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.     

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.     

Seasonal changes in the oviduct of the pied myna (Aves: Sturnidae)

Study of the oviduct of the pied myna (Sturnus contra contra) throughout the year reveals that oviductal weight, length, surface epithelial height and glycogen content are low during August to January (nonbreeding phase), partially increase during February to April (pre-breeding phase), maximally increase in May (breeding phase) and decrease in June and July (post-breeding phase). In the nesting cycle, there is greatest growth in all the regions of the oviduct from early nest-building to the egg-laying period and this is followed by rapid involution during incubation and nestling periods. Some notable features in the oviduct of the pied myna are described: 1) All five regions of the oviduct (infundibulum, magnum, isthmus, uterus, and vagina) are clearly distinguishable when studied from serial sections of the oviduct even during the nonbreeding phase of the annual ovarian cycle. 2) There is a strong correlation between initiation of tubular gland formation and the onset of nestbuilding activity. 3) The distal part of the magnum is differentiated into a 'mucous region' having well developed basal nonciliated cells. 4) A sixth zone can be identified between the magnum and isthmus. Sperm hostlike glands exist at the cranial end of the zone. 5) Several circular epithelial invaginations are evident in the intermucosal folds and their size decreases in centripetal order in the vagina. 6) The pattern and degree of regression are different in various regions of the oviduct. A close synchrony between ovarian and oviducal cycles is indicated in the pied myna (Sturnus contra contra).     

Effect of prostaglandins on oviduct tone in the domestic hen in vivo

The effect of i.v. injected prostaglandins (PG) F₂α and E₂ on intraluminal pressure of the different oviductal parts (infundibulum, magnum, isthmus, uterus and vagina) was investigated in the domestic hen. PGF₂α induced only a pressure rise in all oviduct segments. Administration of PGE₂ resulted in variable changes in oviductal tone: pressure rise in the infundibulum; pressure increase often preceded by a small decrease in the magnum, isthmus and uterus; pressure decrease in the vagina and sometimes in the uterus. Simultaneous i.v. injection of both PG's induced mostly a decrease in vaginal tone. Intraluminal administration of PGF₂α or E₂ resulted only in an increase in uterine pressure.The observed effects on oviduct tone are discussed and a possible in intervention of both PG's in the mechanism of ovum transport and oviposition in the domestic hen is proposed.     

Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Histomorphological changes in the oviduct of the mallard (Aves: Anatidae)

Studies on histomorphometrical changes in different segments (infundibulum, magnum, isthmus, shell gland and vagina) of oviduct of mallard, Anas platyrhynchos during active and quiescent phases of the reproductive cycle have been made. The absolute and per cent length and width of each segment showed a marked change. The magnum showed an increase of 280 per cent. Of all the histological parameters studied the number and height of mucosal folds and mucosal epithelium showed more marked increase in all segments of oviduct. The size of tubular glands and frequency of ciliated and secretory cells were studied in relation to oviductal activity.     

Seasonal variation of the oviduct of the American alligator,Alligator mississippiensis (Reptilia: Crocodylia)

The annual oviductal cycle of the American alligator, Alligator mississippiensis, is described using light and electron microscopy. Previous work done by Palmer and Guillette ([ 1992 ] Biol Reprod 46:39–47) shed some light on the reproductive morphology of the female alligator oviduct; however, their study was limited and did not report details relating to variation across the reproductive season. We recognize six variable regions of the oviduct: infundibulum, tube, isthmus, anterior uterus, posterior uterus, and vagina. Each area shows variation, to some degree, in the histochemistry and ultrastructure of oviductal secretions. Peak secretory activity occurs during the months of May and June, with the greatest variation occurring in the tube and anterior uterus. During the month of May, high densities of neutral carbohydrates and proteins are found within the tubal and anterior uterine glands. The epithelium of the entire oviduct secretes neutral carbohydrates throughout the year, but many regions lack protein secretions, and the posterior uterine glands show little secretory activity of any type throughout the year. After oviposition, secretory activity decreases drastically, andthe oviduct resembles that of the premating season. This study also provides evidence to support the homology between alligator and bird oviducts. Sperm were observed in glands at the tubal‐isthmus and utero‐vaginal junctions in preovulatory, postovulatory and postovipository females. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Regional differences in expression of progesterone receptor in oviduct and uterus of rabbit during early pregnancy

We characterized the expression pattern of progesterone receptor (PR) in two regions of the oviduct (ampullae and isthmus), and the uterus (epithelium and stroma) of the rabbit (Oryctolagus cuniculus) during early pregnancy (1-4 days) by RT-PCR and immunohistochemistry. We observed a significant increase in the expression of PR at mRNA level in the uterus on days 1 and 2 of pregnancy, followed by a decrease on days 3 and 4. These changes were also observed at protein level in the uterine epithelium. Interestingly, PR immunoreactivity decreased in stromal cells in all days of pregnancy as compared with non-pregnant rabbits (NG). In the isthmus PR mRNA expression significantly increased on day 2 of pregnancy and diminished on days 3 and 4, whereas no significant changes were observed in the ampullae. In epithelial and stromal cells of the isthmus, PR immunostaining was reduced through pregnancy as compared with NG group. In contrast, a reduction in PR immunostaining was observed on days 1-3 with an increase on day 4 in epithelial and stromal cells of the ampullae. The overall results suggest that PR exhibit a differential expression pattern in the oviduct and the uterus during early pregnancy of the rabbit, and that these differences are related to different functions of PR in the reproductive tract during early pregnancy.     

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.     

Identification and characterization of telocytes in the uterus of the oviduct in the Chinese soft‐shelled turtle,Pelodiscus sinensis: TEM evidence

Telocytes (Tcs) are cells with telopodes (Tps), which are very long cellular extensions with alternating thin segments (podomers) and dilated bead‐like thick regions known as podoms. Tcs are a distinct category of interstitial cells and have been identified in many mammalian organs including heart, lung and kidney. The present study investigates the existence, ultrastructure, distribution and contacts of Tcs with surrounding cells in the uterus (shell gland) of the oviduct of the Chinese soft‐shelled turtle, Pelodiscus sinensis. Samples from the uterine segment of the oviduct were examined by transmission electron microscopy. Tcs were mainly located in the lamina propria beneath the simple columnar epithelium of the uterus and were situated close to nerve endings, capillaries, collagen fibres and secretory glands. The complete morphology of Tcs and Tps was clearly observed and our data confirmed the existence of Tcs in the uterus of the Chinese soft‐shelled turtle Pelodiscus sinensis. Our results suggest these cells contribute to the function of the secretory glands and contraction of the uterus.     

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen.

The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca2+ pump was not detectable in the infundibulum or the magnum. Calbindin-D28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca2+ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D28k differed, co-localizing with the Ca2+ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca2+ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.     

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen

Summary The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca²⁺ pump was not detectable in the infundibulum or the magnum. Calbindin-D_28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca²⁺ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D_28k differed, co-localizing with the Ca²⁺ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca²⁺ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). II. Annual oviducal cycle

This article is the first ultrastructural study on the annual oviducal cycle in a snake. The ultrastructure of the oviduct was studied in 21 females of the viviparous natricine snake Seminatrix pygaea. Specimens were collected and sacrificed in March, May, June, July, and October from one locale in South Carolina during 1998-1999. The sample included individuals: 1) in an inactive reproductive condition, 2) mated but prior to ovulation, and 3) from early and late periods of gravidity. The oviduct possesses four distinct regions from cranial to caudal: the anterior infundibulum, the posterior infundibulum containing sperm storage tubules (SSTs), the uterus, and the vagina. The epithelium is simple throughout the oviduct and invaginations of the lining form tubular glands in all regions except the anterior infundibulum and the posterior vagina. The tubular glands are not alveolar, as reported in some other snakes, and simply represent a continuation of the oviducal lining with no additional specializations. The anterior infundibulum and vagina show the least amount of variation in relation to season or reproductive condition. In these regions, the epithelium is irregular, varying from squamous to columnar, and cells with elongate cilia alternate with secretory cells. The secretory product of the infundibulum consists largely of lipids, whereas a glycoprotein predominates in the vagina; however, both products are found in these regions and elsewhere in the oviduct. In the SST area and the anterior vagina, tubular glands are compound as well as simple. The epithelium of the SST is most active after mating, and glycoprotein vacuoles and lipid droplets are equally abundant. When present, sperm form tangled masses in the oviducal lumen and glands of the SST area. The glands of the uterus are always simple. During sperm migration, a carrier matrix composed of sloughed epithelial cells, a glycoprotein colloid, lipids, and membranous structures surround sperm in the posterior uterus. During gravidity, tubular glands, cilia, and secretory products diminish with increasing development of the fetus, and numerous capillaries abut the basal lamina of the attenuated epithelial lining of the uterus.     

Differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse,rat, and hamster

Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses. Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms. We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice. In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster. The lactoferrin expression in the cycling adult female rat was also evaluated. Lactoferrin gene polymorphism exists among the different mouse strains. In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse. We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats. Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle. Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium. The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea): Part I. Evidence for oviducal sperm storage

Oviducal sperm storage in the viviparous (lecithotrophic) colubrid snake Seminatrix pygaea was studied by light and electron microscopy. Out of 17 adult snakes examined from May–October, sperm were found in the oviducts of only two specimens. In a preovulatory female sacrificed 14 May, sperm were found in the oviducal lumen and sperm storage tubules (SSTs) of the posterior infundibulum. In a nonvitellogenic female sacrificed 9 June, sperm were found in the lumen and glands of the posterior uterus and anterior vagina, indicating a recent mating. The glands in the posterior infundibulum and vagina were simple or compound tubular, whereas glands in the uterus always were simple tubular. The epithelium of the sperm storage glands was not modified from that lining the rest of the oviduct. The cuboidal or columnar epithelium consisted of alternating ciliated and secretory areas. The secretory product released into the lumen by a merocrine process contained mucoprotein. Lipid droplets also were numerous in the epithelium. Portions of sperm sometimes were embedded in the apical cytoplasm or in secretory material. A carrier matrix containing a mucoid substance, desquamated epithelium, lipids, membranous structures, and possibly phagocytes was found around sperm in the posterior uterus. J. Morphol. 241:1–18, 1999. © 1999 Wiley‐Liss, Inc.     

Effects of sexual maturation and Salmonella infection on the expression of Toll-like receptors in the chicken vagina

Toll-like receptors (TLRs) are a critical component of the innate immune response in many vertebrates, including avian species. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles and in the chicken ovary in vivo, as well as the changes in their expression in response to lipopolysaccharide or Salmonella enteritidis (SE) infection, have broad implications for reproductive physiology and for the prevention of transmission of zoonotic diseases to humans through the consumption of contaminated poultry eggs. Because the main route of egg contamination is from infection of the oviduct and mainly from the vagina, the aim of this study was to investigate the expression of the ten cTLRs identified to date in the chicken oviduct in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the expression of TLRs in the chicken vagina. RNA was extracted from the vagina of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all types of cTLRs apart from TLR1-1 were expressed in the vagina of sexually mature birds. Quantitative real-time PCR analysis revealed that the mRNA abundance of TLR2-1, 2-2 and 4 differ with respect to sexual maturation in the chicken vagina. SE infection resulted in a significant induction of TLR5 and 15 in the vagina of sexually mature birds, and in a significant induction of TLR2-1, 4 and 15 in the vagina of aged birds, while a significant down-regulation was observed for TLR7 in the vagina of sexually mature birds. These findings suggest that a TLR mediated immune response mechanism exists in the chicken vagina, playing a crucial role in preventing microbial pathogens from being incorporated into newly forming eggs.     

Changes in ovalbumin and protein synthesis in vivo in the magnum of laying hens during the egg formation cycle.

1. The present study was carried out to investigate whether or not the rate of synthesis of total protein in various oviducal segments and ovalbumin, a major egg white protein, in the magnum fluctuated during the egg formation cycle in laying hens. 2. Synthesis of total protein and ovalbumin was measured in vivo by the incorporation of [15N]methionine after a primed continuous infusion of tracer for 3 hr. 3. Protein and ovalbumin contents in the magnum and the entire oviduct decreased sharply when an ovum moved down from the magnum to the isthmus, probably due to the secretion of egg white proteins. 4. In contrast, total protein and ovalbumin synthesis in the magnum was significantly higher when an ovum was in there than when it was in any other segments. Fluctuations of ovalbumin synthesis and total protein synthesis in the magnum were roughly parallel to those of total protein synthesis in the entire oviduct. 5. It was concluded, therefore, that the changes seen in total protein synthesis in the whole oviduct during the egg formation cycle were mainly attributable to those in magnum protein synthesis, of which a significant portion was accounted for by the synthesis of ovalbumin.