptations and/or internal design constraints. In salamanders and newts (Urodela), absence of sperm storage in females is the ancestral condition (three families). In the derived condition, sperm storage occurs in cloacal glands called spermathecae, and their possession is a synapomorphy for females in the suborder Salamandroidea (seven families). Salamandroids are the only vertebrates with cloacal sperm storage glands. In this paper, a phenetic analysis of variation in spermathecal characters reveals patterns of convergence in certain spermathecal characters in unrelated taxa that breed in similar habitats. In the family Salamandridae, a role in sperm nutrition for the spermathecal epithelium is questioned, and the widespread occurrence of spermiophagy is related to other reproductive strategies. I propose how the packaging of sperm in structurally different types of spermathecae may influence male paternity.     

Sperm size and sperm competition in birds.

In a sample of 20 species of North American passerine birds we found no relation between sperm size and mating system like that previously reported in mammals (Gomendio & Roldan (Proc. R. Soc. Lond. B 243, 181 (1991)). Instead, we found a positive correlation between sperm length and the length of female sperm storage tubules (SSTS) and a negative correlation between sperm length and the number of SSTS. Both of these correlations suggest that the more than fivefold variation in sperm size we found among species can be explained by sperm competition for access to storage sites (SSTS) in females. As longer sperm appear to be able to swim faster, selection should favour long sperm when SSTS are in short supply; sperm long enough to fill an SST might also prevent access to SSTS by the sperm of other males. Conversely, selection should favour shorter sperm when there is an advantage to sperm layering within an SST promoting a last-male mating advantage. Although we conclude that sperm competition influences sperm size in birds, little is known about the interactions between sperm and SSTS. It seems clear, however, that detailed study of this interaction will provide a new dimension to the study of avian mating systems.     

COMPARATIVE EVIDENCE FOR THE EVOLUTION OF SPERM SWIMMING SPEED BY SPERM COMPETITION AND FEMALE SPERM STORAGE DURATION IN PASSERINE BIRDS

Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3) increased sperm length. We recorded sperm swimming speed in 42 North American and European free-living passerine bird species, representing 35 genera and 16 families. We found that sperm swimming speed was positively related to the frequency of extrapair paternity (a proxy for the risk of sperm competition) and negatively associated with clutch size (a proxy for the duration of female sperm storage). Sperm swimming speed was unrelated to sperm length, although sperm length also increased with the frequency of extrapair paternity. These results suggest that sperm swimming speed and sperm length are not closely associated traits and evolve independently in response to sperm competition in passerine birds. Our findings emphasize the significance of both sperm competition and female sperm storage duration as evolutionary forces driving sperm swimming speed.     

Coevolution of male and female reproductive traits in a simultaneously hermaphroditic land snail

Inter- and intraspecific studies in gonochoristic animals reveal a covariation between sperm characteristics and the size of the female reproductive tract, indicating a rapid evolutionary divergence, which is consistent with the theory of post-copulatory sexual selection. Simultaneous hermaphrodites differ from species with separate sexes (gonochorists) in that they possess both functional male and female reproductive organs at the same time. We investigated whether in hermaphroditic animals intraspecific variation in reproductive traits results from divergent coevolution, by quantifying the variation in male and female traits among six natural populations of the snail Arianta arbustorum and examining the covariation in interacting traits. There was a significant among-population variation in spermatophore volume, number of sperm transferred and sperm length, as well as in volume of the sperm storage organ (spermatheca) and number of tubules, but not in spermatheca length. We found a positive association between sperm number transferred and spermatheca volume. This result suggests that the same post-copulatory mechanisms as in gonochorists drive the correlated evolution of reproductive characters in hermaphrodites.     

Morphological structures for potential sperm storage in poeciliid fishes. Does superfetation matter?

Sperm storage within the female reproductive tract has been reported as a reproductive strategy in several species of vertebrates and invertebrates. However, the morphological structures that allow for sperm to be stored and kept viable for long periods are relatively unknown in osteichthyes. We use histological and stereological tools to identify and quantify sperm storage structures (spermathecae) in 12 species of viviparous Poeciliidae. We found spermathecae in nine species, six of which exhibit superfetation (the ability of females to simultaneously carry within the ovary two or more broods of embryos at different stages of development). These spermathecae are folds of ovarian tissue that close around spermatozoa. We compared the number and size (volume) of spermathecae between species with and without superfetation. Species that exhibit superfetation had a significantly higher number of spermathecae than species that do not exhibit this reproductive strategy. In addition, we found that the mean volume of spermathecae and total volume of spermathecae present in the ovary are marginally higher in species with superfetation. Our results contribute to the understanding of the morphological structures that allow for sperm storage in viviparous osteichthyes and suggest a positive relationship between superfetation and the capacity of females to store sperm.     

Microscopic organization of the sperm storage tubules in the oviducal gland of the female gummy shark (Mustelus antarcticus), with observations on sperm distribution and storage

Oviducal gland morphology, the microscopic organization of the terminal zone, and sperm storage were described in the female gummy shark (Mustelus antarcticus). Mustelus antarcticus is a nonplacental viviparous hound shark, which displays minimal histotrophy during embryonic development. The animals examined represented all stages of maturity and gestation. The oviducal gland was found to have the same fundamental zonation as in most chondrichthyans. Using recent terminology, the oviducal gland of chondrichthyans has an anterior club zone, followed by a papillary zone, both of which produce jelly that surrounds the egg, a baffle zone that elaborates the tertiary egg envelope and a terminal zone, where sperm storage occurs. Each zone is composed of simple tubular glands that connect to transverse grooves, which extend the full width of the gland. The exception is the terminal zone, which does not have transverse grooves but consists of individual tubules. The microscopic organization and histochemical nature of the zones display similar patterns to those of other chondrichthyan genera. Tubules of the terminal zone contain four types of cell: ciliated cells, alcian blue‐positive secretory cells, periodic acid‐Schiff and alcian blue‐negative secretory cells, and secretory columnar cells. These tubules end in recesses, the sperm storage tubules, which extend beyond the periphery of the baffle zone. Sperm were stored in the sperm storage tubules of all maturing and mature animals examined. Of note is the observation of stored sperm in an animal 1 year prior to first ovulation. Sperm were also observed throughout the uterine sphincter, body of the uterus, isthmus, and oviduct of maturing and mature animals, and in the uterine sphincter of an immature animal. These sperm represent immediately postcopulation aggregations of sperm and sperm in the process of migrating to the site of storage or to the site of fertilization. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Functional association between female sperm storage organs and male sperm removal organs in calopterygid damselflies

Female damselflies in the family Calopterygidae have two sperm storage organs: a spherical bursa copulatrix and a tubular spermatheca. Male flies have a peculiar aedeagus with a recurved head with which to remove bursal sperm, and lateral spiny processes to remove spermathecal sperm. The lateral processes differ among species and populations in terms of their width relative to the spermathecal duct: the narrower processes are physically able to access spermathecal sperm, while the wider ones are not. In the present study, sperm storage patterns and aedeagal structures were compared between two calopterygid species with different spermathecal structures –Calopteryx cornelia and Mnais pruinosa– with respect to not only sperm quantity (number) but also sperm quality (viability), by using a recently developed method based on live/dead dual fluorescence. Calopteryx cornelia is a typical spermathecal sperm remover. In this species, viability was similar between bursal and spermathecal sperm. In contrast, in M. pruinosa, the spermatheca was much smaller than the bursa and often contained no sperm. Even when the spermatheca of this species did contain sperm, a high percentage of it was dead. Although the spermatheca of M. pruinosa has such atrophic tendencies, males have nevertheless developed long and spiny lateral processes similar to those of C. cornelia, suggesting the processes have functions other than spermathecal sperm removal. They possibly function as stoppers or guides for manipulating the aedeagal head to remove the sperm mass from the bursa.     

Sperm aggregations in female Agkistrodon piscivorus (Reptilia:Squamata): a histological and ultrastructural investigation

Upon copulation in female Agkistrodon piscivorus, sperm migrate up the oviduct to sperm storage tubules (SSTs) in the posterior infundibulum. The epithelium of the SSTs is composed of ciliated and secretory cells and differs ultrastructurally from that of the epithelium lining the lumen of the posterior infundibulum. Sperm pass through an area composed primarily of ciliated cells at the opening of each gland before aligning themselves in parallel arrays with their nuclei facing an area composed primarily of secretory cells at the base of the tubules. Sperm are also found embedded inter- and intracellularly in the SSTs. The secretory vacuoles in the SSTs become highly electron dense after the start of the fall mating season along with the synthesis of lipid droplets. Histochemical analysis reveals that the alteration in secretory material density is caused by the production of neutral carbohydrates. Some sperm remain in aggregates in the nonglandular section of the posterior uterus until the time of ovulation. However, ultrastructural evidence indicates these sperm degrade before ovulation. Therefore, sperm in posterior aggregates have no role in fertilization of ovulated ova. The data presented here support the hypothesis that infundibular sperm storage is the mode that snakes utilize to sequester viable sperm until ovulation.