Functional and morphological diversity of sperm in Drosophila

Unlike mammals, where the males produce huge quantities of tiny spermatozoa, insects, and Drosophila in particular, exhibit a wide range of reproductive strategies. Sperm gigantism in Drosophila deviates from the rules that normally govern anisogamy, i.e. differences in the size and quantity of male and female gametes. Sperm gigantism has driven anatomical, physiological and cytological adaptations that affect the correlated evolution of the male and female reproductive systems, and has led to the evolution of a new structure, the roller, located between the testis and the seminal vesicle, and to sperm coiling to form pellets. The diversification of sperm strategy is investigated in the light of sexual selection processes that occur in the female genital tract after copulation. These processes, which bias paternity, result from interactions either between spermatozoa from different males, or between the spermatozoa and the environment within the female reproductive tract. In Drosophila, increased sperm size does not confer any reproductive advantage on the male. The evolution of sperm gigantism does not seem to be attributable to competition between spermatozoa from different males, as has been shown to occur in some vertebrate species. Alternative mechanisms, such as interactions between spermatozoa and the female reproductive system, are therefore currently viewed as being more likely explanations. In particular, the impact of sperm size on female reproductive physiology is being investigated to find out whether having large spermatozoa increases the likelihood of male reproductive success. Correlated adaptations of the spermatozoa and female storage organs also seem to be a major factor in determining sperm success, and their role in male-female conflicts is discussed briefly.     

Comparative studies of testicular structure and sperm morphology among copulatory and non-copulatory sculpins (Cottidae: Scorpaeniformes: Teleostei)

Testicular structure of 9 species and sperm head morphology of 19 species of Cottidae were observed in order to clarify relationships between morphological characteristics of the male reproductive organ and reproductive mode (copulation or non-copulation). Morphological structure of the testis was divided into the following five types based on the sperm transfer and reservoir system: (1) a non-duct type in which the sperm duct is not a distinct exterior structure, but the tube for sperm transport traverses along the testis as an interior structure; (2) an anterior duct type with distinct anterior sperm ducts traversing along the testis; (3) a posterior duct type with distinct anterior sperm ducts traversing along the dorsal hilus of testis and posterior sperm ducts extending to the rear of the testis; (4) an anterior duct posterior vesicle type with distinct anterior sperm ducts traversing along the testis, and the right and left sperm ducts fusing in the rear of testis, forming the seminal vesicle; (5) a non-duct posterior vesicle type in which sperm ducts do not accompany the testis, and the testis and seminal vesicle are connected directly or through posterior sperm ducts. It is thought that in Cottidae the non-duct type of reproductive organ is primitive, and the anterior duct type is common to all non-copulating species. The testes and accompanying seminal vesicle were seen only in copulating species. Sperm head morphology was divided into three types according to the length/width ratio: oval type ≤2, intermediate type >2 and ≤3, and slender type >3. The type of sperm head corresponded closely to the reproductive mode; non-copulating species had oval sperm head, and copulating species had intermediate or slender ones. These results suggest that the structure of the testis and the morphology of the sperm head evolved from testes with anterior sperm ducts and oval sperm heads to testes with an associated seminal vesicle and slender sperm heads in association with the evolution from non-copulatory to copulatory reproduction in Cottidae.     

Adaptation to long sperm in Drosophila: correlated development of the sperm roller and sperm packaging

Sperm are generally small and produced in huge numbers, but some species combine exaggerated sperm length with extremely limited numbers of sperm, an evolutionary trend that deviates from the theory of anisogamy. Sperm gigantism has arisen recurrently in various species, but insects exhibit the longest sperm, with some species of the Drosophilidae family producing sperm up to 6 cm in length. The anatomical, cytological, and physiological requirements for males to cope with these giant sperm were hitherto poorly understood. In this paper, we investigate the internal morphology of the male reproductive tract, and highlight specific features that may be linked to this increase in sperm size. We focus on species in the repleta group, within which sperm length varies by a factor of 35. An associated development of the sperm roller, a special twisting device inserted between the testis and the seminal vesicle, is demonstrated. Its length and the number of coils involved increase with sperm size, and it allows individual sperm to swell and roll into a spermatic pellet before reaching the seminal vesicle. This process occurs independently of and in addition to the sperm bundle coiling that takes place at the base of the testis. It is suggested that the emergence and development of the sperm roller may be a male adaptation to sperm gigantism.     

Coevolution of sperm and female reproductive tract morphology in stalk-eyed flies

Sperm and female reproductive tract morphology are among the most rapidly evolving characters known in insects. To investigate whether interspecific variation in these traits results from divergent coevolution we examined testis size, sperm length and female reproductive tract morphology for evidence of correlated evolution using 13 species of diopsid stalk-eyed flies. We found that sperm dimorphism (the simultaneous production of two size classes of sperm by individual males) is ancestral and occurs in four genera while sperm monomorphism evolved once and persists in one genus. The length of ''long-sperm'' types, though unrelated to male body or testis size, exhibits correlated evolution with two regions of the female reproductive tract, the spermathecae and ventral receptacle, where sperm are typically stored and used for fertilization, respectively. Two lines of evidence indicate that ''short sperm'', which are probably incapable of fertilization, coevolve with spermathecae. First, loss of sperm dimorphism coincides phylogenetically with reduction or loss of spermathecae. Second, evolutionary change in short-sperm length correlates with change in spermathecal size but not spermathecal duct length or ventral receptacle length. Morphological coevolution between sperm and female reproductive tracts is consistent with a history of female-mediated selection on sperm length.     

多肠目薄背涡虫生殖系统结构及一氧化氮合酶组化研究

运用常规组织学方法和NADPH-d组织化学方法,研究了薄背涡虫 Notoplana humilis 生殖系统的组织结构和一氧化氮合酶的分布.其雄性生殖系统包括精巢、储精囊、阴茎、雄性生殖孔,精巢壁由一薄层薄膜组成,每个精巢内都含有不同发育时期的雄性生殖细胞,且精子发育无明显同步性;储精囊呈螺旋状排列在雄性生殖孔附近,囊壁由单层扁平上皮组成;阴茎为粗大的球形,外壁由柱状上皮细胞和数层肌细胞组成.雌性生殖系统包括输卵管、生殖腔、雌性生殖孔和受精囊,但不形成集中的卵巢和卵黄腺.雌雄生殖孔、生殖腔、受精囊、阴茎等部位呈NADPH-d强阳性反应.     

Elucidation of the protein composition of mouse seminal vesicle fluid

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.     

Effects of larval exposure to sublethal concentrations of the ecdysteroid agonists RH-5849 and tebufenozide (RH-5992) on male reproductive physiology in Spodoptera litura

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.     

平疣桑椹石磺生殖系统结构及精子储存场所

雌雄同体贝类精子的储存和利用规律一直是国内外贝类生物学研究的难点之一,本文利用活体解剖、显微观察、组织切片和扫描电镜技术,综合研究了平疣桑椹石磺(Platevindex mortoni)的生殖系统及精子储存场所。结果显示,其生殖系统包括生殖器本部、雌性生殖部分和雄性生殖部分。生殖器本部由两性腺、两性输送管、蛋白腺、黏液腺、支囊组成;雌性生殖部分包括输卵管、受精囊、阴道,位于身体中后方体腔内;雄性生殖部分包括输精管、刺激器、阴茎、阴茎鞘和阴茎牵引肌,位于身体前端右侧体腔内;其阴茎有阴茎鞘,阴茎表面布满倒刺。平疣桑椹石磺阴茎为直线状,无雄性附属腺。未交配的性成熟个体支囊内充满细长精子,受精囊内无精子;而交配后充当雌性个体的支囊内均为细长的自体精子,受精囊内有大量活力较强的粗短精子,其支囊为自体精子的存储场所,而受精囊为异体精子的存储场所。其精子储运情况为:两性腺内精子成熟后暂存于支囊,交配时通过输精管运输至阴茎,由阴茎输送精子至对方的阴道,异体精子进入受精囊内存储待用。     

Sperm stock and mating of males in a parasitoid wasp

Hymenoptera are haplodiploid insects, consequently sex ratio depends on female's sperm management which itself arises from the reproductive capacity of neighbouring males. To study the influence of ageing on male reproductive potential, laboratory experiments were conducted on Dinarmus basalis (Hymenoptera, Pteromalidae) males, a tropical wasp in which sperm counts are known to constrain sex ratio. Two groups of virgin males were compared: 1-day and 30-days old. Parameters recorded were sperm quantity and viability in seminal vesicles, shape of testis, mating ability in both individual and competitive situations and sperm stored by females after male multiple mating. Older males had twice as much sperm as young males, but their reproductive capacities did not differ. They were able to copulate with 20 successive virgin females in a short period. Sperm stored in spermathecae decreased with female mating order. In competition, old and young males had the same access to females. The difference between old and young males was visible at the level of reproductive tract: young males have functional testis and old males have empty non-functional testis. Spermatozoa are kept viable in male seminal vesicles for long periods. In this species, the reproductive potential of males is not altered by ageing. At the population level this may represent an adaptation for maintaining continuous reserves of sperm at the disposal of females.     

The structural mechanism of trypsin-induced intrinsic motility in Manduca sexta spermatozoa in vitro

Lepidopteran males produce eupyrene (nucleate) and apyrene (anucleate) spermatozoa, but in the female only eupyrene spermatozoa leave the spermatheca and fertilize the eggs. Both kinds of spermatozoa lack intrinsic motility in the male genital duct. They become motile in the spermatophore, in a process involving proteases from the male duct. In vitro, trypsin induces immotile spermatozoa to become motile. We studied the changes spermatozoa of Manduca sexta undergo during trypsin-induced motility and found that (a) they mimick rather closely those occurring in vivo during normal sperm maturation in genital ducts and (b) they are time- and dose-dependent. As in vivo, they comprise, successively, (a) disappearance of an extracellular matrix that maintains the integrity of eupyrene bundles in the seminal vesicle, (b) dispersion of the eupyrene bundles and intermingling of eupyrene and apyrene spermatozoa and (c) "hatching" of eupyrene spermatozoa from individual enclosing envelopes that are formed in the seminal vesicle. "Hatching" may not directly be related to motility since eupyrene spermatozoa become motile before "hatching" and motile apyrene spermatozoa never "hatch". Rather "hatching" may be related to the capacitation of eupyrene spermatozoa to either leave the spermatheca or fertilize the eggs, or both, as neither apyrene spermatozoa, nor those eupyrene spermatozoa that fail to "hatch", leave the spermatheca.     

Effects of age and repeated mating on male sperm supply and paternity in a parasitoid wasp

Post-copulatory paternity biases after female multiple mating are major constraints on both male and female reproductive systems. The outcome of paternity in certain situations is only controlled directly by male sperm stock. This was tested experimentally in the parasitoid wasp Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), in which sperm stocks are small (several hundred) and the fertilizing efficiency of stored sperm is high (the ratio of sperm stored/fertilized eggs is about 0.75). Sperm in seminal vesicles and paternity of males of different status (virgin young, virgin old, or young previously mated) were measured after female single and double mating. The amount of sperm in the seminal vesicle differed according to male status (increasing from previously mated males to old males), but there was no difference in sperm stored by females after a single mating. In double mating experiments with two males of different status, paternity increased linearly with the relative amount of sperm in seminal vesicles. Paternity distribution conforms to 'a fair raffle' of sperm from both donors following complete mixing of sperm prior to fertilization. Thus, in a female multiple mating context, male fitness depends principally on their sperm stock, which in turn depends on life history parameters, such as age and previous mating.     

Spermatogenesis, changes in reproductive structures, and time constraint associated with insemination in Podisus nigrispinus

Males of the predatory stinkbug, Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae), accomplish long and multiple matings. We hypothesize that this behavior is due to time requirement for spermatozoa production and their transference to the females. Thus, this work investigated the effect of mating status of males and mating duration on spermatozoa transference to the females and the location of spermatozoa in the male reproductive tract during mating. On females, morphological alterations on female spermatheca and associated structures during a mating were investigated. Analyses of male reproductive tract showed presence of spermatozoa in the lumen of vas deferens was independent of mating status (ca. virgin, 0, 12 and 24 h after having a full mating), indicating continuous spermatogenesis which is supported by the absence of a seminal vesicle for spermatozoa storage. Female spermatheca had no changes associated with the duration of mating. However, females exhibited spermathecal elastic duct swelling by 30 min of mating duration. The success of males in filling the female spermatheca with spermatozoa depends on duration of mating. Thus, the results indicate that multiple mating is a requirement for reproductive success in the species by transference of spermatozoa and accessory substances stored in the female spermathecal duct. Likewise, the long mating is a male requirement to transfer materials in appropriate amount to the female but it is not dependent on spermatozoa alone.     

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters,morphology and function of testis and accessory glands

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.     

The morphology of the male reproductive system,spermatogenesis and the spermatozoon of Daphnia magna (Crustacea: Branchiopoda)

This study analyses the histological and cellular morphology of the testis and sperm development in the male Daphnia magna Straus 1820. Due to the rarity of males and predominately parthenogenetic lifecycle of Daphnia , there has been limited detailed information on males in contrast to the well‐studied female. Using light and electron microscopy approaches, we describe the morphology of the testis during the progression from an immature to mature testis. The testis has an encasing muscular mesh sheath outside the basal lamina, beneath which is a thin somatic epithelial cell layer. Internal to the epithelium are the spermatogonial stem cells and subsequent syncytial clusters of the germ cells as they progress through spermatogenesis; spermatozoa occupy the entire testis in sexually mature D. magna . We describe the structure of developing and mature spermatozoa; mature spermatozoa are non‐flagellated, ovoid in shape with plasmalemma filapodia and are encased in an extracellular capsule.     

Longer exaggerated male genitalia confer defensive sperm-competitive benefits in an earwig

As evidence mounts that male genitalia can affect relative fertilisation success, the role that sexual selection has played in the rapid and divergent evolution of genitalia is becoming increasingly recognized. Unfortunately, the limited functional understanding of these complex structures and their interactions with the female reproductive tract often limit interpretation regarding their evolution. Here, we address this issue using the earwig Euborellia brunneri, where both the male intromittent organ and the female spermatheca are highly exaggerated in length yet structurally simple. In a double mating design, we use the sterile male technique to study how sperm precedence patterns are affected by male genital length, male age, and the size of the male sperm storage organ, the seminal vesicle. Relative fertilisation success exhibited considerable variation around modest last-male paternity. Only an interaction between first and second male genital length affected paternity, where males gained reduced paternity when preceded by rivals with longer genitalia. Longer genitalia confer defensive benefits in sperm competition by apparently depositing ejaculate deeper in the tubular spermatheca, safe from removal by rivals. Paternity similarly depended on an interaction between the ages of both males, likely mediated by sperm traits as testes size decreased with age. Seminal vesicle size showed positive allometry but did not affect paternity; instead, greater seminal vesicle size in last males expedited oviposition. The exaggerated yet relatively simple genitalia of E. brunneri facilitate an unusually clear example of post-copulatory selection on phenotypic variation in multiple reproductive traits.     

The influence of mating system on seminal vesicle variability among gobies (Teleostei, Gobiidae)

A variety of sexual selection mechanisms have been implicated to drive the variability of the male reproductive tract in internal fertilizers, while studies on external fertilizers have been largely limited to exploring the influence of sperm competition on testis size and sperm number. Males in the Gobiidae, a speciose teleost family of demersal spawners with external fertilization, are known to be characterized by accessory structures to the sperm duct called seminal vesicles. These seminal vesicles secrete a mucus-enriched seminal fluid. Seminal vesicle size and function have been demonstrated to be influenced by sperm competition at the intraspecific level. With the aim to test the factors influencing the development of these male organs at the interspecific level, an independent contrast analysis was performed on 12 species, differing in mating system type, sperm competition risk, and duration of egg deposition. The type of mating system appears to be the main factor significantly affecting development of seminal vesicles, with males of monogamous species completely lacking or having extremely reduced organs.     

Comparative Analysis of the Male Reproductive System in Bothriuridae Scorpions: Structures Associated with the Paraxial Organs and the Presence of Sperm Packages (Chelicerata, Scorpiones)

The male reproductive system of seven species of the family Bothriuridae are compared. These scorpions are Bothriurus flavidus Kraepelin, B. cordubensis Acosta, B. bonariensis (C. L. Koch), B. chacoensis Maury & Acosta, Brachistosternus ferrugineus (Thorell), Timogenes dorbignyi (Guérin-Méneville), T. elegans (Mello-Leitão) and Urophonius brachycentrus Pocock (Bothriuridae). Additional comparisons are made with the buthid Zabius fuscus (Thorell). Observations on the structures associated with the paraxial organs (testis, seminal vesicle and accessory glands) are given. Sperm obtained from the male reproductive tract and fresh spermatophores as well as from the female's genital atrium and seminal receptacles are examined. Accessory glands occur in six out of eight studied bothriurids and in the buthid Z. fuscus. In most species the distal portion of vas deferens has a developed ampulla. All structures vary in size and shape depending on species. Sperm packages were observed in all bothriurids. In contrast, there is no packaged spermatozoa in Z. fuscus. Each sperm package consists of many spermatozoa surrounded by a common membrane that breaks after the spermatophore capsule is everted into the female genital atrium, releasing the spermatozoa. One hour after insemination, the spermatozoa are found in the atrium and in the seminal receptacles of B. flavidus females, but after 24h spermatozoa are found only in the seminal receptacles. The functional significance of the accessory glands and the presence-absence of sperm packages are discussed.     

Quantitative Proteomic Analysis of Seminal Plasma,Sperm Membrane Proteins,and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction‐specific proteins that show demonstrable links to sperm fertility. Many of these membrane‐bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.     

Zika virus persistence in the male macaque reproductive tract

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.