Histology and histochemistry of the oviductal sperm storage pockets of the agamid lizard Calotes versicolor

Some reptiles are known to possess sperm storage in the female genital tract. Examination of the oviduct of the lizard Calotes versicolor shows the presence of sperm in storage pockets of the uterovaginal region throughout the reproductive cycle. Histochemical studies indicate that the epithelium of these pockets resembles the local oviductal epithelium and that a considerable amount of activity of steroidogenic and metabolic enzymes exists.     

Sperm transport and storage and its relation to the annual sexual cycle of the female red-sided garter snake,Thamnophis sirtalis parietalis

Female red-sided garter snakes (Thamnophis sirtalis parietalis) store sperm from both late-summer and spring matings. Before winter dormancy, sperm are stored in specialized furrows in the vaginal portion of the oviduct, 3–6 cm anterior to the vent. After 6 weeks in dormancy, the epithelial cells lining this vaginal region hypertrophy and stain strongly with periodic acid-Schiff (PAS). This PAS+ epithelial border sloughs and associates with sperm. These aggregations of PAS+ material, which will be referred to as carrier matrices, move anteriorly through the oviduct. After 20 weeks in dormancy, most sperm are found in specialized infundibular storage regions. Spring mating induces a rapid degeneration of winter-stored sperm. Stored sperm are evacuated from infundibular storage receptacles within 6 hours after mating. Yet sperm from the spring mating are not evident in the oviduct until 24 hours after mating. Carrier matrices begin to form at this time. At 48 hours after mating, sperm, often associated with carrier matrices, appear in the infundibulum. At 40 days after mating, most sperm have moved into infundibular storage receptacles. Evidence suggests that carrier matrices not only facilitate the transport of sperm anteriorly from vaginal to infundibular regions, but also function as nutritional stores.     

Seasonal changes of sperm storage and correlative structures in male and female soft-shelled turtles, Trionyx sinensis

Reproductive ducts of male and female soft-shelled turtles, Trionyx sinensis were examined throughout the year (March, May, September, December) using brightfield and electron microscopes (TEM and SEM), to determine the location and histomorphological characteristics of sperm storage structures as well as their changes at different phases of the seasonal reproductive cycle. Sperm stored in the epididymis were also examined. In the male, spermatogenesis is initiated in spring (May), and then the mature sperm are released in autumn as an episodic event. Spermatogenesis is inactive in winter. However, in this species, the epididymis contains sperm throughout the entire year. Sperm observed in the epididymis are intact and some structures are uniquely different from other reptiles, and is characterized by 35–40 concentric mitochondria with a dense core in the centre. Many glycogen granules are observed in the cytoplasm of the midpiece. However, the epithelial cell type of epididymal duct change in different seasons. The cells are fully developed with a highly secretory activity in September. The materials secreted from the epithelium might have the function as nourishment for the stored sperm. Sperm storage structures in the form of tubules are observed in the wall of the isthmus of the oviduct in hibernating females but are absent in the groups of May and September. These tubules develop either by folding or fusion of the oviductal mucosal folds and are lined by both ciliated and secretory cells. These tubules might provide a microenvironment for the sperm to enable its long-term storage. After being separated 4 months (December–March) from the male, sperm are observed in the tubules of the isthmus of the oviduct. The unique character of the sperm combined with the special sperm storage structures enable the sperm to maintain fertility and activity during their storage.     

Oviductal sperm storage structure and their changes during the seasonal (dissociated) reproductive cycle in the soft-shelled turtle Lissemys punctata punctata

The oviduct of the Indian fresh water soft-shelled turtle Lissemys punctata punctata was examined throughout the year under light and scanning electron microscopes to determine the location, histomorphological characteristics, and function of sperm storage structure, as well as their changes at different phases of the seasonal reproductive cycle. Sperm storage structures in the form of tubules were observed in the wall of isthmus throughout the year. These tubules developed either by folding or fusion of the oviductal mucosal folds and were lined by both ciliated and nonciliated epithelial cells. The height and secretory activities of the epithelia were markedly high during the breeding phase (August to September) but low in the nonbreeding phase (October to June). A few short tubules lined by cuboidal epithelium appear in the wall of infundibulum only during the breeding phase. Following mating (May), inseminated sperm were stored within the tubules of isthmus up to the pre-ovulatory stage (August). Thereafter, sperm associated with PAS-positive materials secreted from the epithelium (referred to as a carrier matrix) moved forward to the infundibulum and were stored within the storage tubules of the infundibulum for a short time. Subsequently, sperm evacuated the storage tubules and entered the oviductal lumen to fertilize the subsequently ovulated eggs during or prior to ovulation. The isthmus-tubules become shorter and narrower in the regressive phase (October to November) and remained so until the early preparatory phase (April). Sperm release might have been stimulated by estrogen secreted from the ovarian follicles of pre-ovulatory turtles. Stored sperm not utilized for fertilization remained viable not less than six months in the present turtle species.     

Sperm storage in the oviduct of the American alligator

Oviducts of the American alligator (Alligator mississippiensis) were examined histologically for the presence of stored sperm. Two regions containing sperm were identified, one at the junction of the posterior uterus and the vagina (UVJ) and the other at the junction of the tube and isthmus (TIJ). In these areas, sperm were found in the lumina of oviductal glands. The glands in these areas of the oviduct are diffuse and shallow and appear to allow better access to sperm than glands located elsewhere. Histochemically, the glands of the UVJ reacted weakly for carbohydrates and proteins, whereas those of the TIJ reacted strongly for these same two components, secretions of which are associated with sperm storage structures in other reptiles. Sperm were not in contact with the glandular epithelium, and glands at the UVJ contained more sperm than those at the TIJ. Oviductal sperm storage was observed not only in recently mated females but in all females possessing uterine eggs as well as all females known to be associated with a nest. We conclude that female alligators are capable of storing sperm in their oviductal glands, but not from one year to the next.     

Sperm aggregations in the spermatheca of the red back salamander (Plethodon cinereus)

The spermatheca of Plethodon cinereus is a compound tubular gland that stores sperm from mating in early spring (March–April) to oviposition in summer (June–July). The seasonal variation of sperm storage in this species has previously been studied by light and transmission electron microscopy. In this paper, sperm aggregations, interaction of sperm with the spermathecal epithelium, and spermathecal secretions are studied using scanning electron microscopy. Within spermathecal tubules, relatively small groups of sperm are aligned along their entire lengths in parallel arrays. This pattern is similar to other plethdontids with complex spermathecae. Lumina of spermathecal tubules are filled with secretory material in April prior to the arrival of sperm, and after sperm appear, a coating of secretory material persists on the apices of the spermathecal epithelium. Sperm peripheral to the central luminal mass can become embedded in the secretory matrix or pushed deeper into the spermathecal epithelium. The spermathecal secretions may serve to attract and prolong the viability of sperm, but sperm that become enmeshed in the secretions or epithelium are phagocytized. Sperm and spermathecal secretions are largely absent after ovulation and in summer months, and new secretory vacuoles are formed in fall, although mating does not occur until spring.     

A histological and ultrastructural investigation of the female reproductive system of the water snake (Erythrolamprus miliaris): Oviductal cycle and sperm storage

We studied the structural and cellular organisation of the oviduct of Erythrolamprus miliaris including its morphological variation during the reproductive cycle using light microscopy, scanning electron microscopy and transmission electron microscopy. Four anatomically distinct regions compose the oviduct of E. miliaris including the anterior and posterior infundibulum, glandular uterus, non-glandular uterus and pouch. The cells of the oviductal epithelium secrete material by apocrine and merocrine processes, which vary between the anatomical regions and according to each phase of the reproductive cycle. The infundibular epithelium secretes electron dense vacuoles, which suggests the production of lipids, whereas the epithelial secretion of the glandular uterus, non-glandular uterus and pouch creates lucent and slightly electron dense vacuoles, indicating the production of glycoproteins. The timing of mating, vitellogenesis and sperm storage directly influences the morphofunctional alterations in the oviducts of E. miliaris. Sperm storage occurs only in the infundibular receptacles with increased production of the neutral carbohydrates in the presence of male gametes. Sperm storage happens in vitellogenic, non-vitellogenic and pregnant females of E. miliaris. Thus, females may be able to produce multiple clutches at different seasons of the year regardless of mating during autumn.     

Histology and functional morphology of the female reproductive tract of the tortoise Gopherus polyphemus

The oviducts of 25 tortoises (Gopherus polyphemus) were examined by using histology and scanning electron microscopy to determine oviductal functional morphology. Oviductal formation of albumen and eggshell was of particular interest. The oviduct is composed of 5 morphologically distinct regions; infundibulum, uterine tube, isthmus, uterus, and vagina. The epithelium consists of ciliated cells and microvillous secretory cells throughout the oviduct, whereas bleb secretory cells are unique to the infundibulum. The epithelium and endometrial glands of the uterine tube histologically resemble those of the avian magnum which produce egg albumen and may be functionally homologous. The isthmus is a short, nonglandular region of the oviduct and appears to contribute little to either albumen or eggshell formation. The uterus retains the eggs until oviposition and may form both the fibrous and calcareous eggshell. The endometrial glands are histologically similar to the endometrial glands of the isthmus of birds, which are known to secrete the fibers of the eggshell. These glands hypertrophy during vitellogenesis but become depleted during gravidity. The uterine epithelium may supply "plumping water" to the egg albumen as well as transport calcium ions for eggshell formation. The vagina is extremely muscular and serves as a sphincter to retain the eggs until oviposition. Sperm are found within the oviductal lumen and endometrial glands from the posterior tube to the anterior uterus throughout the reproductive cycle. This indicates sperm storage within the female tract, although the viability and reproductive significance of these sperm are unknown.     

Regulation of sperm storage and movement in the mammalian oviduct

The oviduct plays a vital role in ensuring successful fertilization and normal early embryonic development. The male inseminates many thousands or even millions of sperm, but this alone does not ensure that fertilization will be successful. The female tract, particularly the oviduct, provides filters that select for normal vigorously motile sperm. In conjunction with molecules in the seminal plasma and on sperm, the female tract regulates how and when sperm pass though the tract to reach the site of fertilization. Various regulatory processes control sperm passage into and through the oviduct. In some species, the uterotubal junction opens and closes to regulate when sperm may enter; furthermore, passage through the junction requires certain proteins on the sperm surface. Most of the sperm that manage to enter the oviduct soon become trapped and held in a reservoir. In marsupials and insectivores, this involves trapping sperm in mucosal crypts; while in most other mammalian species, this involves binding sperm to the oviductal epithelium. As the time of ovulation approaches, the sperm in the reservoir undergo capacitation, including motility hyperactivation. Capacitating sperm shed proteins that bind them to the mucosal epithelium, while hyperactivation assists the sperm in pulling off of the epithelium and escaping out of mucosal pockets. The process of sperm release is gradual, reducing chances of polyspermic fertilization. Released sperm may be guided towards the oocyte by secretions of the oviduct, cumulus cells, or oocyte. Hyperactivation likely assists sperm in penetrating the cumulus matrix and is absolutely required for penetrating the oocyte zona pellucida and achieving fertilization.     

Mechanism of internal fertilization in Pegea socia (Tunicata,Thaliacea), a salp with a solid oviduct

The ovary of the salp Pegea socia (Bosc, 1802) is located at the end of an atrial diverticulum. The ovary consists of a single oocyte encased in a layer of follicle cells and is connected to the atrial epithelium by an oviduct. Transmission electron microscopy shows that the oocyte lacks a vitelline layer, cortical granules, and yolk granules and that the oviduct lacks a continuous lumen. What previous authors thought was a lumen is a line of dense intercellular junctions running down the center of the oviduct. The sperm nucleus in this species, as in other salps, is elongate. The tubular mitochondrion spirals about the sperm nucleus giving it a corkscrew-shape appearance. Sperm reach the ovary when the oocyte is still at the germinal vesicle stage. Many sperm swim up the atrial diverticulum and burrow through the cells of the atrial epithelium, oviduct, and follicular epithelium. Thus oviduct shortening, which occurs when the oocyte is in the meiotic divisions, is evidently unrelated to sperm moving up the oviduct. All previous authors, who argued either that a continuous lumen is necessary for sperm to move up the oviduct or that sperm bypass the oviduct, were incorrect. © 1994 Wiley-Liss, Inc.     

Sperm competition and reproductive success in the decapod Inachus phalangium (Majidae): a male ghost spider crab that seals off rivals' sperm

Parker's (1970a) hypothesis that the overlap of multiple mating sperm in the female's storage organs promotes sperm competition is tested here for the first time in Crustacea: specifically, the mechanisms and consequences of sperm competition are detailed for the spider crab Inachus phalangium . Females of this species store ejaculates from successive copulations with different males discretely and consecutively in sac-like twin seminal receptacles. During copulation males transfer a large quantity of a sperm-free seminal plasma, followed by the sperm which is stored in small spermatophores and forms a densely-packed sperm packet. It was shown, using ³H-thymidine-labelled ejaculate, that the last male to mate displaces the ejaculate of his predecessors dorsally into the apex of the receptacle. Sperm of previous matings are sealed in with the hardening seminal plasma (sperm gel) and are thus prevented from being used to fertilize eggs, while the last male to mate places his sperm closest to the oviduct and vaginal openings. In experiments using the 'sterile-male' method, sperm from the last male to mate gained all fertilizations in subsequent broods. The seminal plasma forms the sperm gel in ghost spider crabs which is used for displacement of previously stored sperm, whereas various other brachyuran taxa use seminal plasma to produce the sperm plug, which prevents a male's sperm from being displaced.     

Sperm release and use at fertilization by yellow dung fly females (Scathophaga stercoraria)

Females of many species mate multiple times and store transferred sperm in storage organs. The mechanisms underlying sperm release from the stores at fertilization remain poorly understood, although they are central to an understanding of the female influence on post-copulatory male competition. Using double-mated females of the yellow dung fly, we counted the sperm sticking to the surface of deposited eggs of two successive clutches to obtain insight into the physiological processes associated with fertilization. The number of sperm released to fertilize an egg decreased between the first and second clutches, as well as within clutches from early to late eggs. These results indicate that: (1) sperm are lost from the stores over time independent of egg laying and (2) the number of sperm released depends on the amount of sperm stored. The lower number of sperm on eggs of the second clutches was accompanied by a strong increase of the proportion of sperm adhering to the micropyle region, suggesting that sperm use is more efficient and sperm release better controlled when sperm supply is substantially reduced. Finally, our approach indicates that sperm storage capacity of the female is higher than assumed from counts of spermathecal sperm.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 511–518.     

Morphology of the female reproductive system of European pea crabs (Crustacea,Decapoda, Brachyura,Pinnotheridae)

Commensal pea crabs inhabiting bivalves have a high reproductive output due to the extension andfecundity of the ovary. We studied the underlying morphology of the female reproductive system in the Pinnotheridae Pinnotheres pisum, Pinnotheres pectunculi and Nepinnotheres pinnotheres using light microscopy and transmission electron microscopy (TEM). Eubrachyura have internal fertilization: the paired vaginas enlarge into storage structures, the spermathecae, which are connected to the ovaries by oviducts. Sperm is stored inside the spermathecae until the oocytes are mature. The oocytes are transported by oviducts into the spermathecae where fertilization takes place. In the investigated pinnotherids, the vagina is of the “concave pattern” (sensu Hartnoll 1968 ): musculature is attached alongside flexible parts of the vagina wall that controls the dimension of its lumen. The genital opening is closed by a muscular mobile operculum. The spermatheca can be divided into two distinct regions by function and morphology. The ventral part includes the connection with vagina and oviduct and is regarded as the zone where fertilization takes place. It is lined with cuticle except where the oviduct enters the spermatheca by the “holocrine transfer tissue.” At ovulation, the oocytes have to pass through this multilayered glandular epithelium performing holocrine secretion. The dorsal part of the spermatheca is considered as the main sperm storage area. It is lined by a highly secretory apocrine glandular epithelium. Thus, two different forms of secretion occur in the spermathecae of pinnotherids. The definite role of secretion in sperm storage and fertilization is not yet resolved, but it is notable that structure and function of spermathecal secretion are more complex in pinnotherids, and probably more efficient, than in other brachyuran crabs. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Sperm transfer and storage in the lizard,Anolis carolinensis

The relationship of the hemipenis to the cloaca in copula and sperm storage and transport in the female oviduct were studied in Anolis carolinensis using light and scanning electron microscopy. During copulation, the hemipenis does not penetrate beyond the cloaca, but the two apical openings of the bifurcate sulcus spermaticus appose the openings of the oviducts from the cloaca. Sperm enter the sperm storage tubules between 2 and 6 hr after insemination and small amounts of sperm reach the infundibulum 6 to 24 hr following mating. Sperm storage tubules are embedded in the wall of the utero-vaginal transition, and are formed by the folding and fusion of the oviducal epithelium. The importance of the hemipenile-cloacal relationship and the role of sperm storage in the life history of A. carolinensis are discussed.     

Sperm precedence in the dragonfly Erythemis simplicicollis

Females of the dragonfly Erythemis simplicicollis (Say) (Odonata, Libellulidae) store enough sperm to fertilize 6–13 clutches of eggs laid on consecutive days. Nonetheless, they usually mate one or more times per day. Males wait for females at ponds containing surface vegetation on which the females lay eggs. Some males defend vegetation while other act as satellites. After mating, both types of males attempt to guard females against takeover by other males. Sperm precedence by male E. simplicicollis was studied using sterility produced by gamma irradiation to label sperm. After a dose-response analysis, males receiving a dose of 25 kiloroentgens (>99.9% sterile) were returned to their home pond as territory residents and satellites. Both types of males fertilized an average of 99.5% (range 97.3–100%) of the female's remaining clutch. After mating with a sterile male, females were isolated in a large cage, and eggs collected for several consecutive days. These clutches revealed that sperm mixing in the bursa of the females is essentially complete after 24 to 48 h and that the last male to mate had replaced an average of more than 57–75% of the sperm stored by female from previous matings. Thus, the last sperm in is the first sperm out fertilizing essentially all of the eggs laid soon (5–6 min) after the mating. Sperm from the most recent mating competes for fertilizations with sperm stored from previous matings only if the female oviposits on the following day without remating.     

Sperm storage within the oviduct of turtles

Tubules containing sperm were identified by light microscopy in the oviducts from 11 species of turtles representing six different families. Sperm storage tubules were found in a small region of the posterior portion of the egg albumin-secreting section of the oviduct located between the infundibulum and the uterus. This location of storage tubules, midway between the ovary and vagina, is unique among vertebrates. Ducts, restricted to the posterior albumin region, connect the tubules to the oviduct lumen, allow entrance of sperm to the tubules. Sperm were identified in tubules of female turtles isolated from males for as long as 423 days.     

Sperm storage and spermatozoa interaction with epithelial cells in oviduct of Chinese soft‐shelled turtle,Pelodiscus sinensis

Spermatozoa are known to be stored within the female genital tract after mating in various species to optimize timing of reproductive events such as copulation, fertilization, and ovulation. The mechanism supporting long‐term sperm storage is still unclear in turtles. The aim of this study was to investigate the interaction between the spermatozoa and oviduct in Chinese soft‐shelled turtle by light and electron microscopy to reveal the potential cytological mechanism of long‐term sperm storage. Spermatozoa were stored in isthmus, uterine, and vagina of the oviduct throughout the year, indicating long‐term sperm storage in vivo. Sperm heads were always embedded among the cilia and even intercalated into the apical hollowness of the ciliated cells in the oviduct mucosal epithelium. The stored spermatozoa could also gather in the gland conduit. There was no lysosome distribution around the hollowness of the ciliated cell, suggesting that the ciliated cells of the oviduct can support the spermatozoa instead of phagocytosing them in the oviduct. Immune cells were sparse in the epithelium and lamina propria of oviduct, although few were found inside the blood vessel of mucosa, which may be an indication of immune tolerance during sperm storage in the oviduct of the soft‐shelled turtle. These characteristics developed in the turtle benefited spermatozoa survival for a long time as extraneous cells in the oviduct of this species. These findings would help to improve the understanding of reproductive regularity and develop strategies of species conservation in the turtle. The Chinese soft‐shelled turtle may be a potential model for uncovering the mechanism behind the sperm storage phenomenon.     

Sperm aggregations in female Agkistrodon piscivorus (Reptilia:Squamata): a histological and ultrastructural investigation

Upon copulation in female Agkistrodon piscivorus, sperm migrate up the oviduct to sperm storage tubules (SSTs) in the posterior infundibulum. The epithelium of the SSTs is composed of ciliated and secretory cells and differs ultrastructurally from that of the epithelium lining the lumen of the posterior infundibulum. Sperm pass through an area composed primarily of ciliated cells at the opening of each gland before aligning themselves in parallel arrays with their nuclei facing an area composed primarily of secretory cells at the base of the tubules. Sperm are also found embedded inter- and intracellularly in the SSTs. The secretory vacuoles in the SSTs become highly electron dense after the start of the fall mating season along with the synthesis of lipid droplets. Histochemical analysis reveals that the alteration in secretory material density is caused by the production of neutral carbohydrates. Some sperm remain in aggregates in the nonglandular section of the posterior uterus until the time of ovulation. However, ultrastructural evidence indicates these sperm degrade before ovulation. Therefore, sperm in posterior aggregates have no role in fertilization of ovulated ova. The data presented here support the hypothesis that infundibular sperm storage is the mode that snakes utilize to sequester viable sperm until ovulation.     

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica , comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.