Spermatozoa transport in the oviduct of the chicken and the turkey

Recent studies of the storage and release of spermatozoa from utero-vaginal glands in birds have shown that: following intra-vaginal insemination, storage is completed within 2-3 d (domestic hen) or 1-5 d (turkey) and not within a few minutes or hours as previously described; as spermatozoa can be recovered from any segment of the oviduct during the egg formation cycle, it seems unlikely that sperm release from the utero-vaginal glands is directly dependent upon the egg formation cycle. The progressive inability of spermatozoa to agglutinate may be part of this mechanism.     

Development of sperm storage tubules in the quail during sexual maturation.

The development of the utero-vaginal sperm storage tubules (SST) in the quail during sexual maturation was studied using light microscopy and image analysis. SST development starts at around 28 days with low columnar cells, 10.9 +/- 0.7 microm high, found at the base of mucosal folds in the distal uterus. Seven days later, small bud-like invaginations consisting of columnar, 16.4 +/- 1.1 microm high cells with basal nuclei were found in this region. An extremely rapid growth of the oviduct occurred at approximately 40-42 days of age with considerable variation in oviductal length between birds, coefficient of variation (CV) 64.8% and 66.2%, respectively. Two of these birds had SST containing spermatozoa but were not laying. At 49 days, oviductal length was 24 +/- 0.5 cm (CV 2.0%), and all birds had functional SST with spermatozoa and had started to lay. Mature SST consist of columnar, nonciliated cells, 19.8 +/- 0.7 microm high. Although development of SST in the quail, to a large extent, coincides with the development of the rest of the oviduct, the present findings suggest that utero-vaginal sperm storage is possible before the complete maturation of the oviduct and subsequent onset of lay.     

Histochemistry of the utero-vaginal junction with special reference to the sperm-host glands in the oviduct of the domestic duck.

The "utero-vaginal junction" that connects the uterus and vagina of duck oviduct differs histologically and histochemically from the adjacent zones. The region is characterised by low, somewhat longitudinally arranged mucosal folds, lined by tall columner cilliated cells with apical nuclei alternating with mucous secreting goblet cells containing basal nuclei. Within the propria of the mucosa there are numerous tubular glands--the sperm host glands--which are responsible for sperm storage after copulation. The goblet cells of the utero-vaginal junction contain an admixture of neutral, sulfated and nonsulfated acid mucopolysaccharides, while the sperm host glands are devoid of any mucopolysaccharide and secrete complex lipoidal materials and exhibit intense acid phosphatase activity. The functional significance of the secretory materials of the sperm host glands have been discussed in the light of sperm release mechanism.     

Changes in the expression of estrogen receptor mRNA in the utero-vaginal junction containing sperm storage tubules in laying hens after repeated artificial insemination

The objective was to determine whether expression of estrogen receptor (ER) mRNA in the utero-vaginal junction (UVJ) of laying hens was altered after repeated artificial insemination (AI). Semi-quantitative RT-PCR was used to determine the expression of mRNA of the two types of receptor, ERalpha and ERbeta. Only ERalpha mRNA was expressed in all segments of the oviducts of both virgin and artificially inseminated birds, whereas ERbeta mRNA was expressed in ovarian follicles but not in the oviduct. The expression of ERalpha mRNA in the UVJ was significantly decreased after repeated AI, whereas that in the uterus was not significantly different between virgin and inseminated birds. Since estrogen may be involved in maintaining the sperm storage function of sperm storage tubules, the decreased expression of ERalpha mRNA in the UVJ after repeated AI may contribute to reduced fertility in these birds.     

Antioxidant status of the lower oviduct in the chicken varies with age and dietary vitamin E supplementation

Protection of sperm membranes against lipid peroxidation is a pre-requisite to prolonged sperm storage, both in vivo and in vitro. As females from avian species can store spermatozoa in the utero-vaginal junction (UVJ) for prolonged periods, we investigated the mechanisms involved in antioxidative protection of the plasma membrane of chicken sperm in this region. Comparisons of concentrations in nonenzymatic (alpha-tocopherol, ascorbic acid, and GSH) and enzymatic (GSH-Px, SOD) antioxidants among the vagina, UVJ and uterus of sexually mature chicken hens revealed tissue-specific profiles, with higher ascorbic acid content and increased GSH-Px and SOD activity in the UVJ compared to other regions of the lower oviduct (vagina, uterus). Deterioration of the antioxidant profile in the UVJ was observed in aging hens, but it was partially compensated by dietary supplementation with vitamin E (130 ppm). It is concluded that the chicken UVJ provides a complex defense barrier against lipid peroxidation of the sperm membrane during in vivo storage, which can be partially improved by dietary supplementation with vitamin E. The protective effects of this barrier decline over time during the reproductive season.     

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.     

The oviduct as a complex mediator of mammalian sperm function and selection

The fallopian tube, or oviduct, is no longer considered merely a conduit that joins the uterine horns and the ovaries, being recognised as a venue for the capacitation of spermatozoa and fertilisation. However, recent evidence has implicated the oviduct in the stringent selection of spermatozoa prior to fertilisation, sperm storage prior to fertilisation, the regulation of sperm motility and possibly the guidance of spermatozoa towards the egg. Moreover, the arrival of spermatozoa within the oviduct is now known to regulate gene expression in oviductal epithelial cells with the consequent up- and downregulation of various proteins. In this review, we examine the emerging significance of sperm-oviduct interactions, as they relate to both physiological functions and the likelihood that the oviduct has a role in post-copulatory sperm selection by females (cryptic female choice) under conditions of sperm competition. The mechanisms by which sperm selection might operate still remain a mystery, especially when the underlying rationale for such mechanism appears to require the recognition by the female tract of sperm qualities related to the intrinsic integrity and information content of the sperm DNA. The oviduct not only selects against spermatozoa containing fragmented DNA but also imposes selection related to the fitness or quality of individual males. This implies the existence of, as yet unrecognised, mechanisms for the detection and interpretation of sperm-surface markers that link phenotypic and genotypic qualities of each individual cell.     

Turkey breeder hen infertility associated with plasma cells in the uterovaginal sperm storage glands

Uterovaginal sperm storage glands taken from fertile and infertile turkey breeder hens were analyzed morphologically using transmission electron microscopy. Sperm storage glands from the infertile hens were generally devoid of spermatozoa, while glands from the fertile hens contained many such cells. Extensive lymphocytic infiltration into the basolateral clefts between contiguous cells of the sperm glands was found in both fertile and infertile hens. Plasma cell infiltration into these intracellular clefts was also found in infertile turkeys. Plasma cells were not found, however, in the glandular clefts of fertile hens.Lymphocytes present in the sperm storage glands of fertile hens are theorized to be regulatory suppressor T-cells, which could explain the hen's immunological tolerance to continual exposure to antigenic spermatozoa. Conversely, the presence of antibody-producing plasma cells in the sperm storage glands of infertile hens could explain the absence of stored spermatozoa and the reduced fecundity of these hens.     

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.     

Female sperm storage in reptiles.

Internal fertilization and oviparity most likely are symplesiomorphies for modern reptiles, and viviparity has evolved independently numerous times in Sauria and Serpentes. Oviducal sperm storage is known in females of all taxa except Amphisbaenia. However, in Rhynchocephalia and Crocodilia, sperm storage is poorly studied, and specialized sperm storage tubules (Ssts) are unknown. We use the molecular phylogenetic hypothesis [(Chelonia+Archosauria) (Squamata)] to trace evolution of sperm storage characters. Ssts arose independently in Chelonia and Squamata. Turtles possess albumen-secreting glands in the anterior half of the oviduct (the tuba or isthmus), and the most distal of these glands also serve as Ssts; in addition, some turtles possess Ssts in the adjacent segment of the oviduct, the uterus. Squamates lack albumen-secreting glands, and the ancestral state is possession of Ssts in the posterior infundibulum (uterine tube). Secondarily, iguanids have evolved vaginal Ssts. In this paper, we present the first ultrastructural observations on vaginal Ssts in lizards, using Anolis sagrei (Polychrotidae). Proximally, the neck of these simple tubular glands continues the alternation of ciliated and secretory cells lining the lumen of the vagina. However, the epithelial cells of the distal sperm storage area are neither secretory nor ciliated. The Ssts of Anolis are more similar to those of birds more than to infundibular receptacles in snakes and lizards.     

Sperm release from the uterovaginal storage gland of the chicken: A perfusion study

In vitro and in vivo intraluminal perfusions of the uterovaginal junction of the oviduct were performed in an attempt to quantitate sperm release from the uterovaginal sperm host glands (UV-SHG) of breeder hens. Spermatozoa were present in the perfusate at all time periods examined. However, the quantity of spermatozoa recovered showed a significant (P<0.0001) decline over a 2-h perfusion period in all experiments. Furthermore, histological examination of the perfused oviduct revealed significantly lower percentages (P<0.05) of UV-SHG containing spermatozoa compared to unperfused control oviducts. The perfusion techniques used in this study seemed to influence the pattern of sperm release from the storage glands.     

Effect of gamma-radiation on fowl sperm function in vitro and in vivo

Doses of up to 300 Gy of ionizing radiation had little effect on fowl sperm morphology, ATP content and motility when measured in vitro. Fertility of eggs from hens inseminated with spermatozoa receiving 50 Gy, in terms of post-oviducal development, was less than 4%. However, 35% appeared 'fertile' by macroscopic examination of the germinal disc of unincubated eggs. These contained few embryonic cells, although the vitelline membrane contained many trapped spermatozoa. After doses of 100 Gy or more, inseminated spermatozoa were not found in the vitelline membrane and no fertile or apparently fertile eggs were produced; nor did such spermatozoa enter the utero-vaginal sperm-host glands. Genetic transformation using fowl spermatozoa irradiated with doses in excess of 100 Gy appears to be an unlikely prospect.     

Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.     

金丝雀输卵管内贮精腺体的组织学及组织化学的研究

本文对产蛋期金丝雀（Ｓｅｒｉｎｕｓｃａｎａｒｉａ）输卵管内的贮精腺体进行了组织学和组织化学观察。结果表明,贮精腺体分布于子宫－阴道交接处阴道一侧长度为０．２１±０．０７ｃｍ区域（以下简称ＵＶ区域）内的固有膜中。该腺体为管状腺,少数具有分枝,由单层柱状上皮细胞构成。腺细胞顶部无纤毛,细胞核排列整齐,位于细胞的基部。贮精腺体呈ＰＡＳ阳性,糖原及酸性粘多糖的含量极微或未能检出。脂类含量较为丰富。酸性磷酸酶活性很高,不含碱性磷酸酶。在组织学及组织化学特征上,ＵＶ区域与毗邻的子宫及阴道区域存在明显差异,贮精腺体细胞与ＵＶ区域粘膜上皮细胞之间的差异也较为显著。     

Numbers and size of sperm storage tubules and the duration of sperm storage in birds: a comparative study

Estimates of the numbers of sperm storage tubules (SSTs) in the utero-vaginal junction of 11 bird species are presented. Numbers of SSTs varied by a factor of 40 between species, and ranged from 500 to 20000. Body mass accounted for over 50% of the variation in SST mumbers. SST length was positively correlated with the length of spermatozoa across species. The duration of sperm storage was not correlated with the number of SSTs or the volume of sperm storage tissue. However, the number of 'active' SSTs appears to vary between species and it was not possible to make allowance for this. Sperm storage duration was weakly, positively correlated wth clutch size, but showed a significant positive relationship with the number of days over which laying occurred. The number of SSTs was also positively correlated with the number of sperm per ejaculate. The best predictor of sperm storage duration was a multiple regression equation using the spread of laying and the length of sperm storage tubules. The duration of sperm storage in birds which remain together during the pre-laying period is such that a single insemination immediately before the start of laying could fertilize the entire clutch.     

Immunolocalization of cubilin, megalin, apolipoprotein J, and apolipoprotein A-I in the uterus and oviduct

Spermatozoa maturation and capacitation occurring in the male and female reproductive tracts, respectively, involves the remodeling of the spermatozoa plasma membrane. Apolipoprotein J (apoJ) and apolipoprotein A-I (apoA-I) have been implicated in the process of lipid exchange from the spermatozoa plasma membrane to epithelial cells lining the male reproductive tract. Evidence suggests that this process is mediated by the cooperative action of the endocytic lipoprotein receptors megalin and cubilin, which are expressed at the apical surface of absorptive epithelia in various tissues, including the efferent ducts and epididymis. Here, we investigated the possibility that these receptors and their lipid-binding ligands, apoJ and apoA-I, might function similarly in the female reproductive tract. We show that megalin and cubilin are expressed in the uterine epithelium at all stages of the estrous cycle, maximally during estrous and metestrous stages. In the oviduct, there is pronounced expression of both megalin and cubilin in the nonciliated cells of the proximal oviduct and epithelial cells of the distal oviduct, particularly during estrous and metestrous stages. In both uterine and oviduct epithelial cells, megalin and cubilin were located on the apical regions of the cells, consistent with a distribution at the cell surface and in endosomes. ApoJ and apoA-I were both detected in apical regions of uterine and oviduct epithelial cells. Secretory cells of the uterine glands were found to express apoJ and apoA-I suggesting that the glands are a site of synthesis for both proteins. In summary, our findings indicate that megalin and cubilin function within the female reproductive tract, possibly mediating uterine and oviduct epithelial cell endocytosis of apoJ/apoA-I-lipid complexes and thus playing a role in lipid efflux from the sperm plasma membrane, a major initiator of capacitation.     

Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis

Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species.     

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.     

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-epithelium relationships in relation to the time of insemination in little brown bats (Myotis lucifugus)

Copulation lasted for up to 46 min in little brown bats. Spermatozoa were stored in both the uterus and the utero-tubal junction, although intimate relationships between spermatozoa and the epithelium were particularly evident in the utero-tubal junction, and were established at the beginning of the period of sperm storage. Polymorphonuclear leucocytes were present in all uteri irrespective of whether or not they had been inseminated but were not generally present in the utero-tubal junction or oviduct. Engulfment of spermatozoa by the epithelial cells of the utero-tubal junction and by polymorphonuclear leucocytes in the uterine glands was evident soon after copulation. It is suggested that this may effect the removal of defective spermatozoa and allow luminal spermatozoa access to the spatially restricted storage sites. Uninseminated female bats attempted to elicit copulation from torpid males, and were also observed adjacent to copulating pairs. Female bats also uttered copulation calls.