Site Of Sperm Storage Research Articles

Sperm Dynamics in Spiders (Araneae): Ultrastructural Analysis of the Sperm Activation Process in the Garden Spider Argiope bruennichi (Scopoli, 1772)

Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.

Changes in glucose and carnitine levels and their transporters in utero‐tubal junction in relation to sperm storage in the vespertilionid bat, Scotophilus heathi

Prolonged sperm storage over winter is a common feature of reproduction in some bats. In order to understand how sperm storage in the female genital tract of the vespertilionid bat, Scotophilus heathi (Greater yellow bat), is controlled, we compared concentrations of glucose and the fatty-acid carrier carnitine in the blood, and carnitine concentrations and levels of expression of the glucose transporters (GLUTs) and the carnitine transporter OCTN2 in the utero-tubal junction of females during non-storage (early winter) and sperm-storage periods (late winter-early spring). During the sperm-storage period (December-January) blood glucose concentrations declined, as did the expression of GLUT3 and GLUT5 in the utero-tubal junction. At the same time there were increases in the concentration of carnitine, and expression of OCTN2 and hormone-sensitive lipase (HSL) in the utero-tubal junction. These results suggest that prolonged sperm storage is enhanced by decreased glucose availability but increased free fatty acid availability at the site of sperm storage. Increases in expression of GLUT4 and GLUT8 in late winter suggest a role for these GLUTs in increasing sperm motility prior to fertilization.

Postnatal development of mononuclear phagocytes in the mouse epididymis

The epithelium lining the epididymis (site of sperm maturation and storage) is surrounded by mononuclear phagocytes (MPs, expressing dendritic cell and macrophage markers) that closely interact with adjacent epithelial cells. We mapped here their distribution during postnatal development in CD11c‐EYFP and CX3CR1‐GFP mice. MPs were present in the epididymis as early as 1 week after birth. The number of epididymal MPs gradually increased and, between week 4 and week 5 (puberty), they underwent major morphological changes. MPs progressively acquired a stellate/dendriform morphology and relocated from the interstitium to the peritubular region. Cellular bodies and lateral extensions were located at the base of the epithelium, while, in the initial segment, slender dendrites extended between epithelial cells towards the lumen. At week 5, MPs had acquired the overall appearance that we described previously in adult mice. In contrast, the peritubular region of the vas deferens (VD) was heavily populated by MPs during the early stages of postnatal development, long before puberty, possibly to prevent ascending pathogens from infecting the developing epididymis and testis. Our data will help better understanding the complex relationships between the immune and reproductive systems during development of the excurrent duct.Supported by NIH‐NICHD grant 1R01HD069623.

Epithelial surface changes and spermatozoa storage in the reproductive tract of the bitch

Spermatozoa are known to bind to the epithelial cells lining the uterine tube in various species, but information in canids is conflicting and sparse. The first aim of this study was to measure the epithelial surface outline (ESO) of different regions of the canine uterine tube in the four stages of the oestrous cycle as an indicator of a changing potential reservoir for spermatozoa. The second aim was to identify the site of sperm storage in the bitch after natural mating. Reproductive tracts were collected from bitches undergoing routine ovariohysterectomy. Histological analysis showed that, when corrected for uterine tube size, the ESO of pro-oestrus (P<0.005) and oestrus (P<0.05) tubes were larger than anoestrus, but not metoestrus, tubes. The second study examined reproductive tracts from 12 Beagle bitches at 6, 12, 24 and 48h after mating. Light and electron microscopy revealed large numbers of spermatozoa in the proximal regions of the uterus and particularly the distal utero-tubal junction (UTJ), with few present in the proximal UTJ or uterine tubes. Spermatozoa were bound by their heads to microvilli on the epithelial surface of the uterine lumen and to ciliated cells in the distal UTJ. This is the first report to measure and document differences in potential epithelial attachment sites of the uterine tubes at different stages of the oestrous cycle and to provide compelling evidence that the main spermatozoal storage site in the reproductive tract of the bitch is the distal UTJ.

Proliferative enteropathy in foals: Disease, diagnostics and transmission

Spermatozoa are known to bind to the epithelial cells lining the uterine tube in various species, but information in canids is conflicting and sparse. The first aim of this study was to measure the epithelial surface outline (ESO) of different regions of the canine uterine tube in the four stages of the oestrous cycle as an indicator of a changing potential reservoir for spermatozoa. The second aim was to identify the site of sperm storage in the bitch after natural mating. Reproductive tracts were collected from bitches undergoing routine ovariohysterectomy.Histological analysis showed that, when corrected for uterine tube size, the ESO of pro-oestrus (P < 0.005) and oestrus (P < 0.05) tubes were larger than anoestrus, but not metoestrus, tubes. The second study examined reproductive tracts from 12 Beagle bitches at 6, 12, 24 and 48 h after mating. Light and electron microscopy revealed large numbers of spermatozoa in the proximal regions of the uterus and particularly the distal utero-tubal junction (UTJ), with few present in the proximal UTJ or uterine tubes. Spermatozoa were bound by their heads to microvilli on the epithelial surface of the uterine lumen and to ciliated cells in the distal UTJ. This is the first report to measure and document differences in potential epithelial attachment sites of the uterine tubes at different stages of the oestrous cycle and to provide compelling evidence that the main spermatozoal storage site in the reproductive tract of the bitch is the distal UTJ.

Rhox5 Rules in an Evolving Saga of Reproductive Diversity

Rhox5 Rules in an Evolving Saga of Reproductive Diversity

The Rhox5 Homeobox Gene Regulates the Region-Specific Expression of Its Paralogs in the Rodent Epididymis1

The mechanisms by which the region-specific expression patterns of clustered genes evolve are poorly understood. The epididymis is an ideal organ to examine this, as it is a highly segmented tissue that differs significantly in structure between closely related species. Here we examined this issue through analysis of the rapidly evolving X-linked reproductive homeobox (Rhox) gene cluster, the largest known homeobox gene cluster in metazoans. In the mouse, we found that most Rhox genes are expressed primarily in the caput region of the epididymis, a site where sperm mature and begin acquiring forward motility. This region-specific expression pattern depends, in part, on the founding member of the Rhox cluster--Rhox5--as targeted mutation of Rhox5 greatly diminishes the expression of several other family members in the caput region. In the rat, Rhox5 expression switches from the caput to the site of sperm storage: the cauda. All Rhox genes under the control of Rhox5 in the mouse epididymis display a concomitant change in their regional expression in the rat epididymis. Our results lead us to propose that widespread changes in the region-specific expression pattern of genes over evolutionary time can be the result of alterations of one or only a few master regulatory genes.

Changes in the expression of HSL and OCTN2 in the female reproductive tract of the bat, Scotophilus heathii in relation to sperm storage

The aim of this study was to compare the changes in the expression of hormone sensitive lipase (HSL) and organic cation transporter 2 (OCTN2) in different regions of the female reproductive tract of bats (Scotophilus heathii) during the period of sperm storage. Western blot analysis revealed that both HSL and OCTN2 showed elevated expression in the utero-tubal junction, the site of sperm storage, compared to the vagina, cervix and uterus. An immunohistochemical study showed localization of HSL and OCTN2 in the cytoplasm of luminal epithelial cells in the utero-tubal junction of S. heathii, which suggests a role of fatty acids metabolism at the site of sperm storage. Furthermore, this study showed the presence OCTN2 immunostaining in the tail of stored sperm, which suggests a direct role of carnitine in sperm physiology. However, the role of carnitine in sperm storage needs further investigation. It is hypothesized that the utero-tubal junction may be finely tuned with fat depletion to support sperm storage in the female genital tract of S. heathii.

Insulin/FOXO Signaling Regulates Ovarian Prostaglandins Critical for Reproduction

Abnormalities in insulin/IGF-1 signaling are associated with infertility, but the molecular mechanisms are not well understood. Here we use liquid chromatography with electrospray ionization tandem mass spectrometry to show that the C. elegans insulin/FOXO pathway regulates the metabolism of locally acting lipid hormones called prostaglandins. C. elegans prostaglandins are synthesized without prostaglandin G/H synthase homologs, the targets of nonsteroidal anti-inflammatory drugs. Our results support the model that insulin signaling promotes the conversion of oocyte polyunsaturated fatty acids (PUFAs) into F-series prostaglandins that guide sperm to the fertilization site. Reduction in insulin signaling activates DAF-16/FOXO, which represses the transcription of germline and intestinal genes required to deliver PUFAs to oocytes in lipoprotein complexes. Nutritional and neuroendocrine cues target this mechanism to control prostaglandin metabolism and reproductive output. Prostaglandins may be conserved sperm guidance factors and widespread downstream effectors of insulin actions that influence both reproductive and nonreproductive processes.

Inbreeding avoidance in a poeciliid fish (Heterandria formosa)

Polyandry is hypothesized to give females an opportunity to avoid inbreeding through postcopulatory selection mechanisms if precopulatory inbreeding avoidance is not possible, for example, because of forced matings. Here, we report a postcopulatory, prefertilization, inbreeding avoidance mechanism in the least killifish, Heterandria formosa, a species in which males can force matings. Females had 50% less sperm in their oviducts and ovarian cavities after mating with a sibling compared to a mating with a nonsibling male. Neither sex showed inbreeding avoidance in a dichotomous precopulatory mate choice test or during mating trials. Females in this species invest substantially in each offspring after fertilization (matrotrophy), whereas males invest little more than sperm. Based on theory, females should therefore be more likely than males to avoid inbreeding in this species. We suggest that females do this by reducing the amount of sibling sperm in their reproductive system. However, the possibility that males invested more sperm to nonsiblings could not be ruled out. In a fertilization success experiment, the first male to mate with a female sired all the offspring in most cases, even if it was a sibling. However, large females were more likely to carry offspring of multiple males. Possibly female sperm storage sites were filled by the first male, and only large females had space for the second male's sperm.

Mechanisms of last male precedence in a moth: sperm displacement at ejaculation and storage sites

In many polyandrous species, the last male to mate sires most of the offspring. This phenomenon has been explained as the result of male manipulation, postcopulatory female choice, or both. However, in most previous studies, direct evidence for these explanations is lacking. Here, we report our work on a polyandrous moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). We show that the last male sperm precedence occurs by sperm displacement operating at both sperm ejaculation and storage sites. Thus, the second male physically displaces the first male’s spermatophore with his own in the bursa copulatrix. However, females usually allow most sperm of the first males to move to the spermatheca before they accept a second copulation. The intermating duration, which is positively correlated with the number of the first male’s sperm moving to the spermatheca, may be the product of mate choice and sexual conflict between the sexes. After the female accepts the second mating, she ejects ’50% of the first male’s sperm from the spermatheca to accommodate the second male’s sperm. Spermathecal contractions appear to be the mechanism for sperm ejection. Before oviposition occurs, ’75% of eupyrene sperm in the spermatheca of the twice-mated female are from the second male. The outcome of sperm displacement before and after sperm storage appears to be the result of male 3 female interactions. We demonstrate that ejaculate size in the bursa copulatrix by the second male, and sperm stratification and flushing in the spermatheca, do not play a role in last male sperm precedence. Key words: bursa copulatrix, last male precedence, sperm displacement, spermatheca. [Behav Ecol]

Complex female genitalia indicate sperm dumping in armored goblin spiders (Arachnida, Araneae, Oonopidae)

In promiscuous females, sperm ejection from the sperm storage site can be a strong mechanism to influence sperm priority patterns. Sperm dumping is reported from different animals including birds, insects, and humans. In spiders, it has been documented for four species including the oonopid Silhouettella loricatula. Oonopidae are a diverse spider family comprising many species with peculiar female genitalia. Especially in species where studies of mating behavior are difficult, morphological investigations of the genitalia help to understand their function and evolution. In the present study, the genitalia of the oonopids Myrmopopaea sp., Grymeus sp., and Lionneta sp. are investigated by means of histological serial sections and scanning electron microscopy (SEM). The results are compared with previous findings on S. loricatula. In Myrmopopaea sp. and Grymeus sp., the same morphological components are present that are involved in sperm dumping in S. loricatula. Inside the receptaculum, sperm are enclosed in a secretory sac which can be moved to the genital opening and dumped during copulation by muscle contractions. The female genitalia of Lionneta sp. are asymmetric. They show the same characteristics as S. loricatula but all the investigated females were unmated. The results strongly suggest that sperm dumping occurs in Myrmopopaea sp., Grymeus sp., and Lionneta sp. and happens by the same mechanism as in S. loricatula. Sperm dumping might even be common within a clade of oonopids. As in S. loricatula, the sperm transfer forms in the investigated species consist of several spermatozoa. Papillae with unknown function occur on the receptacula of all females.

Evidence of androgen-dependent sperm storage in female reproductive tract of Scotophilus heathi

The aim of present study was to investigate the role of androgen in sperm storage in the female genital tract of Scotophilus heathi. Spermatozoa were observed in the distal part of oviduct and utero-tubal junction of all the female bats collected between January and early March. Increase in circulating testosterone level coincided with the arrangement of sperm with their head oriented towards the epithelial lining of reproductive tract. Immunocytochemical and Western blot analysis revealed the presence of androgen receptor (AR) only in the distal part of the oviduct and utero-tubal junction, the site of sperm storage. Localization of AR in the cytoplasm of luminal epithelial cells in utero-tubal junction of S. heathi suggests non-genomic action of androgen at the site of sperm storage. Further study showed the presence of intense immunoreactivity of androgen binding protein (ABP) in the glandular epithelial cells of utero-tubal junction . It is hypothesized that androgen creates a unique microenvironment e.g. secretion of ABP within lumen of utero-tubal junction which helps to store spermatozoa for prolonged period in the female genital tract of S. heathi.

Turkey Sperm Reside in the Tubular Glands in the Urodeum Following Artificial Insemination

A turkey hen in egg production requires 48 h after the last insemination to maximize the number of sperm in the uterovaginal junction sperm-storage tubules. Where the sperm that continue to fill the oviductal sperm-storage sites during this 48-h period reside remains unknown. Histological sections of the juncture of the vagina with the urodeum, the central compartment of the cloaca, revealed deep tubular glands containing periodic acid-Schiff-positive secretory material. When examined 36 h after the last artificial insemination, sperm were observed in the lumen of the tubular glands associated with the urodeum. We suggest that sperm reside in the tubular glands within the urodeum and are released in association with the secretory activity of the tubular glands. These sperm then may ascend the vagina to continue to populate the sperm-storage tubules. Alternatively, the sperm in the tubular glands of the urodeum may be evidence of spermatorrhea and have no functional role in the fertilization process.

The Caenorhabditis elegans NR4A nuclear receptor is required for spermatheca morphogenesis

The gene nhr-6 encodes the Caenorhabditis elegans ortholog of the NR4A nuclear receptor. We determined the biological functions of NHR-6 through the isolation and characterization of a deletion allele of nhr-6, lg6001. We demonstrate that nhr-6 has an essential role in the development of the C. elegans somatic gonad. Specifically, nhr-6 is required for the development of the hermaphrodite spermatheca, a somatic gonad organ that serves as the site of sperm storage and oocyte fertilization. Using a variety of spermatheca cell markers, we have determined that loss of nhr-6 function causes severe morphological defects in the spermatheca and associated spermathecal valves. This appears to be due to specific requirements for nhr-6 in regulating cell proliferation and cell differentiation during development of these structures. The improper development of these structures in nhr-6(lg6001) mutants leads to defects in ovulation and significantly reduced fecundity of C. elegans hermaphrodites. The phenotypes of nhr-6(lg6001) mutants are consistent with a role for nhr-6 in organogenesis, similar to the functions of its mammalian homologs.

Threshold dimorphism in ejaculate characteristics in the squid Loligo bleekeri

Male dimorphism has been thought to correlate with alternative reproductive behav- iors. Alternative reproductive behaviors promote asymmetry in sperm competition, and the differ- ences in fertilization success could promote adaptations in ejaculate characteristics in relation to each reproductive behavior. Using allometric analysis, we show that ejaculate dimorphism clearly exists in males of the squid Loligo bleekeri, a cephalopod species with body size-related alternative mating behaviors. A morphological switch point was detected for internal characters: larger individuals pro- duced discontinuously longer spermatophores than did smaller individuals, although no switch point was detected for external characteristics (fin length, fin width, head width, mantle width, tentacle length and hectocotylus length) except for bimodal body size. This clear internal switch point could be an adaptation to the characteristic alternative mating behaviors of loliginid squid, in which males use different mating tactics to pass spermatophores to different sperm storage sites in and on the females. Our results indicated that alternative reproductive behaviors can result in morphological adjustment in internal characteristics.

Sperm dumping in a haplogyne spider

AbstractThe present study shows that females of Silhouettella loricatula (Arachnida: Araneae: Oonopidae) manage to process sperm in an unusual and previously unknown way. The male ejaculate consisting of spermatozoa and globular secretion is enclosed in a secretory sac. This may avoid the mixing of sperm from different males and at least severely limit sperm competition. The process of sperm enclosure occurs within the female's sperm storage site (receptaculum) as the ejaculate is not surrounded by a sac inside the male's sperm‐transferring organs (palpal bulbs). The secretion forming the sac is produced by glands adjoining the receptaculum. The possibility that globular secretions in the male palpal bulbs partly contribute to the sac cannot be ruled out completely. It is suggested that in S. loricatula, the main function of sperm enclosure in a sac is enabling females to dump the ejaculate of a male. The present study represents the first report on sperm dumping in the family Oonopidae. During five first and three second copulations in the laboratory, the dumping of a sac was observed. One dumped sac was sectioned and contained spermatozoa. Two couples were flash‐fixed with liquid nitrogen early during copulation, which revealed the mechanism of the sac dumping. By muscle contractions, the receptaculum is bent backwards and the sac moved into the genital opening. The actual sac dumping occurs most probably in cooperation with the male, which moves his pedipalps rhythmically during the entire copulation. Extensions and furrows on the emboli suggest that they may additionally be used as copulatory courtship devices. The enclosure of sperm from the current male in secretion takes place during or immediately following copulation as all mated females sacrificed after copulation had a new sac containing spermatozoa in the receptaculum. Dumping sperm of a previous male during the next copulation may allow females to bias sperm precedence.

The role of gap junctions in Caenorhabditis elegans oocyte maturation and fertilization

We have investigated the role of gap junctions in Caenorhabditis elegans oocyte maturation and fertilization. Gap junctions are observed between oocytes and the surrounding ovarian sheath cells in wild-type gonads. The sheath transcription factor CEH-18 is required to negatively regulate oocyte maturation, mitogen-activated protein kinase (MAPK) activation, and ovulation. Transmission electron microscopy (TEM) indicates that sheath/oocyte gap junctions are rare or absent in ceh-18(mg57) null mutant gonads. To test the hypothesis that gap junctions negatively regulate oocyte maturation, we performed an RNAi screen of innexin genes, which encode channel-forming proteins. Here we show that INX-14 and INX-22 are required in the female germ line to inhibit oocyte maturation, MAPK activation, and ovulation. Genetic analysis and TEM are consistent with INX-14 and INX-22 being components of sheath/oocyte gap junctions. Our results support the hypothesis that gap junctions maintain oocytes in meiotic prophase I when sperm are absent. We also implicate these channels in regulating sheath cell contractile activity and sperm recruitment to the spermatheca, the site of sperm storage and fertilization. Together with previous studies, our results help establish the C. elegans gonad as a model system for investigating the molecular mechanism(s) by which gap junctions regulate meiosis and fertilization.

Structural investigation of the female genitalia and sperm-storage sites in the terrestrial isopod Armadillidium vulgare (Crustacea, Isopoda)

The cuticular genitalia of the terrestrial isopod, Armadillidium vulgare, have two distinct states during the reproductive cycle of the females. The structural differences between the reproductive and non-reproductive states, and the structure of the sperm storage sites were investigated employing electron and light microscopy. In both states the genitalia consist of a distal segment that connects to the gonopore, and a cuticular tube-like structure lining the lumen of the oviduct in the middle region of the oviduct. Sheath-like projections, apparently consisting of cuticular material, extend laterally along two sides of the cuticular tube. In the proximal region of the oviduct cuticular structures are lacking. In the non-reproductive state the distal segment consists of endo-, exo- and epicuticle. The exocuticle is three layered with unusual spongy and dense layers at the distal side. On one side the endocuticle doubles in thickness to form a cuticular bulge that fills the lumen of the distal segment leaving just a narrow U-shaped space. The cuticular tube consists of endo- and epicuticle only. In the reproductive state the distal segment is funnel-shaped and forms branched cuticular folds that increase in complexity from distal to proximal. In the cuticular tube these folds tightly fill the lumen of the oviduct. At the confluence of the oviduct with the ovary spermatozoa are stored in a seminal receptacle.

Evolution: Sex and Cannibalism in Redback Spiders

Female redback spiders cannibalise mating males. New research has shown they have evolved two separate sperm storage organs, allowing them to make post-copulatory paternity choices. Counter-adaptation by males, to maximise paternity after cannibalism, has led to the evolution of an abdominal constricting mechanism, which enhances short-term survival and mating success after attack by the female and maximises the chances of inseminating both sperm storage sites.