A structural analysis of the freshly extruded spermatophore from the lobster,Homarus americanus

Freshly extruded spermatophores from the lobster, Homarus americanus, were examined using light microscopy and scanning and transmission electron microscopy. The tubular spermatophore is trifoil in shape with two lobes tapered laterally from a third lobe situated ventrally. It is comprised of sperm surrounded by three acellular investments: (1) a primary spermatophore layer, (2) an intermediate layer, and (3) an outer bounding layer. The sperm are packed into a continuous tube contained largely within the ventral lobe and are embedded in a matrix of moderate electron density. The primary spermatophore layer is uniformly thick around the sperm mass and contains at its peripheral margins both ring structures and crystals in an amorphous matrix. The intermediate layer is thicker dorsally than ventrally. Dense granules dominate the ventral half of the intermediate layer while inclusions populate the dorsal half; both react positively to the periodic acid-Schiff (PAS) technique. The innermost portion of the outer bounding layer is composed of parallel fibrils; a flocculent material is present peripherally. This flocculent substance is presumed to impart stickiness to freshly extruded spermatophores. These observations provide a basis for the future understanding of the mechanisms involved in long-term storage of sperm in spermatophores.     

Ultrastructural and histochemical observations on electroejaculated spermatophores of the palaemonid shrimp, Macrobrachium rosenbergii

Spermatophores obtained by electroejaculation from mature males of Macrobrachium rosenbergii were studied by light microscopy and by transmission and scanning electron microscopy. Upon electrical stimulation, single Spermatophores were usually extruded simultaneously from each gonopore and frequently became firmly adherent to one another forming a compound spermatophore. Individual Spermatophores were pod-shaped. 0.5-1 cm long, 2-3 mum in diameter and consisted of a lateral sperm mass, a medial mucus mass, and a noncellular capsule. The capsule was a single-layered PAS-reactive structure composed of fine, intertwining fibrils and almost completely surrounded the sperm and mucus masses. On its outer surface were large. AB-reactive globules of flocculent material partially surrounded by fingerlike extensions of a canaliculated reticulum composed of fine, intertwinning fibrils. The canaliculated reticulum may permit fluid imbibition by the spermatophore. Forming a central band within the capsule were many small membrane-less AB/PAS-reactive granules. The lateral sperm masses consisted of unistellatc spermatozoa embedded in an AB/PAS-reaetive matrix composed of globules of flocculent. AB-rcactive material and a highly plastic canaliculated and thread-filled reticulum. The reticulum itself consisted of fine, intertwining fibrils. The threads within the interstices of the reticulum were long, curving structures, 30-36 nm in diameter. The function(s) of the canaliculated and thread-filled reticulum within the sperm mass is not known at this time. The medial mucus mass was similar in fine structure and histochemical reactivity to the sperm mass, except that spermatozoa were lacking. It is apparent that the spermatophore of M. rosenbergii consists of relatively few complexly arranged structural and macromolecular components designed to transport and protect the spermatozoa.     

Detailed developmental morphology of the spermatophore of the Mediterranean field cricket, Gryllus bimaculatus (De Geer) (Orthoptera: Gryllidae)

The detailed developmental morphology of the spermatophore of a cricket (Gryllus bimaculatus, De Geer) is described for the first time. A specific behavioural reference point is identified from which time developmental processes proceed in a more or less robotic manner. The structures previously identified as forming part of the fully formed spermatophore, that is, the ampulla, the sperm tube and the attachment plate are all identified in detail during their development. Interestingly the attachment plate material is extruded as an amorphous mass from the ejaculatory duct prior to its insertion into the dorsal pouch, the sperm do not enter the sperm sac until the end of spermatophore formation, and the sperm tube itself is formed from a flattened layer of material which rolls up into a tube and involves the use of a die mechanism.     

Morphological and morphometric appraisal of the spermatophore of the southern hermit crab Isocheles sawayai (Anomura: Diogenidae), with comments on gonopores in both sexes

The spermatophore morphology of the hermit crab Isocheles sawayai from southwestern Atlantic (Brazil) is described. The spermatophores show similarities with those described for other members of the family Diogenidae, especially with the recently described Loxopagurus loxochelis. The spermatophore is composed of three major regions: a sperm filled head or ampulla, a columnar stalk and a foot or pedestal. The spermatophores show specific morphology in having a circular ampulla, and a constriction or neck between the ampulla (100 μm) and the thin (27 μm), long stalk (500 μm). The stalk penetrates less than half way into the spermatophore head. Most spermatophores show one of the small posterior projections on the underside of the ampulla as being bigger than the other, making it asymmetrical. The size of the spermatophore is related to hermit crab size with direct relationships found between spermatophore ampulla width, total length, and peduncle length with shield length of the hermit crab. The morphological characteristics of the spermatophore of I. sawayai are species-specific distinguishing it from other members of the family, and are useful to infer further phylogenetic relationships.     

Scanning electron microscopic investigation of the spermatophore and spermatozoa of the shrimp Farfantepenaeus subtilis (Decapoda: Penaeidae)

Scanning electron microscopy was used to describe the structure of the spermatozoon and spermatophore of Farfantepenaeus subtilis. The spermatophore showed characteristics similar to those of members of the subgenus Farfantepenaeus. This included an extensive glandular epithelium and a lack of a wing. The sperm mass, which was distributed at the periphery of the spermatophore, was surrounded by a large amount of acellular material. The spermatozoon has a spherical main body and a well-defined acrosomal region with a single spike, which was bent in some cells. The immotile sperm cells have an average length of 7.1?±?0.6?μm. Information on sperm location within the spermatophore will assist in the efficient extraction of the sperm mass during dissection.     

褶纹冠蚌精子球的形态和超微结构观察

利用光镜和电镜技术研究了褶纹冠蚌精子球的形态和超微结构.结果表明:精子球呈球形,直径约65-70μm,容纳2300多个精子.从外向内,精子球由非细胞层、表面层和内部区组成.非细胞层薄,厚约0.6μm,易碎.表面层厚约7.5μm,被分隔成许多辐射状排列的小室,单个精子头部位于小室内,指向精子球的中央,而精子的单个鞭毛由精子球的表面伸出.精子质膜在鞭毛领处与表面层相连,相邻精子间无细胞质桥相连.内部区呈球状,内含絮状物质.精子球从雄蚌出水管排出后,位于精子球外周的鞭毛沿固定方向不停地摆动,精子球翻滚着向前运动,且单个精子依次从精子球上脱落下来,最后精子球成为一中空的球,历时120h.       

Spermatophores of the salamander Ambystoma texanum

Living spermatozoa were observed in freshly deposited spermatophores and in fluid from vasa deferentia. In the distal, but not proximal, vas deferens spermatozoa moved together in whorls with heads and tails in alignment. Around the entire periphery of the spermatophore cap, similar slowly undulating groups of spermatozoa had their heads aligned and directed outward. Over time, some individual spermatozoa left the cap of the spermatophore and moved into the surrounding water (cap deterioration). Microscopical observations were made on spermatophore squashes and paraffin sections of spermatophores and vasa deferentia. Spermatozoa around the periphery of the cap were underlain by a PAS-positive membrane-like material. Cytoplasmic droplets, which were attached to spermatozoan necks in the vas deferens, were accumulated in the center of the spermatophore cap deep to the PAS-positive membrane. Spermatophore stalks were strongly PAS and Alcian blue positive and showed positive reaction for RNA. Tests for lipids and proteins were negative in the whole spermatophore. Electron microscopic observations showed the stalk of the spermatophore to be composed of rounded ‘balls’ of fibrous material. At the juncture of the stalk and cap a less dense fibrous material impacted the stalk enclosing many sperm tails and some heads and, although no attachment devices were visualized, the sperm were closely apposed to this material as was the spermatophore stalk. This finely filamentous material encircled the cap and was more prominent in some regions than others. The PAS-positive material detected with the light microscope was also observed with the electron microscope. It was circumferentially oriented and was composed of 200 Å packed filamentous densities. Sperm heads and tails were found lying external to the membrane, whereas only tails and cytoplasmic droplets occupied the core of the spermatophore. Cytoplasmic droplets were usually free of the sperm tail and contained membranous sacs and two types of nuage material.     

Ultrastructure and role of the lobster vas deferens in spermatophore formation: The proximal segment

We have examined the anatomy of the vas deferens of the lobster Homarus americanus and have described the structure of the proximal vas deferens (segments one and two). The two tubes of segment one descend from the testes and gradually merge into segment two. The epithelium of segment one has synthetic activity and appears to contribute to the sperm-supporting matrix by exocytotic release of granules through its apical surface. The epithelium of segment two is also highly synthetic and secretes the primary spermatophore layer and part of the intermediate layer that surround the sperm mass. The trifoil shape of the extruded spermatophore is established through a change in height of some of the cells lining the lumen in segment two. Connective tissue and circular bands of striated muscle surround the epithelium of both segments.     

The sclerotized spermatophore of the barklouse Lepinotus patruelis

Lepinotus patruelis Pearman (Psocoptera: Trogiidae) has a very unusual spermatophore that is a permanent, hardened structure which is deposited and retained in the spermatheca. This study elaborates on the structure of the spermatophore, shows for the first time that the spermatophore is sclerotized, and provides information on its composition and development over time. The spermatophore is produced within the ventral seminal vesicles, and transferred to the female as a semi-solid, bullet shaped object. It is composed of five lamina which undergo several transformations during the first 24 h after copulation. A sample of three newly formed spermatophores were found to contain 18, 40 and 55 sperm. The possible function of the sclerotized spermatophore wall is discussed.     

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.     

日本沼虾雄性生殖系统的研究：Ⅲ.输精管内精荚的结构与形成

于光镜和电镜下研究日本沼虾输精管内精荚的结构与形成。结果显示：粗荚呈索状,由精子群、精荚基质、粘液团和荚壁组成;精荚基质与粘液团内均含有交织的纤丝,其中散布着絮状泡和同心圆形泡,精荚壁呈“Ｃ”型,单层,由致密纤丝、絮状泡和沟形的网状结构组成,包裹精子群和粘液团;前、中、后输精管的上皮细胞均具合成、分泌精荚形成物质的功能。     

The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size

Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity.     

Fine structure of spermatophores of some Oribatid mites (Acari,Arachnida)

The ultrastructure of spermatophores was studied in seven species of Oribatei. Each spermatophore is composed of a head and a stalk which is attached to the substratum. The spherical head consists of two distinct portions: (1) the sperm package; and (2) the head matrix. Position and structure of the sperm package are described in the different species, as well as the various structures in the head matrix. The sperm package contains sperm embedded in secretory products. The most characteristic feature of the sperm is an electron-dense chromatin body. Mitochondria lie adjacent to it or are partly or completely incorporated into the chromatin body. The cytoplasmic surface of the plasma membrane regularly bears an electron-dense layer; the plasma membrane is covered by a secretory sheath. The spermatophores of the Oribatei are structurally complicated and not uniform. The possible role of the spermatophore's structural elements is discussed and an attempt is made to evaluate characteristic features taxonomically.     

A model to explain spermatophore implantation in cephalopods (Mollusca: Cephalopoda) and a discussion on its evolutionary origins and significance

Male coleoid cephalopods produce spermatophores that can attach autonomously on the female's body during a complex process of evagination called the ‘spermatophoric reaction’, during which the ejaculatory apparatus and spiral filament of the spermatophore are everted and exposed to the external milieu. In some deepwater cephalopods, the reaction leads to the intradermal implantation of the spermatophore, a hitherto enigmatic phenomenon. The present study builds upon several lines of evidence to propose that spermatophore implantation is probably achieved through the combination of (1) an ‘evaginating‐tube’ mechanism performed by the everting ejaculatory apparatus and (2) the anchorage provided by the spiral filament's stellate particles. The proposed theoretical model assumes that, as it is exposed to the external milieu, each whorl of the spiral filament anchors to the surrounding tissue by means of its sharp stellate particles. As the ejaculatory apparatus tip continues evaginating, it grows in diameter and stretches lengthwise, enlarging the diameter of the whorl and propelling it, consequently tearing and pushing the anchored tissue outward and backward, and opening space for the next whorl to attach. After the ejaculatory apparatus has been everted and has perforated tissue, the cement body is extruded, possibly aiding in final attachment, and the sperm mass comes to lie inside the female tissue, encompassed by the everted ejaculatory apparatus tube. It is proposed that this unique, efficient spermatophore attachment mechanism possibly evolved in intimate relationship with the adoption of an active mode of life by coleoids. The possible roles of predation pressure and sperm competition in the evolution of this mechanism are also discussed. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 711–726.     

Spernathecal morphology,sperm transfer and a novel mechanism of sperm displacement in the rove beetle,Aleochara curtula (Coleoptera,Staphylinidae)

Summary The mechanism by which sperm are transferred from the male's spermatophore to the female's storing cage is described for the rove beetle Aleochara curtula, emphasizing a novel mechanism of sperm displacement by competing males. The cuticular, U-shaped spermatheca is equipped with a valve structure and two sclerotized teeth. The tube of the spermatophore extends into the spermathecal duct through the guidance of the flagellum of the male endophallus. Further elongation of the spermatophore tube, however, occurs only after separation of the pair. A primary tube bursts at its tip after passing through the valve. Within the lumen of the primary tube, a second tube passes through the valve and continues to extend up to the apical bulb of the spermatheca, doubles back on itself and swells to form a balloon filling most of the spermatheca. The balloon of the spermatophore is pierced within the spermatheca by tooth-like structures pressed against the spermatophore through contraction of the spermathecal muscle. The same process of spermatophore growing and swelling is also observed in mated females. Sperm from previous copulations are backflushed through the valve and the spermathecal duct, indicative of last-male sperm predominance.Abbreviations ad adhesive secretion covering the sperm - sac am amorphous secretion of the spermatophore - as ascending portion of the spermatophore - ds descending portion of the spermatophore - end parts of the male endophallus - ext extended tube - f flagellum - gs genital segment - lt large tooth - m muscle of the spermatheca - nsc non sclerotized cuticle - op opening of the spermathecal gland - pt primary tube - sc sclerotized cuticle - sd spermathecal duct - se secretion of the spermathecal gland - sf secretion flowing out of the primary tube - sg spermathecal gland - sm sperm - smt small tooth - sp spermatheca - ss sperm sac - st secondary tube - vm vaginal muscle     

Structure of the vector tissue in piscicolid leeches (Annelida, Hirudinea, Rhynchobdellida, Piscicolidae)

Hypodermic insemination occurs in piscicolid leeches (Hirudinea, Rhynchobdellida, Piscicolidae). The spermatophore is implanted in a specialized region of the leech body, the copulatory area. Just beneath the copulatory area, there is a specialized connective tissue (vector tissue) that is considered to guide the sperm toward the ovaries. In this study, we show that the vector tissue in the four species of the genus Piscicola is composed of a mass of cells located directly beneath the copulatory area, and two thin strands extend toward the ovaries. The ultrastructure of the vector tissue has been described for the first time. Four cell types were identified, constructing the vector tissue. The envelope of this tissue is made up of extracellular fibrous matrix and two types of cells: vesicular and flat envelope cells, which are embedded within the matrix. The rest of the tissue is formed of granular and plasmatic cells. Both of these last cell types have prominent cytoplasmic projections, filled with a filamentous material. However, only granular cells have numerous small electron-dense granules in their cytoplasm. The vector tissue was described prior, during and following copulation. Sperm passes within free spaces between the granular and plasmatic cells. Characteristic vector tissue cells also occur within the ovary wall and inside the ovary lumen. This supports earlier data, which postulated that the vector tissue appears to be an outgrowth of the ovary wall.     

Purification and characterization of a proline-rich secretory protein that is a precursor to a structural protein of an insect spermatophore

The spermatophore or sperm sac of Tenebrio molitor (yellow mealworm beetle) is an acellular structure composed mostly of structural proteins, termed spermatophorins. The proteins are derived from the bean-shaped accessory reproductive glands of the male and are assembled into the multilayered structure within the ejaculatory duct. Homogenates of the secretory plug from this gland were used as immunogens for the production of monoclonal antibodies, including one identified as PL 21.1 which recognizes an antigen in the gland and the spermatophore. With the aid of gel filtration and immunoaffinity chromatography with a PL 21.1, we isolated a glandular secretory protein that is a precursor to a spermatophorin with similar electrophoretic mobility. On native polyacrylamide gels, the antigen from gland homogenates has an apparent molecular mass of 370 kDa. On sodium dodecyl sulfate gels, the antigen from the gland and that from the spermatophore have apparent molecular masses of 23 kDa. According to immunoblots of sodium dodecyl sulfate gels, the 23-kDa glandular antigen is organ-specific and adult-specific. By immunocytochemistry with PL 21.1, we found the antigens to be restricted to secretory vesicles of only one cell type in the gland and to a discrete layer in the outer wall of the spermatophore. The 23-kDa secretory antigen is distinguished by being high in glutamic acid/glutamine (15.4%) and in proline (25.2%).     

Insemination of the monogenean Neobenedenia girellae (Capsalidae,Benedeniinae)

In vitro spermatophore formation and insemination of Neobenedenia girellae (Monogenea: Capsalidae, Benedeniinae) were recorded on video and described for the first time. Upon contact of two individuals, the anterior adhesive discs of the donor firmly attached to the dorsal tegument of the recipient and the donor's fore body strongly contracted such that the genital pore region protruded and the penis was pushed anteriorly to protrude through the genital pore. It is hypothesised that the donor penis mechanically damaged the tegument of the recipient. The sperm and spermatophore matrix were released together through the penis, which was placed under the left anterior attachment disc immediately behind the adhesive pad. The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. When observed under scanning electron microscopy, the spermatophores were irregularly shaped, with a diameter of 52–83 μm. Under light microscopy they consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing spermatozoa. Both parts were enclosed with a thin outer casing. Insemination occurred during and after spermatophore formation. Three types of insemination were recorded, unilateral and mutual insemination and self-insemination. The presence of self-insemination indicates that even a single N. girellae on a cultured fish may cause a significant parasite infection in the entire aquaculture system.     

A comparison of the spermatozoa of OratosquiMa stephensoni and Squilla mantis (Crustacea, Stomatopoda) with comments on the phylogeny of the Malacostraca

Each stomatopod sperm, aflagellate and obovoid, is surrounded by an electron dense coat. A spermatophore is absent. The discus-shaped acrosome vesicle is penetrated and underlain by a straight, slender acrosome rod (perforatorium) ensheathed, below the vesicle, in subacrosomal material. Feulgen-positive granular material, indicating chromatin, fills most of the length of the cell but there is no certain nuclear membrane. Two centrioles, consisting of doublets each with a radial “foot” as in decapods and peracarids, occur near the acrosome and like it are embedded in the chromatin. Myelin-like membranes are associated with degenerating mitochondria in the posterior region of the cell. Thiéry-positive granules are aggregated as a glycogen body posteriorly in the cell. Spermatozoa1 ultrastructure confirms monophyly of the mysid–amphipod–isopod– cumacean section of the Peracarida but affinities of the tanaids are uncertain. A closer phylogenetic relationship of Phyllocarida to the Branchiopoda than to the Malacostraca is suggested. A weak similarity (synapomorphy?) of syncarids and peracarids is the filiform perforatorium bipassing the nucleus. Stomatopods resemble decapods in their diffuse sperm chromatin but are placed below the syncarid–peracarid–decapod assemblage.     

Cytodiffrentiation in the accessory glands of Tenebrio molitor. VI. A congruent map of cells and their secretions in the layered elastic product of the male bean-shaped gland

The morphology of the bean-shaped accessory glands (BAGs) of males of Tenebrio molitor is described. All cells in the secretory epithelium are long and narrow (300–400 mμ × 5 mμ). The seven types of secretory cells are distinguished from one another by the morphology of their secretory granules. Granule substructure varies from simple spheres with homogeneous electrondense contents to complex forms with thickened exterior walls or with crystalline and membranous contents. Individual cell types were mapped by staining whole glands with Oil Red O, and the cell distributions were confirmed by wax histology and ultramicroscopy. The secretions of all seven cell types form a secretory plug composed of seven layers. During mating, the secretory plug from each BAG is forced into the ejaculatory duct by contractions of a sheath of circular muscle. The mirror image plugs from symmetrical BAGs fuse and are transformed into the wall of the spermatophore.