Male internal reproductive structures of european pea crabs (Crustacea,Decapoda, Brachyura,Pinnotheridae): Vas deferens morphology and spermatozoal ultrastructure

Pea crabs of the subfamily Pinnotherinae (Pinnotheridae) have a high investment in reproduction and an outstanding reproductive output, probably as an adaptation to the required increase in reproductive rate due to the pinnotherids small size and their parasitic, host‐dependant way of life. In the present study, we investigate the male internal reproductive structures and the ultrastructure of spermatozoa of Pinnotheres pisum and Nepinnotheres pinnotheres by histological methods and both scanning‐ and transmission electron microscopy. In the Brachyura, the male internal reproductive systems generally consist of paired testes and corresponding vasa deferentia where spermatozoa develop and mature. Spermatozoal ultrastructure of the investigated pinnotherids conforms to the thoracotreme type, however, N. pinnotheres has an accessory opercular ring and a periopercular rim, neither of which are present in spermatozoa of P. pisum. Spermatozoa are enclosed within spermatophores in the secretory proximal vas deferens. Two types of secretions were observed in P. pisum and N. pinnotheres: an electron dense substance secreted in the proximal vas deferens involved in spermatophore formation, and large electron‐luscent vesicles constituting the seminal plasma in the medial and distal vas deferens. The medial vas deferens is strongly widened compared to other brachyurans to purpose storing spermatophores embedded in seminal plasma. Tubular appendices, which produce and store large amounts of seminal plasma, arise from the distal region of the vas deferens. The appendices extend into the ventral cephalothorax and also in the first pleomere. The latter being an exceptional location for reproductive structures among male brachyurans. J. Morphol. 274:1312–1322, 2013. © 2013 Wiley Periodicals, Inc.     

Morphological changes in eupyrene and apyrene spermatozoa in the reproductive tract of the male butterfly Atrophaneura alcinous Klug

Summary

Eupyrene and apyrene spermatozoa are contained in separate cysts in the testis of the butterfly Atrophaneura alcinous. Spermatozoa of both types from various parts of the male reproductive tract were examined with particular reference to their morphological characteristics. All spermatozoa collected from the vas deferens and the vesicula seminalis were found to be immotile under a dissecting microscope. No spermatozoa of either type were recognized in any part of the ejaculatory duct. Within the testis, eupyrene spermatozoa are present in bundles and each spermatozoon has a slender nucleus with an acrosome and a long flagellum containing mitochondrial derivatives. Two kinds of appendages, lacinate and reticular, are present on the surface of the sperm membrane. They are replaced with an extracellular sheath during passage through the vas deferens. In contrast, apyrene spermatozoa have neither nucleus nor acrosome, whereas a cup-shaped structure was found at the sperm tip instead of the acrosome. Unlike eupyrene spermatozoa, they are surrounded by a concentric sheath outside the sperm membrane in the vas deferens. Individual apyrene spermatozoa and coiled bundles of eupyrene spermatozoa were both found to accumulate in the vesicula seminalis before mating. These morphological changes during passage through the male reproductive tract suggests the occurrence of a kind of maturation and capacitation process reminiscent of mammalian spermatozoa.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Ultrastructure and function of the seminal vesicle of Bittacidae (Insecta: Mecoptera)

The fine structure of the seminal vesicle and reproductive accessory glands was investigated in Bittacidae of Mecoptera using light and transmission electron microscopy. The male reproductive system of Bittacidae mainly consists of a pair of testes, a pair of vasa deferentia, and an ejaculatory sac. The vas deferens is greatly expanded for its middle and medio-posterior parts to form a well-developed seminal vesicle. The seminal vesicle is composed of layers of developed muscles and a mono-layered epithelium surrounding the small central lumen. The epithelium is rich in rough endoplasmic reticulum and mitochondria, and secretes vesicles and granules into the central lumen by merocrine mechanisms. A pair of elongate mesodermal accessory glands opens into the lateral side of the seminal vesicles. The accessory glands are similar to the seminal vesicle in structure, also consisting of layers of muscle fibres and a mono-layered elongated epithelium, the cells of which contain numerous cisterns of rough endoplasmic reticulum and mitochondria, and a few Golgi complexes. The epithelial cells of accessory glands extrude secretions via apocrine and merocrine processes. The seminal vesicles mainly serve the function of secretion rather than temporarily storing spermatozoa. The sperm instead are temporarily stored in the epididymis, the greatly coiled distal portion of the vas deferens.     

Spermatogenesis and spermatophore production in the hawaiian red lobster Enoplometopus occidentalis (Randall) (Crustacea,nephropidae)

Light microscopy of the male reproductive tract of the Hawaiian red lobster Enoplometopus occidentalis documented the cyclic nature of spermatogenesis and spermatophore formation. Testes are composed of a convoluted collecting tubule bearing many spermatogenic follicles, all within a supporting mesentery. Spermatogonia are restricted to the basal side of the follicular epithelium and proliferate at onset of spermateleosis within the same follicle. Two generations of spermatogenic cells thus occupy each follicle, and accessory cells in the follicle form a basophilic epithelium between them. These accessory cells may detach with the spermatozoa at spermiation. The vas deferens lies outside the testicular mesentery and consists of a coiled proximal portion in which spermatophore production commences. Clusters of spermatozoa are here surrounded by a PAS-positive primary spermatophore layer, and a PAS-negative outer bounding layer is initiated. Completed further distally in the vas deferens, the outer bounding layer is thinner on the side of the spermatophore which adheres to the substratum after ejaculation; the thick side of this layer forms a broad cap. Outer circular and inner longitudinal muscular layers become well developed in the distal loop and descending portions of the vas deferens. The terminal portion of this duct contains no spermatophore prior to ejaculation. It has a longitudinally folded epithelium and an attached tubular gland which produces an extra-spermatophoral, gelatinous secretion. The androgenic gland is associated with this terminal segment of the vas deferens. These features are compared with those reported for other lobsters.     

Internal anatomy and ultrastructure of the male reproductive organization of the Sesarmid crab Muradium tetragonum (1798)—(Decapoda: Brachyura)

Sesarmid crab, Muradium tetragonum, considered a key detritus consumer plays a significant role in the nutrient cycling and energy flow in most of the mangrove environments. Morphological and ultrastructural organization of the M. tetragonum male reproductive system are characterized through transmission electron microscopic studies. Adult males (3.2–4.2 cm) with dark violet carapace and white-tipped cheliped were procured alongside the coastal areas of Tanjavur district, Tamil Nadu, India. The morphological analysis highlights the male gonads to be bilaterally symmetrical and anterolaterally located inside the cephalothorax. A pair of elongated testes lying attached to the hypodermis of the carapace leads to a long highly coiled vas deferens categorized into three distinct regions (Proximal vas deferens, Middle vas deferens and distal vas deferens) structurally and functionally with Posterior vas deferens receiving sac-like accessory glands. It gets followed by an ejaculatory duct and ends with the penile papillae at the coxae's base of the fifth peripod. Structural modifications were observed in the ultrastructure of vas deferens envisage (considering) its functional role in storing spermatophores, active absorption and assisting the secretory activity. Spermatophores, witnessed as spherical bodies are bounded by a dense double wall. Aflagellate, immotile and spherical spermatozoa that measuring 3.6 μm in diameter encompasses a complex acrosome cupped by a nucleus. Moreover, perforatorium and the extending nuclear arms with chromatin, as displayed in the experimental organism M. tetragonum, are in synergy with that of certain brachyurans as specified in the study. Hence, the current study assessing the morphology and ultrastructure parameters of the male gonads could be useful in understanding the physiology of sexual maturation, annual cyclic changes, tracing the phylogenetic relationship among species and enhancing the brood-stock management.     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acrosomes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.     

Structure of the male reproductive system of the blue swimmer crab Portunus pelagicus (Decapoda: Portunidae)

An attempt was made here to study the structure of the male reproductive system of Portunus pelagicus, which would improve the knowledge base on the reproductive biology of the species and also help in the maintenance of broodstock under controlled conditions. Male P. pelagicus of different sizes were collected from the Palk Bay off Mandapam (9°17′ N, 79°9′ E) and maintained under controlled conditions for the study. Tissues from testis, anterior vas deferens (AVD), median vas deferens (MVD), posterior vas deferens (PVD), ejaculatory duct and penis were fixed in Bouin's fluid and 2.5% buffered glutaraldehyde separately and processed for light and electron microscopic studies, respectively. The reproductive system consisted of testis, commissure, vas deferens, ejaculatory duct and penis. The vas deferens was divided based on the morphology and/or histology into AVD, MVD and PVD. The AVD was further divided based on histology into proximal and distal regions, and the MVD, based on diameter into major and minor coils. The testicular lobe had several lobules with a central seminiferous tubule, which continued till the penis. The seminiferous tubule was lined by a layer of cuboidal or columnar epithelium. The lining of the central tubule of the vas deferens formed several ‘folds’, which at times formed ‘pouches’. High incidence of cell organelles in the columnar epithelial cells, aggregations of vesicles and occurrence of blebs at the luminal periphery and the projection of numerous microvilli containing electron‐dense materials into the lumen from the cell lining denoted high secretory activity of the epithelial cells.     

Fine structure of the mature spermatozoon of rhynchocinetes typus,crustacea decapoda

Rhynchocinetes typus spermatozoa obtained from the vas deferens have the shape of a round-headed nail. The head measures 30 μm in diameter and 14 μm of height. At the center of the flat face of the head emerges a single rigid spike of 53 μm in length. Cross sections of this spike show that it has a wall of 0.4 μm in thickness and a core of 0.6 to 0.8 μm. The outer surface of the spike has a longitudinal striation. When the spermatozoa are placed in sea water it is possible to observe the unfolding of rays. The number of rays in different spermatozoa of the same individual varies from 9 to 13. Each ray is formed by a channel-like sheath that contains a rigid rod that occupies about 1/3 the length of the ray. This rod has a transverse striation with a periodicity of 185A. The rays are bound among them by a thin membranous sheet that is highly folded in vas deferens spermatozoa. At the distal end of each ray there is a rigid spine of 50 μm in length. The nucleus is coplanar to the radial plane and it extends through the rays. The structure and ultrastructure of R typus spermatozoa depart from that reported for spermatozoa of other Caridea species.     

The histochemical localization of prostaglandin synthetase activity in reproductive tract of the male rat.

PG synthetase activity was assessed histochemically in the reproductive tract of male rats. Moderate activity was observed in tails of spermatozoa within the corpus and cauda epididymidis but there was no activity in the caput epididymidis or the seminiferous tubules. The sperm tail activity was maximal for cells within the vas deferens. PG synthetase activity was also observed in individual adipose cells adhering to the testicular capsule, epididymis and vas deferens, and in isolated interstitial cells of the testis and the caput, corpus and cauda epididymidis. Specific cells in the capsules of the testes, epididymis and vas deferens also produced PGs. The activity observed in the interstitial cells of the testis and the caput epididymidis was less than that for the other tissues in terms of the proportion of possible cells. The demonstration of PG synthetase activity paralleled to known loss of arachidonic acid from the phospholipids of the spermatozoa as they pass through the male tract. Endogenous substrate was not limiting in the assay system, even in the testis and caput epididymidis where PG synthesis was not normally observed, indicating that a PG synthesis inhibitor may be present in these two tissues. PG synthetase activity within teased seminiferous tubules was markedly increased by physical trauma. Indomethacin diminished but did not eliminate synthesis.     

Immunolocalization of Aquaporin-1, -5, and -7 in the Avian Testis and Vas Deferens

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been found in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. The localization of AQP subtypes (AQP1, 2, 3, 4, 5, 7, 8, 9, and 11) in the goose testis and vas deferens has been studied through immunohistochemistry and immunobloting. Interestingly, the testicular and deferential tissues were positive for AQP1, -5, and -7 but not the others. AQP1 immunoreactivity was detected in the capillary endothelial cells of testis and vas deferens. AQP5 was localized in the interstitial tissue of the testis, including Leydig cells, as well as in the basal cells of vas deferens. Double-labeling confocal microscopy revealed coexpression of AQP5 with capillary AQP1 in the testis. AQP7 was expressed in elongated spermatid and spermatozoa tails in the testis, as well as spermatozoa tails in the vas deferens. These results suggest that several subtypes of AQPs are involved in the regulation of water homeostasis in the goose male reproductive system. (J Histochem Cytochem 57:915–922, 2009)     

Male reproductive system of the arrow crab Stenorhynchus seticornis (Inachoididae)

Our aim was to describe the reproductive system of males and the formation of sperm packages in the seminal receptacle (SR) of recently mated females of the arrow crab Stenorhynchus seticornis. The male reproductive system was analyzed, and was described using light microscopy and histological and histochemical methods. The first pair of gonopods was described by means of scanning electron microscopy. Additionally, the dehiscence of spermatophores was tested using samples obtained from the vas deferens of males and from the seminal receptacle of recently mated females. Testes were tubular type, and each vas deferens consisted of three regions: the anterior vas deferens (AVD), including a proximal portion that was filled with free spermatozoa and a distal portion contained developing spermatophores; the median vas deferens (MVD) that contained completely formed spermatophores; and the posterior vas deferens (PVD), which contained only granular secretions. The accessory gland, which was filled with secretions, was located in the transition region between the MVD and the PVD. The spermatophores from the MVD were of different sizes, and none of them showed dehiscence in seawater, whereas those spermatophores in contact with the seminal receptacle were immediately broken. The ultrastructure of the gonopods revealed the presence of denticles at the distal portion, which contribute to the mechanical rupture of the spermatophore wall during the transfer of sperm. The contents of the PVD and accessory gland of males are transferred together with the spermatophores, and are responsible for the secretions observed among the sperm packets in the SR of the female. We suggest that these secretions formed the layers found in the SR of recently mated females, and may play a role in sperm competition in arrow crabs.     

日本沼虾输精管的结构及其在精荚形成中作用的研究

应用光镜和透射电镜技术研究了日本沼虾输精管的结构及其在精荚形成中的作用。结果表明,日本沼虾输精管从形态结构上可分为近端输精管、卷曲输精管、远端输精管和膨大的远端输精管四部分。各部分的管壁皆由分泌上皮、基膜、肌肉层和结缔组织构成,其中分泌上皮包括高度明显不同的低柱状上皮和高拄状上皮两部分。输精管各部分管腔内含有处于不同形成阶段的精荚。进入近端输精管内的精子被支撑在一种嗜酸性基质中。近端输精管的分泌物主要帮助形成精子团,同时形成精荚壁的极小部分。卷曲和远端输精管分泌形成精荚壁的绝大部分,其分泌物由细胞顶端通过外排作用和顶泌机制分泌产生。膨大的远端输精管具有贮存精荚的作用,其分泌上皮也通过外排作用和顶泌机制产生分泌物包裹在已基本形成的精荚外侧,管壁肌肉层在雌雄交配时将管腔内的精荚切割成适宜长度并排出体外。       

Circadian rhythm of sperm release in the gypsy moth,Lymantria dispar: ultrastructural study of transepithelial penetration of sperm bundles

Release of mature bundles of spermatozoa from the testis into the vas deferens is a critical but poorly understood step in male insect reproduction. In moths, the release of sperm bundles is controlled by a circadian clock which imposes a temporal gate on the daily exit of bundles through the terminal epithelium-a layer of specialized epithelial cells separating testis follicles from the vas deferens. The sequence of cellular events associated with the daily cycle of sperm release was investigated by scanning and transmission electron microscopy. In the hours preceding sperm release, there is a solid barrier between the testis and the vas deferens formed by the interdigitation of cytoplasmic processes of adjacent terminal epithelial cells. At the beginning of the sperm release cycle, sperm bundles protrude through this barrier while the terminal epithelial cells change their shape and position relative to the bundles. Subsequently, the cyst cells enveloping the sperm bundles break down and spermatozoa move out of the testis through the exit channels formed between the epithelial cells. Afterwards, cyst cell remnants and other cellular debris are released into the vas deferens lumen, and the epithelial barrier is reconstructed due to phagocytic activity of its cells. These data provide a foundation on which to build an understanding of the cellular mechanisms of clock-controlled sperm release in insects.     

Female cloacal protuberance of the polygynandrous Alpine Accentor Prunella collaris: histological features and possible functional significance

This paper describes the histological features of the prominent cloacal protuberance (CP) of sexually mature females of the polygynandrous Alpine Accentor Prunella collaris . In the copulation period, the CP of the prelaying mature female developed remarkably and the lips of the vent turned brilliant scarlet. Macroscopic and microscopic examination of the cloacal region from females of different ovarian maturity showed that the formation of the CP was mainly due to the hypertrophy of the cloacal musculature, i.e. the sphincter cloacae muscle and levator cloacae muscle, and to the development of the associated connective tissue in the sexually mature female. The scarlet coloration of the cloacal lips was ascribed exclusively to the development of the dermal vascular plexus accompanied by hyperaemia, as characterized by the dilated blood vessels filled with blood. There was no evidence that skin pigmentation was involved in the scarlet coloration of the cloacal lips. We speculate that the morphological changes in the cloacal lips of the female Alpine Accentor are mediated hormonally by the brain–pituitary–gonadal axis.     

Co-expression and interaction of cubilin and megalin in the adult male rat reproductive system

Cubilin is a peripheral membrane protein that cooperates with the endocytic receptor megalin to mediate endocytosis of ligands in various polarized epithelia. Megalin is expressed in the male reproductive tract where it has been implicated in the process of sperm membrane remodeling. A potential role for cubilin in the male reproductive tract has not been explored. Using RT-PCR, we found that cubilin and megalin mRNAs are expressed in the efferent ducts, corpus and cauda epididymis, and proximal and distal vas deferens. Immunohistological analysis revealed that cubilin was expressed in nonciliated cells of the efferent ducts, principal cells of the corpus and cauda epididymis and vas deferens. Immunogold EM showed cubilin in endocytic pits, endocytic vesicles, and endosomes of these cells. The expression profile of cubilin in the male reproductive tract was coincident with that of megalin except in principal cells of the caput epididymis. Double immunogold labeling showed that cubilin and megalin co-localized within the endocytic apparatus and recycling vesicles of efferent duct cells. Neither protein was found in lysosomes. Injection of RAP, an antagonist of megalin interaction with cubilin, reduced the level of intracellular cubilin in cells of the efferent ducts and vas deferens. In conclusion, cubilin and megalin are co-expressed in cells of the epididymis and vas deferens and the endocytosis of cubilin in these tissues is dependent on megalin. Together, these findings highlight the potential for a joint endocytic role for cubilin and megalin in the male reproductive tract.     

Morphology and development of the male reproductive tract in Callinectes danae (Crustacea: Brachyura)

The aim of this study was to characterize the morphology and function of each section of the reproductive system of male Callinectes danae, as well as the stages of reproductive development and their relation to secondary sexual characteristics. Development of their reproductive system begins after completion of the pubertal moult. The growth of the gonopodium showed negative allometry for both juveniles and adults. The reproductive system is divided into portions with different functions. There is a germinal zone in the testes containing spermatogonia, a zone of maturation containing spermatocytes, spermatids or spermatozoa, as well as a collecting duct, which carries spermatozoa to the vas deferens. There are two matrices in the anterior vas deferens that initiate the separation of spermatozoa groups, one composed of polysaccharide acids (matrix I) and another consisting of neutral polysaccharides (matrix II). In the median vas deferens, the matrix II forms an acellular capsule, which forms the spermatophores. In the posterior vas deferens, the matrices are accumulated, initially with a granular texture and are homogenous for the final portion. The ejaculatory duct and penis have muscle lining to expel the spermatophores at copulation. Even after copulation, males retain a stock of spermatophores, allowing copulation with other females.     

Functional morphology of the male reproductive system in Callichirus major (Crustacea: Decapoda: Axiidea): Evidence of oocytes in the gonad

Callichirus major inhabits the intertidal region of marine ecosystems and it is frequently used as live bait for fishing. This study aimed to describe the functional anatomy of the male reproductive system by microscopic techniques. The animals were collected along the Corujão beach, Piuma—ES, Brazil, and, in laboratory, the males were classified into two phases: immature (IM) and developed (DE) based on the macroscopic characteristics of the gonads. The gonad and vas deferens were dissected for histological routine and histochemical tests. Histologically, it was noted that in both phases, the more distal region of gonads has ovarian characteristics, showing developing oocytes. Also, different male germ cells were identified: spermatogonium (SPG), spermatocytes I and II (SPTCI, SPTCII), initial and final spermatid (IS, FS) and sperm (SPZ). Accessory cells with spherical or pyramidal nuclei were also present inside the testicular lobules. According to the vas deferens structure, three regions can be characterized: proximal (PVD), middle (MVD) and distal (DVD). In the lumen of the vas deferens, a spermatophoric matrix highly reactive for histochemical tests was observed. The presence of female germ cells in males suggests the occurrence of intersexuality or hermaphroditism in this species.