Segmental and cellular expression of aquaporins in the male excurrent duct

The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Spermatozoa undergo their final steps of maturation as they pass through the male excurrent duct, which includes efferent ducts, the epididymis and the vas deferens. The composition of the luminal environment in these organs is tightly regulated. Major fluid reabsorption occurs in efferent ducts and in the epididymis, and leads to a significant increase in sperm concentration. In the distal epididymis and vas deferens, fluid secretion controls the final fluidity of the luminal content. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. Aquaporins contribute to transepithelial water transport in many tissues, including the kidney, the brain, the eye and the respiratory tract. The present article reviews our current knowledge regarding the distribution and function of aquaporins in the male excurrent duct.     

Segmental and cellular expression of aquaporins in the male excurrent duct

The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Spermatozoa undergo their final steps of maturation as they pass through the male excurrent duct, which includes efferent ducts, the epididymis and the vas deferens. The composition of the luminal environment in these organs is tightly regulated. Major fluid reabsorption occurs in efferent ducts and in the epididymis, and leads to a significant increase in sperm concentration. In the distal epididymis and vas deferens, fluid secretion controls the final fluidity of the luminal content. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. Aquaporins contribute to transepithelial water transport in many tissues, including the kidney, the brain, the eye and the respiratory tract. The present article reviews our current knowledge regarding the distribution and function of aquaporins in the male excurrent duct.     

Modulation of the oviductal environment by gametes

The notion of a gamete recognition system that alerts females to the presence of gametes in their reproductive tract profoundly influences our understanding of the physiology of events leading to conception and the bearing of offspring. Here, we show that the female responds to gametes within her tract by modulating the environment in which pregnancy is initially established. We found distinct alterations in oviductal gene expression as a result of sperm and oocyte arrival in the oviduct, which led directly to distinct alterations to the composition of oviductal fluid in vivo. This suggests that either gamete activates a cell-type-specific signal transduction pathway within the oviduct. This gamete recognition system presents a mechanism for immediate and local control of the oviductal microenvironment in which sperm transport, sperm binding and release, capacitation, transport of oocytes, fertilization, and early cleavage-stage embryonic development occur. This may explain the mechanisms involved in postcopulatory sexual selection, where there is evidence suggesting that the female reproductive tract can bias spermatozoa from different males in the favour of the more biologically attractive male. In addition, the presence of a gamete recognition system explains the oviduct's ability to tolerate spermatozoa while remaining intolerant to pathogens.     

Fundamental cryobiology of reproductive cells and tissues

During the last half of the 20th century there have been considerable advancements in mammalian reproductive technologies, including in vitro production of pre-implantation embryos and embryo sexing, and even cloning in some species. However, in most cases, management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor-recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Therefore, there is an urgent need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. Today frozen-thawed spermatozoa and embryos have become an integral component of animal agriculture, laboratory animal genome banking, and human sperm banking and infertility programs. However, although widely implemented, the protocols currently used to cryopreserve bull sperm, for example, are still suboptimal, and cannot readily be extrapolated to other species' sperm. Similarly, embryo-freezing protocols successfully used for mouse and cattle have yielded little success when applied to some other species' embryos, or to a related cell type, oocytes. To date, with the exception of mouse oocytes, almost all mammalian species' oocytes studied have proven very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for these low survival rates in an effort to develop better cryopreservation methods for oocytes. Additionally, there is growing interest in developing technologies for the optimal isolation and cryopreservation of the earliest stage of male (spermatogonia, spermatids) and female (primordial follicle) germ cells, with subsequent maturation to the desired stage in vitro. Female gamete maturation, fertilization, and embryo development entirely under in vitro conditions from primordial follicles has been achieved in mice, however techniques for this and other species are still very early in their development. Furthermore, with the recent advances made in intracytoplasmic sperm injection (ICSI), and gamete isolation and maturation, close attention has been given to cryopreservation of gametes in the form of gonadal tissue (i.e., testicular tissue and ovarian tissue) containing various developmental stages of male (spermatogonia, spermatids, and spermatozoa) and female (primordial, secondary) germ lines.     

Sperm transport and survival in the mare: a review

After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, semen plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mare's reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen/thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes in spermatozoa during cryopreservation, and the removal of semen plasma during cryopreservation of equine semen.     

Sperm transport and survival in the mare

Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, seminal plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mares reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen-thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes of spermatozoa during cryopreservation, and the removal of seminal plasma during cryopreservation of equine semen.     

Proteins implicated in sperm capacitation

Spermatozoa after being deposited in the female reproductive tract spend a considerable time in this foreign environment prior to fertilization of the oocyte. Chang and Austin independently observed1,2 that this time spent by the spermatozoa in the female tract is not consequential but a necessary event in the life cycle of the male gamete, and Austin2 first called this maturation period of spermatozoa as 'Capacitation'. Ever since, attempts have been made to understand and unravel the molecular mechanism of capacitation. Based on the results obtained so far, it is clear that capacitation is guided by novel signal transduction pathways influencing varied aspects of spermatozoa. Capacitation could be, thus, defined as the cumulative molecular, cellular and physiological changes that occur in spermatozoa in the female reproductive tract to achieve the final competence to fertilize the oocyte. This review is structured so as to first understand the key features of capacitation and then to survey the players which bring about these changes during capacitation.     

Sperm migration and selection in the reproductive tract of female mice is mostly affected by male genotype

The aim of the present study is to assess the influence of male and female genotypes on the transport of sperm to the site of fertilization. We mated B10.BR, B10.BR-Ydel and BALB/c males with B10.BR and BALB/c females and then analyzed the quality and quantity of spermatozoa found five hours post coitus in three successive parts of the female reproductive tract. We found that B10.BR and B10.BR-Ydel spermatozoa are very effectively selected by the uterotubaljunction and other barriers of the female genital tract. On the contrary, severely deformed BALB/c spermatozoa appeared to be able to cross selective barriers and were present both in oviducts and in cumulus oophorus. It cannot be excluded that these morphologically abnormal male gametes take part in fertilization. B10.BR-Ydel spermatozoa were very rarely observed above the uterotubaljunction. This shows that in vivo they migrate with delay and with difficulties pass the border between uterus and oviducts. This finding is in agreement with previous in vitro analyzes, which revealed many irregularities in movement of B10.BR-Ydel spermatozoa. Sperm quality and quantity in the reproductive tracts of B10.BR and BALB/c females were convergent if they were mated with males belonging to one strain, proving that migration and selection of spermatozoa in the female genital tract depend mostly on male genotype.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

Aquaporin 9 expression along the male reproductive tract

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.     

Resorption of unemitted gametes in Lithognathus mormyrus (Sparidae,Teleostei): a possible synergic action of somatic and immune cells

The resorption of unemitted gametes during the post-spawning period of the male and female reproductive cycles in Lithognathus mormyrus was studied by histochemical, histological and cytological methods. The resorption of residual spermatozoa involved the phagocytotic activity of Sertoli cells bounding the seminiferous cysts of spermatozoa, and those associated with spermatogonia lining the lobular lumen. Spermatozoa remaining in the sperm duct were phagocytozed by the lining epithelial cells. Eosinophilic granulocytes and macrophages were identified in the vicinity of residual spermatozoa. The remnants of oocytes underwent an atretic phenomenon in which follicle cells were firstly involved, inducing a progressive fragmentation of the oocyte cytoplasm. Subsequently, eosinophilic granulocytes invaded oocyte degenerative areas and clung to the remaining vitelline inclusions ensuring their biotransformation into waste products (brown bodies). The analogy of the resorption processes of both male and female unemitted gametes during the post-spawning period of natural reproductive cycle, involving first the enveloping somatic cells and then immune cells, is emphasized.     

Dimorphism of Spermatozoa in the Sea Urchin Strongylocentrotus nudus

A phenomenon of dimorphism in spermatozoa has been revealed for the sea urchin Strongylocentrotus nudus. The spermatozoa are different in the configuration of the circular mitochondrion. The bulk of male gametes (73.4%) have a symmetrical mitochondrion, whereas the remainder spermatozoa (26.6%) have an asymmetrical one. No other ultrastructural differences in the structure of spermatozoa have been revealed. The two types of spermatozoa are supposed to represent different kinds of normal gametes capable of fertilization.     

Structure, function and translational relevance of aquaporin dual water and ion channels

Aquaporins have been assumed to be selective for water alone, and aquaglyceroporins are accepted as carrying water and small uncharged solutes including glycerol. This review presents an expanded view of aquaporins as channels with more complex mechanisms of regulation and diverse repertoires of substrate permeabilities than were originally appreciated in the early establishment of the field. The role of aquaporins as dual water and gated ion channels is likely to have physiological and potentially translational relevance, and can be evaluated with newly developed molecular and pharmacological tools. Ion channel activity has been shown for Aquaporins -0, -1, and -6, Drosphila Big Brain, and plant Nodulin-26. Although the concept of ion channel function in aquaporins remains controversial, research advances are beginning to define not only the ion channel function but also the detailed molecular mechanisms that govern and mediate the multifunctional capabilities. With regard to physiological relevance, the adaptive benefit of expression of ion channel activity in aquaporins, implied by amino acid sequence conservation of the ion channel gating domains, suggests they provide more than water or glycerol and solute transport. Dual ion and water channels are of interest for understanding the modulation of transmembrane fluid gradients, volume regulation, and possible signal transduction in tissues expressing classes of aquaporins that have the dual function capability. Other aquaporin classes might be found in future work to have ion channel activities, pending identification of the possible signaling pathways that could govern activation.     

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.     

Conservation de tissu testiculaire et maturation in vitro de la lignée germinale pour préservation du potentiel de reproduction avant traitement anticancéreux chez le garçon pré-pubère

Sperm auto-conservation before chimioradiotherapy allows preservation of future reproductive possibilities in case of malignancy in young adult male. Because of the lack of mature spermatozoa, such possibilities cannot be offered for boys before puberty, even though the rate of cure of childhood malignancies is high. This paper reviewed recent advances in reproductive technology, which open the field of withdrawal of immature germ cells in prepubertal boys for in vitro maturation and cryopreservation for future paternity. It has been shown that the main steps of male meiosis have been driven in vitro, allowing to obtain round spermatids from pachytene spermatocytes in the rat. In mice, cryopreserved round spermatids have been used for oocyte fertilization and gave rise to normal living pups. In humans pregnancies and living babies have been reported after microinjection of round spermatids in cases of azoospermia. One pregnancy has been obtained with a cryopreserved spermatid. Thus the project of withdrawal of testicular tissues before sterilizing treatment, in vitro maturation of spermatogonia into round spermatids and cryopreservation of immature germ cells for future use for assisted fertilization does not seem unrealistic since each step has been done individually. However developing animal models is necessary to study not only the efficiency of the whole procedure but also to check its harmlessness before clinical trials.     

Motility of fish spermatozoa: from external signaling to flagella response

For successful fertilization, spermatozoa must access, bind, and penetrate an egg, processes for which activation of spermatozoa motility is a prerequisite. Fish spermatozoa are stored in seminal plasma where they are immotile during transit through the genital tract of most externally fertilizing teleosts and chondrosteans. Under natural conditions, motility is induced immediately following release of spermatozoa from the male genital tract into the aqueous environment. The nature of an external trigger for the initiation of motility is highly dependent on the aquatic environment (fresh or salt water) and the species’ reproductive behavior. Triggering signals include osmotic pressure, ionic and gaseous components of external media and, in some cases, egg-derived substances. Extensive study of environmental factors influencing fish spermatozoa motility has led to the proposal of several mechanisms of activation in freshwater and marine fish. However, the signal transduction pathways initiated by these mechanisms remain clear. This review presents the current knowledge with respect to (1) membrane reception of the activation signal and its transduction through the spermatozoa plasma membrane via the external membrane components, ion channels, and aquaporins; (2) cytoplasmic trafficking of the activation signal; (3) final steps of the signaling, including signal transduction to the axonemal machinery, and activation of axonemal dyneins and regulation of their activity; and (4) pathways supplying energy for flagellar motility.     

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Immunocytochemical localization of alpha-lactalbumin in the male reproductive tract

The immunocytochemical localization of the milk protein alpha-lactalbumin in the male reproductive tract is described. Using a primary antiserum raised against highly purified rat milk alpha-lactalbumin, specific staining was consistently shown in the supranuclear Golgi region of the principal cells of the proximal caput epididymidis but only occasionally in epithelial cells from other regions of the duct. Staining was also found in the epididymal lumen and associated with spermatozoa. This luminal staining persisted throughout the distal caput, corpus and cauda epididymidis. Staining was rarely associated with spermatozoa in the efferent ducts and initial segment. Alpha-lactalbumin immunoreactivity was also detected in the seminiferous epithelium. Staining was confined to the Golgi-acrosome region of spermatids. These results indicate that an alpha-lactalbumin-like molecule, or molecules, is present in the male reproductive tract and that it is localized specifically in principal cells from the proximal caput epididymidis and germ cells from the seminiferous epithelium.