Sperm survival in the female reproductive tract in the fly Scathophaga stercoraria (L.)

While sperm competition risk favours males transferring many sperm to secure fertilizations, females of a variety of species actively reduce sperm numbers reaching their reproductive tract, e.g. by extrusion or killing. Potential benefits of spermicide to females include nutritional gains, influence over sperm storage and paternity, and the elimination of sperm bearing somatic mutations that would lower zygote fitness.We investigated changes in sperm viability after in vivo and in vitro exposure to the female tract in the polyandrous fly, Scathophaga stercoraria. Sperm viability was significantly lower in the females' spermathecae immediately after mating than in the experimental males' testes. Males also varied significantly in the proportion of live sperm found in storage in vivo. However, the exact mechanism of sperm degradation remains to be clarified. In vitro exposure to extracts of the female reproductive tract, including female accessory glands, failed to significantly lower sperm viability compared to controls. These results are consistent either with postcopulatory sperm mortality in vivo depending entirely on the male (with individual differences in sperm viability, motility or longevity) or with postcopulatory sperm mortality being subtly affected by female effects which were not detected by the in vitro experimental conditions. Importantly, we found no evidence in support of the hypothesis that female accessory glands contribute to sexual conflict via spermicide. Therefore, female muscular control remains to date the only ascertained mechanism of female influence on sperm storage in this species.     

Male and female effects on sperm precedence in the giant sperm species <Emphasis Type="Italic">Drosophila bifurca</Emphasis>

Sperm competition is expected to be a driving force in sexual selection. In internally fertilized organisms, it occurs when ejaculates from more than one male are present simultaneously within the female’s reproductive tract. It has been suggested that greater sperm size may improve the competitive ability of sperm, but studies provide contradictory results depending on the species. More recently, the role of females in the evolution of sperm morphology has been pointed out. We investigate here the male and female effects that influence sperm precedence in the giant sperm species, Drosophila bifurca Patterson & Wheeler. Females were mated with two successive males, and the paternity outcomes for both males were analyzed after determining sperm transfer and storage. We found very high values of last male sperm precedence, suggesting a strong interaction between rival sperm. However, the data also indicate high frequencies of removal of the sperm of the first male from the female reproductive tract prior to any interaction with the second male. This implies that successful paternity depends mainly on successful sperm storage. Knowing what happens to the sperm within females appears to be a prerequisite for disentangling post-copulatory sexual interactions between males and females.     

Sperm storage and viability in Photinus fireflies

In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.     

Function of multiple sperm storage organs in female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae)

Sperm storage organs allow females to temporally separate insemination from fertilization, manipulate ejaculates and control fertilization. In the reproductive tract of female fruit flies (Diptera: Tephritidae), sperm are found in two different organs--a pair or triplet of spermathecae, and a "fertilization chamber". In order to understand the specific function of each of these organs, we tested the following hypotheses: (1) Sperm are distributed equally amongst the various sperm storage organs; (2) Both organ types maintain sperm viability; and (3) Sperm used in fertilization come from the fertilization chamber. We counted sperm in spermathecae and fertilization chamber of Mediterranean fruit flies (Ceratitis capitata) every 3 days for 18 days following insemination, and used a live/dead staining technique to determine the viability of sperm in these organs. Finally, by extirpating spermathecae from inseminated females and allowing them to oviposit, we were able to identify the fertilization chamber as the source of fertilizing sperm. Numbers of sperm in the spermathecae declined from an average of 3575 on the day of copulation to 649, 18 days later. Conversely, the fertilization chamber maintained a fairly constant level of sperms, ranging between an average of 207 cells on day 3 to 115 sperms on day 18. Throughout the period we monitored, we found high levels of sperm viability in both organs (> 80%). Sperm viability was similarly high in the fertilization chambers of females without spermathecae. However, fertility of eggs laid by these females declined rapidly, as did the number of sperm in the fertilization chamber. We conclude that both the spermathecae and the fertilization chamber are active sperm storage organs, with separate functions: the spermathecae for long-term storage and the fertilization chamber, periodically filled by the spermathecae, a staging point for fertilizing sperm. We suggest that the use of both organs by females results in sperm economy, which adaptively prolongs the intervals between copulations.     

The impact of cushioned centrifugation protocols on semen quality of stallions

The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/mL. Sperm recovery rate among centrifugation treatment groups was compared. Motion characteristics, plasma membrane intactness (SMI), and DNA quality (COMPαt) of sperm were compared among treatment groups and uncentrifuged controls immediately following centrifugation (Time 0 h) and following 24 h of cooled storage (Time 24 h). Centrifugation treatment did not affect sperm recovery rate (P > 0.05). At Time 0 h, no differences in experimental end points were detected between cushion-fluid volumes tested (P > 0.05). Values for percent total sperm motility, percent progressive sperm motility, and track straightness were similar between sperm-number treatments subjected to centrifugation (P > 0.05). At Time 24 h, values for all experimental endpoints were similar between centrifugation treatments for cushion volume per tube, and between centrifugation treatments for sperm number per tube (P > 0.05). Centrifugation treatments and control treatments were similar for five of six variables tested (P > 0.05). Sperm storage concentrations of 25 × 10⁶ and 250 × 10⁶/mL yielded similar values for percent total sperm motility, percent progressive sperm motility, percent SMI, and percent COMPαt (P > 0.05). A storage concentration of 250 × 10⁶ sperm/ml yielded higher values for curvilinear velocity, and lower values for straightness, than all other groups (P < 0.05). In conclusion, centrifugation with as little as 1 ml of cushion fluid and a sperm number of up to 3 × 10⁹ sperm in 50-ml conical-bottom centrifuge tubes had no detrimental effect on initial or cool-stored sperm quality. Additionally, storage of centrifuged sperm at a concentration of 250 × 10⁶/mL with 20% seminal plasma (v/v) did not have a detrimental effect on percentages of motile or progressively motile sperm, or sperm DNA quality.     

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster,Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC₅₀), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.     

Transmembrane adenylyl cyclase regulates amphibian sperm motility through protein kinase A activation

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.     

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 μg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.     

Presence of F-actin in sperm head of Armadillidium peraccae (Isopoda, Oniscidea)

Sperm of Armadillidium peraccae have been examined with cytochemical and immunocytochemical methods for fluorescence and electron microscopic visualization of cytoskeleton components. Sperm incubation in an antibody anti-β-tubulin shows only the presence of two centrioles located in the cytoplasmic region above the nucleus; no other microtubules are present in the sperm head. Instead, fluorescence microscopy of sperm incubated in FITC-phalloidin allowed to detect the presence of a large amount of F-actin in the apical region of the sperm head. The incubation of ultrathin sections of sperm embedded in Lowicryl K4M with a phalloidin–gold complex allowed a more precise localization of F-actin in the amorphous part of the acrosome and in the cytoplasmic region between acrosome and nucleus; F-actin is also present in the thin cytoplasmic layer between plasma membrane and nuclear envelope at the apical portion of the nucleus. Although the sperm was always found completely devoid of motility, the discovery of the presence of an actin cytoskeleton leads us to hypothesize a possible acquisition of motility by the sperm at the time of its interaction with the female gamete. Such a hypothesis is supported by what is known for ostracods whose aflagellate sperm implement a type of amoeboid movement only at the time of their interaction with the female gamete.     

Mating strategy in Aleochara bilineata: sperm storage and allocation

Abstract By contrast to females that can maximize reproductive success with only one or a few copulations, males generally increase their fitness with frequency of mating. Sperm storage and allocation is therefore crucial for both male and female fitness. Sperm storage in Aleochara bilineata (Coleoptera; Staphylinidae) is investigated by measuring the number of spermatozoa stored in the female spermatheca after single, double or triple successive copulations with different males. The potential advantages of polyandry are studied in terms of the number of sperm stored by females mated twice with the same male (i.e. repeated copulation), compared with females mated twice with two different virgin males (i.e. polyandry). Level of polygyny is also estimated by measuring sperm allocation when ten successive mates are offered to a virgin male. Aleochara bilineata females store the sperm of the same or different males additively, suggesting no advantage for polyandry in terms of the number of sperm stored. A virgin male is able to inseminate ten different females but the number of sperm transferred decreases linearly. Finally, the latencies and durations of copulations are measured in all experiments to estimate changes according to the male or female status (i.e. virgin or mated). The latency before mating is higher when females are virgin than when females have already mated.     

SPERM PRODUCTION AND STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA PSEUDOOBSCURA

Subspecies of Drosophila pseudoobscura, one occurring in the United States and the other in Bogota, Columbia, exhibit Haldane's Rule in one direction of the cross. Additionally, D. pseudoobscura produces two sperm types: short, sterile sperm and long, fertile, sperm. Here I examine the relationship between the production of short and long sperm and hybrid sterility. Fertile and sterile hybrid males produce a greater proportion of short sperm compared to parental males with sterile hybrids producing mainly short, immotile sperm. Sperm transfer and storage patterns were similar between fertile hybrid and parental strains; and unexpectedly, short, immotile sperm from sterile hybrids were stored. These findings raise the question of whether different genetic mechanisms disrupt both sperm heteromorphic production and sperm motility and whether this indicates that females exert some control over sperm storage.     

Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.     

慢性间歇性缺氧降低SD大鼠精子数量和活力的研究

摘要 目的：通过制备大鼠慢性间歇性缺氧的动物模型,观察间歇性缺氧对雄性大鼠精子数量和活力的影响,并初步探讨其可能的发生机制。方法：16只雄性SD大鼠随机分为对照组和缺氧组。间歇性缺氧8周后分别测定两组大鼠精子活力和精子浓度,睾丸组织中P53的表达量和细胞的凋亡水平,以及血清睾酮（T）、促卵泡素（FSH）和黄体生成素（LH）的浓度。结果：与对照组比较,缺氧组大鼠精子的浓度和活力均降低（P>0.05),并且前向运动的精子数明显减少（P<0.05）。同时,缺氧组睾丸组织P53 mRNA的表达和睾丸组织中凋亡的细胞均多于对照组,血清T、LH的浓度低于对照组（P<0.05）。结论：间歇性缺氧时可能影响了下丘脑-垂体-性腺轴,不仅使睾酮合成减少,还降低了对P53的抑制作用而诱导了大量细胞的凋亡,最终降低了SD大鼠的精子数量和活力。     

Optimization of handling and refrigerated storage of guppy Poecilia reticulata sperm

Sperm collection methods and the effect of osmolality, ions, sugar, temperature, pH and dilution ratio on sperm motility were investigated in guppies Poecilia reticulata. The present study revealed that the sperm was motile in a wide range of osmolalities (200–470 mOsm kg^?1) either in Hanks balanced‐salt solution (HBSS) or in non‐electrolyte solutions such as glucose or sucrose. Sperm collected from crushing testes yielded lower motility and shorter motility duration than samples collected without crushing but gentle disruption. Dilution ratios within the range of 1:50 to 1:500 of sperm to HBSS had minimal effect on sperm motility during extended refrigerated storage. Examination of storage temperature showed that refrigerated storage at 4° C was superior to room temperature (25° C). Sperm was found to tolerate a wide range of pH from 5·6 to 7·8, but motility was affected negatively by pH values >7·8.     

Sperm Storage is not Subject to Cephalic Control in the Caribbean Fruit Fly, Anastrepha suspensa

Male Anastrepha suspensa exhibit a repertoire of “copulatory” behaviors involving the female head. This study examined the effect of female decapitation on copulation duration, and quantity and distribution of sperm in the four storage organs. Copulation duration and sperm storage were highly variable (16 to 160 min and 0 to 2,643 sperm, respectively). Sperm quantity variation was due primarily to storage patterns among three spermathecae. There was no significant correlation (Pearson, P > 0.05) between body size and copulation duration, or sperm stored in relation to both variables. Although mean copulation duration was significantly shorter for non-decapitated females (t-test, t = 8.48, P < 0.001), sperm quantity and storage patterns were not significantly different. These data suggest the near autonomy of the abdominal ganglion’s role in any female mediated effects on sperm storage.     

Sperm displacement behavior of the cuttlefish Sepia esculenta (Cephalopoda: Sepiidae)

Sperm displacement behavior of cuttlefish (Sepia esculenta) was observed in a tank. Before ejaculation, male cuttlefish used their arms III to scrape out sperm masses attached to the buccal membranes of females. The removed sperm mass debris was directly visible and countable. Active sperm were present within the removed sperm debris, implying that the aim of this behavior is to remove competing male sperm. However, many sperm masses remained on the female buccal membrane even after the removal behavior, showing that sperm removal in S. esculenta is incomplete. The duration of sperm removal (an indicator of male investment in that process) was unaffected by the body sizes of mated pair, the duration of spermatangia placement at the current mating (for the hypothesis that the sperm removal serves to creat attachment space of spermatophores), or the estimated amount of sperm masses deposited from previous matings. Moreover, male S. esculenta performed sperm removal regardless of whether the last male to mate with the partner was himself, suggesting males remove not only the sperm of rivals but also their own. Although the number of removed sperm masses increased with the time spent on removal of sperm, male cuttlefish may shorten the duration of sperm removal to avoid the risk of mating interruption. We conclude that this time restriction would likely influence the degree of partial sperm removal in S. esculenta. A digital video image relating to the article is available at .This revised version was published online in April 2005 with corrections in the abstract.     

A species‐specific multigene family mediates differential sperm displacement in Drosophila melanogaster

Sperm competition is a postcopulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. Here, we compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. We found that a less effective removal of first‐to‐mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. Our results highlight the role young genes can play in driving the evolution of sperm competition.     

Sperm numbers,their storage and usage in the fly Dryomyza anilis

In the fly Dryomyza anilis females have two kinds of sperm storage organs: one bursa copulatrix and three spermathecae (two spermathecae with a common duct form the doublet, and the third is a singlet spermathecal unit). At the beginning of a mating the male deposits his sperm in the bursa copulatrix. After sperm transfer the male taps the female''s abdomen with his claspers. This behaviour has been shown to increase the male''s fertilization success. After mating, the female discharges large quantities of sperm before oviposition. To find out where the sperm remaining in the female are stored, I counted the number of sperm in the droplet and in the female''s sperm storage organs after different types of mating. I carried out three mating experiments. In experiment 1, virgin females were mated with one male and the matings were interrupted either immediately after sperm transfer or after several tapping sequences. The results show that during male tapping more sperm moved into the singlet spermatheca. In addition, the total number of sperm correlated with sperm numbers in all sperm storage organs, and male size was positively related to the number of sperm remaining in the bursa. In experiment 2, females mated with several males. The number of sperm increased with increasing number of matings only in the doublet spermatheca. No increase in the number of sperm in the singlet spermatheca during consecutive matings suggests that sperm were replaced or did not reach this sperm storage organ. In experiment 3, virgin females were mated with a single male and half of them were allowed to lay eggs. The experiment showed that during egglaying, females primarily used sperm from their singlet spermatheca. The results from the three experiments suggest that sperm stored in the singlet spermatheca is central for male fertilization success and male tapping is related to sperm storage in the singlet spermatheca. The different female''s sperm storage organs in D. anilis may have separate functions during sperm storage as well as during sperm usage.