Avian Oviduct Research Articles

Application of ultrasonic diagnostics in the study of urogenital organs of birds

Ultrasound examination is one of the most accessible and having a high probability of competence of research of diagnostics of diseases of internal organs, which are carried out mainly in small pets. The aim of the study is to obtain information about parenchymatous organs in birds in norm and in case of pathologic changes for making or confirming clinical diagnosis. Material and methods. Ultrasound diagnostics of avian oviduct was carried out on ultrasonic diagnostic scanner ETS-D-05 "Raskan", ~ 220V, 50 Hz, 100 VA, class 1. The transducer is microconvex 7.5 MHz. Results. At ultrasound examination of the kidneys in the chicken, it was noted that the kidneys had smooth, clear boundaries. The echostructure of cortical and brain layers in the median plane is heterogeneous, echogenicity of parenchyma corresponds to the norm. The capsule is not visualized. The presence of lighter and darker areas in the parenchyma is clearly visible, which is associated with filtration of mineral substances contained in the blood. Ultrasound examination of the genitalia in the chicken was carried out in several stages. At the first stage, the presence of a mature follicle was noted. Localized volumetric formations of rounded shape - follicles at different stages of development were revealed. They have homogeneous echogenicity and echostructure of hyperechogenic character, giving a weak acoustic shadow. In the second stage of the study we noted the presence of yolk covered with vitelline shell, which is located in the central part of the albumen section of the oviduct, has a high density and good echogenicity. In the third stage, the yolk is located in the caudal part of the albumen section. At its periphery, the albumen with several layers is located. Protein section is displaced somewhat to the left, has strongly tortuous loops. The wall of the albumen is well structured, the expression of layers is weakly marked. In the cavity of the oviduct, the content of the oviduct is homogeneous with a small amount of content of liquid consistency. At the fourth stage of the study in the uterus of the oviduct, an anechogenic formation - an egg - is visualized on the echogram. In its center, a hypoechogenic structure with indistinct contours - yolk is detected. The contours of the egg are smooth, borders are indistinct. The egg completely fills the uterine cavity, the lumen between the egg and the uterine wall is absent, the uterine wall contours are clear and even with homogeneous echogenic structure, no nodular formations are detected, uterine vessels are not dilated.

Effect of tamoxifen treatment on catalase (CAT) and superoxide dismutase (SOD) expression and localization in the hen oviduct

The imbalance between free reactive oxygen species (ROS) generation and removal (e.g., by antioxidative enzymes) leads to the damage of important biomolecules and cells. Earlier studies in hens showed that treatment with tamoxifen (TMX; estrogen receptor inhibitor) modulates oxidative stress and causes the reproductive system regression realized by cell apoptosis. The aim of the present study was, therefore, to examine the expression and immunolocalization of the key enzymatic antioxidants, i.e. catalase (CAT) and superoxide dismutase (SOD), in the chicken oviduct following TMX treatment. Laying hens were treated daily with TMX until a pause in egg-laying occurred and then euthanized on day 8 of the experiment. Quantitative real-time PCR and western blot analyses showed the presence of CAT and SOD transcripts and proteins, respectively, in all oviductal segments, i.e., the infundibulum, magnum, isthmus, shell gland and vagina. In control hens (laying), the mRNA expression of CAT was the highest in the shell gland, lower in the isthmus and the lowest in other oviductal parts, whereas protein expression was the highest in the magnum, lower in the isthmus and the lowest in other segments. The SOD transcript and protein abundances only were lower in the magnum than in other segments. Immunoreactive CAT and SOD products were localized in all layers of the oviductal wall, but the intensity of staining depended on the cell type. TMX treatment affected CAT and SOD expression and the effect of TMX depended on gene, protein, cell type and oviductal part. Generally, CAT expression was elevated, while SOD expression was decreased under TMX treatment. These results point to the importance of CAT and SOD in the maintenance of proper oviduct health and function. Changes in ROS scavenging enzymes after estrogen receptor blockage indicate the significance of estrogen in the regulation of oxidative status in the avian oviduct.

Intravaginal injection of Lactobacillus johnsonii may modulates oviductal microbiota and mucosal barrier function of laying hens

The avian oviduct connects to the gastrointestinal tract through cloaca, where it is exposed to pathogenic bacteria from intestinal contents. Therefore, improvement of mucosal barrier function in the oviduct is important for safe poultry production. Lactic acid bacteria are known to contribute to strengthening the mucosal barrier function in the intestinal tract, and a similar effect is expected in the oviduct mucosa of chickens. This study aimed to clarify the effects of vaginal administration of lactic acid bacteria on the mucosal barrier function of the oviduct. White Leghorn laying hens (500-days old) were intravaginally administered 1 mL of Lactobacillus johnsonii suspension (1 × 105 and 1 × 108 cfu/mL: low concentration of Lactobacillus (LL) and high concentration of Lactobacillus (HL) groups, respectively) or without bacteria (control: C group) for 7 d (n = 6). The oviductal magnum, uterus, and vagina were collected for histological observations and mucosal barrier function-related gene expression analysis. Amplicon sequence analysis of oviductal mucus bacteria was also performed. Eggs were collected during the experimental period and their weight was measured. Vaginally administering L. johnsonii for 7 d caused 1) an increase in α-diversity of vaginal mucosa microbiota with an increase in the abundance ratio of beneficial bacteria and a decrease in pathogenic bacteria, 2) enhanced claudin (CLA) 1 and 3 gene expression in the magnum and vaginal mucosa, and 3) a decrease in avian β-defensin (AvBD) 10, 11, and 12 gene expression in the magnum, uterus, and vaginal mucosa. These results suggest that transvaginal administration of L. johnsonii contributes to protection against infection in the oviduct by improving the microflora of the oviductal mucosa and strengthening the mechanical barrier function of the tight junctions. In contrast, transvaginal administration of lactic acid bacteria does not enhance the production of AvBD10, 11, and 12 in the oviduct.

Short Communication: Polymorphism of collagen type X (COLX) gene and their association with egg production traits and egg weight in Alabio and Mojosari ducks

Abstract. Kumalawati DS, Maharani D, Sari APZNL, Pratomo GH, Sasongko H, Fathoni A, Susanti T. 2023. Short Communication: Polymorphism of collagen type X (COLX) gene and their association with egg production traits and egg weight in Alabio and Mojosari ducks. Biodiversitas 24: 1518-1523. Type X collagen (COLX) is non-fibrillar collagen expressed in the white isthmus of the avian oviduct and plays a role in egg formation. This study aimed to identify COLX gene polymorphism and its association with egg production and egg weight in Alabio (Anas platyrhynchos Borneo) and Mojosari ducks (Anas platyrhynchos Javanicus). Blood samples of 100 female ducks were collected, and their deoxyribonucleic acid (DNA) was extracted. In addition, a 220 bp fragment of COLX gene was amplified using the polymerase chain reaction (PCR) method and sequenced for single nucleotide polymorphism (SNP) identification and genotyping. Data analysis was performed to calculate genotype and allele frequencies, and an independent sample t-test was used to examine the association between genotype and egg production and egg weight of each individual. Three genotypes (CC, TT, and CT) of SNP g.74C/T were detected in both breeds. Allele C (0.77) was dominant in Alabio population; otherwise, allele T (0.81) dominated Mojosari population. The frequency of CC (0.59) was highest in Alabio ducks, followed by CT (0.37) and TT (0.04), while TT (0.65) was highest in Mojosari ducks, followed by CT (0.33) and CC (0.02). Meanwhile, association analysis showed that ducks with CC genotype had significantly higher egg production at 28 and 34 weeks in Alabio than those with CT genotype (p<0.05). These results indicate that COLX is an effective genetic marker for a breeding program to increase egg production in Alabio ducks.

Sperm Gatekeeping: 3D Imaging Reveals a Constricted Entrance to Zebra Finch Sperm Storage Tubules

Females across many internally fertilizing taxa store sperm, often in specialized storage organs in their reproductive tracts. In birds, several hundred sperm storage tubules exist in the utero-vaginal junction of the oviduct, and there is growing evidence that sperm storage in these tubules is selective. The mechanisms underlying female sperm storage in birds remain unknown because of our limited ability to make three-dimensional, live observations inside the large, muscular avian oviduct. Here, we describe a new application of fluorescence selective plane illumination microscopy to optically section oviduct tissue from zebra finch Taeniopygia guttata females label free by harnessing tissue autofluorescence. Our data provide the first description of the three-dimensional structure of sperm storage organs in any vertebrate to the best of our knowledge and reveal the presence of gate-like constricted openings that may play a role in sperm selection.

Changes in proliferating and apoptotic markers in the oviductal magnum of chickens during sexual maturation

The avian oviduct is characterized by dynamic hormonal, biochemical, and cellular changes during its development. To better understand the molecular mechanisms regulating proper development of this organ in birds, the rate of cell proliferation and apoptosis as well as these processes-related gene expressions in the chicken oviduct during the sexual maturation were examined. The oviducts were isolated from Hy-Line Brown chickens at 2-week intervals from 10 to 16 weeks of age, and at 17 weeks, i.e. just after the onset of egg laying. In the tissue from the middle part of the oviduct (the magnum) the following parameters were tested: (1) proliferating (proliferating cell nuclear antigen [PCNA]-positive) and apoptotic (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive) cells, (2) mRNA expression of bcl-2, caspases 2, 3, 8, and 9, PCNA, survivin-142, and ovalbumin by quantitative real-time polymerase chain reaction, (3) protein expression of Bcl-2, PCNA, and caspases 3 and 9 by Western blot, (4) activity of caspases 2, 3, 8, and 9 by fluorometric method, and (5) localization of Bcl-2 and caspases by immunohistochemistry. It was found that the number of proliferating cells per unit area did not change during the examined period. The number of apoptotic cells in the oviductal wall remained on the same level until 14 weeks of age followed by a gradual decrease, reaching the lowest number at 17 weeks. The mRNA expression of all caspases and Bcl-2 gradually decreased during maturation, and PCNA decreased after 14 weeks of age. Survivin-142 mRNA level increased in 14-week-old chickens and then diminished, whereas ovalbumin expression was dramatically elevated in birds 16 weeks old and older. Patterns of protein expression of Bcl-2, PCNA, and caspases and activity of caspases were similar to mRNA, although not as pronounced. In the wall of the magnum the apoptotic cells and examined proteins were localized predominantly in the mucosa (surface epithelium and tubular glands). In summary, the results obtained provide some evidence of changes in selected proliferation- and apoptosis-related gene expression, alterations in activity of multiple apoptotic markers, and differences in the frequency of proliferating and apoptotic markers between mucosa and stroma in the oviductal magnum during the sexual maturation. Concluding, we suggest that Bcl-2, PCNA, survivin-142, and some caspases may cooperatively orchestrate a cascade of events mainly related to the cell proliferation, apoptosis, and differentiation in the chicken oviduct over the course of its development.

Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during maturation.

Although participation of matrix metalloproteinases (MMPs) in reproductive tract remodeling has been strongly suggested in mammalian species, the role of MMPs in the avian oviduct has received little attention. To gain a better understanding of the potential role of the MMP system in avian oviduct development, mRNA and protein expression, localization of selected MMPs and their tissue inhibitors (TIMPs), and gelatinolytic activity in the oviduct of growing chickens were examined. The oviducts were collected from Hy-Line Brown hens before (10, 12, 14 and 16 weeks of age) and after (week 17) the onset of egg laying. The MMP-2, -7, -9 and TIMP-2 and -3 genes were found to be differentially expressed in all examined oviductal sections: the infundibulum, magnum, isthmus and shell gland on both mRNA (by real time polymerase chain reaction) and protein (by western blotting and immunohistochemistry) levels. In the course of oviduct development, the relative expression of all genes decreased in most sections. Protein level of MMP-9 was diminished, while MMP-7 and TIMP-3 were elevated in the oviduct of growing birds. MMP-2 and TIMP-2 protein levels remained constant, with a slight increase in MMP-2 concentration just before reaching maturity. The relative activity of MMP-2 and -9 (assessed by gelatin zymography) was higher (P < 0.05, P < 0.01) in immature birds compared with adults. Immunohistochemistry demonstrated cell- and tissue-specific localization of MMPs and TIMPs in the wall of the chicken oviduct. We concluded that changes in the expression of examined MMPs and their inhibitors, as well as alterations in MMP activity occurring simultaneously with changes in the morphology of the chicken oviduct, suggest the involvement of the MMP system in the proper development and functioning of this organ. Mechanisms regulating the expression and activity of MMPs require further clarification.

Ovodefensins, an Oviduct-Specific Antimicrobial Gene Family, Have Evolved in Birds and Reptiles to Protect the Egg by Both Sequence and Intra-Six-Cysteine Sequence Motif Spacing.

Ovodefensins are a novel beta defensin-related family of antimicrobial peptides containing conserved glycine and six cysteine residues. Originally thought to be restricted to the albumen-producing region of the avian oviduct, expression was found in chicken, turkey, duck, and zebra finch in large quantities in many parts of the oviduct, but this varied between species and between gene forms in the same species. Using new search strategies, the ovodefensin family now has 35 members, including reptiles, but no representatives outside birds and reptiles have been found. Analysis of their evolution shows that ovodefensins divide into six groups based on the intra-cysteine amino acid spacing, representing a unique mechanism alongside traditional evolution of sequence. The groups have been used to base a nomenclature for the family. Antimicrobial activity for three ovodefensins from chicken and duck was confirmed against Escherichia coli and a pathogenic E. coli strain as well as a Gram-positive organism, Staphylococcus aureus, for the first time. However, activity varied greatly between peptides, with Gallus gallus OvoDA1 being the most potent, suggesting a link with the different structures. Expression of Gallus gallus OvoDA1 (gallin) in the oviduct was increased by estrogen and progesterone and in the reproductive state. Overall, the results support the hypothesis that ovodefensins evolved to protect the egg, but they are not necessarily restricted to the egg white. Therefore, divergent motif structure and sequence present an interesting area of research for antimicrobial peptide design and understanding protection of the cleidoic egg.

Prevalence and pathology of oviduct impaction in commercial white leghorn layer chicken in Namakkal region of India

Aim : The avian oviduct is a tubular organ responsible for fertilization, secretion of the components surrounding the yolk and transport of egg in the reproductive tract. Disorders of oviduct may have a great bearing on production potential and incur a heavy loss. A study was undertaken to assess the prevalence and pathological changes of impacted oviduct in commercial white leghorn layer chicken in Namakkal region of India for a period of four years from 2006 to 2009. : A total of 5145 carcasses of white leghorn layers, above 20 weeks age from 255 flocks were examined for various oviduct abnormalities. Heart blood, liver and oviduct swabs collected upon necropsy from 45 layer chicken from six flocks with oviduct impaction were screened for bacterial agents. Pooled tissue (trachea, lung, spleen, caecal tonsil, kidney and oviduct) samples from impacted oviduct cases were screened for viral agents. Serum samples collected from affected flocks were screened for Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and egg drop syndrome - 76 (EDS-76) virus by haemagglutination inhibition (HI) and (Mg) and (Ms) by ELISA. Flock details and pathological changes were recorded in affected flocks to assess the prevalence and impact of oviduct impaction on commercial layer chicken. : The results of the present investigation indicated that the oviduct impaction was responsible for 0.87% of the reproductive tract abnormalities in commercial layers between 21 and 80 wk of age. Egg production drop, morbidity and mortality recorded in the affected flocks were varied from 3 to 8, 0.4 to 1.2 and 0.2 to 0.5 % respectively. The oviduct impaction was commonly noticed above 40 wk old layers and predominantly during colder months. Serum samples collected from three flocks with oviduct impaction were found positive for Mg and Ms infection in ELISA test. was isolated as a pure culture in 29 birds and concurrent with other bacterial agents in 16 birds. Serotyping of isolates revealed that O , O and O were the predominant types. Necropsy examination of carcasses with impacted oviduct showed emaciation, peritonitis, regression of ovarian follicles and distension of oviduct commonly in the infundibulum and magnum region. The exudate was either large mass with concentric layering on cut section or numerous caesous masses with varying size and shape often mixed with creamy or serosangunious fluid. Histopathological examination of oviduct revealed the presence of lymphoid foci in the epithelial layer with marked atrophy and degeneration of mucosal folds. : Impacted oviduct constituted 0.87 % of oviduct abnormality in commercial layer chicken with an overall mortality of 0.5 %. The findings of this study showed that the oviduct impaction might be caused by in concurrence with spp.

The expression of p63 and Ck HMW in magnum and infundibulum of Gallus domesticus oviduct.

The potential for proliferation and differentiation has a critical meaning in terms of the long-term in vitro culture of oviductal target cells. Therefore, it is important to characterize the oviduct epithelial cells, using approved markers. There is scarce data describing the epithelial cells lining the avian oviduct, most of it referring only to the magnum section of the oviduct. This study presents a comprehensive analysis of both magnum and infundibulum tissues, as the most preferred sources of epithelial cells for research on production of recombinant proteins in oviducts of birds. The main objective was to evaluate the expression of p63 and high molecular weight cytokeratins (anti- p63 antibody and anti- High Molecular Weight Cytokeratins) in epithelial cells (EC) of 2 oviduct sections: magnum (proximal and middle) and infundibulum (distal). IHC analysis and western blotting were performed using the mouse monoclonal anti- p63 antibody and anti-Ck HMW. Immunoreactivity was quantified based on the Remmele - Stegner scoring system (0-12). The expression of p63 in nuclei of luminal cells was significantly higher in the infundibulum (P < 0.05), compared to the magnum section. Cytokeratins were also highly expressed in the infundibulum, but the difference was non-significant. These findings reveal new characteristics of the oviduct EC and designate the location of the source of cells in the oviduct tissue for in vitro culture.

Avian SERPINB12 expression in the avian oviduct is regulated by estrogen and up-regulated in epithelial cell-derived ovarian carcinomas of laying hens.

Serine protease inhibitors (SERPINs) are involved in a variety of biological processes such as blood clotting, angiogenesis, immune system, and embryogenesis. Although, of these, SERPINB12 is identified as the latest member of clade B in humans, little is known of it in chickens. Thus, in this study, we investigated SERPINB12 expression profiles in various tissues of chickens and focused on effects of steroid hormone regulation of its expression. In the chicken oviduct, SERPINB12 mRNA and protein are abundant in the luminal (LE) and glandular (GE) epithelia of the magnum in response to endogenous or exogenous estrogen. Furthermore, SERPINB12 mRNA and protein increase significantly in GE of cancerous ovaries of laying hens with epithelia-derived ovarian cancer. Collectively, these results indicate that SERPINB12 is a novel estrogen-stimulated gene that is up-regulated by estrogen in epithelial cells of the chicken oviduct and that it is a potential biomarker for early detection of ovarian carcinomas in laying hens and women.

Hormonal regulation of beta-catenin during development of the avian oviduct and its expression in epithelial cell-derived ovarian carcinogenesis

Beta-catenin (CTNNB1) is a dual function molecule that acts as a key component of the cadherin complex and WNT signaling pathway. It has a crucial role in embryogenesis, tumorigenesis, angiogenesis and progression of metastasis. Recently, it has been suggested that the CTNNB1 complex is a major regulator of development of the mouse oviduct and uterus. However, little is known about the CTNNB1 gene in chickens. Therefore, in this study, we focused on the CTNNB1 gene in the chicken reproductive tract and hormonal control of its expression in the chicken oviduct. CTNNB1 was localized specifically to the luminal and glandular epithelium of the four segments of chicken oviduct and DES (diethylstilbestrol, a synthetic non-steroidal estrogen) increased its expression primarily in LE of the magnum. In addition, CTNNB1 mRNA and protein were expressed abundantly in glandular epithelium of endometrioid-type ovarian carcinoma, but not in normal ovaries. Moreover, CTNNB1 expression was post-transcriptionally regulated via its 3′-UTR by binding with target miRNAs including miR-217, miR-1467, miR-1623 and miR-1697. Collectively, these results indicate that CTNNB1 is a novel gene regulated by estrogen in epithelial cells of the chicken oviduct and that it is also abundantly expressed in epithelial cells of endometrioid-type ovarian carcinoma suggesting that it could be used as a marker for diagnosis of ovarian cancer in laying hens and women.

In vitro Effects of TCDD, PCB126 and PCB153 on Estrogen Receptors, Caspases and Metalloproteinase-2 mRNA Expression in the Chicken Shell Gland

Among the environmental chemicals which disturb endocrine functions, dioxins and polychlorinated biphenyls (PCBs) are known as the most toxic. Numerous studies in mammals revealed that dioxins and PCBs disrupt functions of the uterus, delay implantation and increase embryo loss. The direct effect of these chemicals on the avian oviduct is not known. Therefore, in the study chicken shell gland tissues were used to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar PCB126 and non-coplanar PCB153 on estrogen receptors (ERs), initiator caspase-1, executioner caspase-3 and metalloproteinase-2 (MMP-2) mRNA expression. Fragments of shell gland tissue isolated from the laying chicken were incubated for 24h with TCDD (100nM), PCB126 (100nM) or PCB153 (100 microM). Quantitative PCR analysis showed that: (1) TCDD increased ER beta (ERbeta) mRNA expression, (2) PCB126 increased ER alpha (ERalpha), ERbeta and caspase-1, and decreased MMP-2 mRNA expression, (3) PCB153 elevated the ERbeta and caspase-1 expression levels and (4) expression of caspase-3 was not altered by any investigated xenobiotics. The results obtained using the shell gland explants model indicate that dioxins and PCBs have a direct effect on the chicken oviduct, especially the shell gland, by affecting the expression of genes involved in the function of this oviductal segment. It is suggested that coplanar PCBs such as PCB126, by changing cellular and extracellular regulators gene expression, may lead to disruption of shell gland activity and impair egg components formed in this organ.

AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor-binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3′-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway.

Ultrastructural identification of interstitial cells of Cajal in hen oviduct

The interstitial cells of Cajal (ICC) are widely believed to be neuroeffector cells of smooth muscle activity in all tubular organs, including the oviduct. The avian oviduct involves the secretion, sheathing, and transportation of a large-sized egg, but there is no information available on ICC in this special organ. We have demonstrated the presence of ICC in different segments throughout the oviduct in the laying hen and provided details on their ultrastructure by transmission electron microscopy technique, for the first time. The observed ICC appeared bipolar and multipolar cells of different shapes, with varying nuclear morphologies, a thin rim of electron-dense cytoplasm, and an infrequent basal lamina. They showed moniliform primary processes with one or 2 secondary or terminal processes. We found ICC near smooth muscle cells, nerve fibers, and the epithelia, where they make specialized contacts in the form of close membrane associations or gap-like junctions and peg-and-socket-like junctions. Intricate labyrinthine-type networking contacts were also present in ICC processes. Moreover, we report for the first time, that ICC in avian oviduct make interdigitating contacts with the epithelium. Cytoplasmic organelles identified in ICC include numerous well-developed mitochondria, abundant rough endoplasmic reticulum, and dispersed intermediate filaments. Many caveolae and vesicles were also present. Golgi bodies and centrioles were rare. Fibroblasts, on the other hand, were distinct cells with larger cytoplasmic area, more rough endoplasmic reticulum, and less mitochondrial content. No basal lamina, intermediate filaments, or caveolae were present in fibroblasts. Their processes were shorter and showed no contacts with smooth muscle cells or nerves. We conclude that these ICC might also have a key role in the regulatory mechanisms of motility and transportation in the hen oviduct, as already proved in mammalian oviduct. Such role of ICC might also be responsible for the function of the muscular infundibulum, where the fertilization takes place, and that moves to surround the released ovum, failure of which results in the internal laying.

Mechanisms of Sperm Storage in the Female Reproductive Tract: an Interspecies Comparison

Once semen has been collected for artificial insemination, it is diluted into extenders designed to prevent its deterioration over the period prior to insemination. If the semen is not frozen, the extenders provide protection for a period of a few hours to a few days, depending on species. Despite the efforts of biotechnologists to increase the duration of storage without compromising fertility, there has been relatively little progress for many years. However, comparative studies in diverse species have revealed that long-term sperm storage (up to months and years) within the female reproductive tract is relatively commonplace in reptiles, fishes, birds and amphibians. Even among mammals, some species of bat have evolved mechanisms for storing spermatozoa for several months in the uterus or oviduct so that they can mate in the autumn but postpone fertilization until the spring. We currently know little about the mechanisms that support such long-term sperm storage, mainly because evidence from such species is either absent or fragmentary. Nevertheless, parallels between mammalian and other systems, where spermatozoa are sequestered in sperm storage tubules, suggest that the enclosure of spermatozoa within pockets of epithelial cells may be sufficient to achieve long-term sperm storage. In addition, recent evidence from sperm-storing bats has suggested an alternative, or additional, hypothesis that the modulation of apoptosis within epithelial cells is important in controlling sperm survival. Despite a lack of direct experimental evidence from a wide variety of species, I propose that there is now enough evidence to warrant investigation of these hypotheses.

A novel disulfide-rich protein motif from avian eggshell membranes.

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X4-C-X5-C-X8-C-X6 pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

New hypotheses on the function of the avian shell gland derived from microarray analysis comparing tissue from juvenile and sexually mature hens

Activation of the shell gland region of the avian oviduct is mediated by ovarian steroids. To understand more extensively how shell glands are maintained and function, we have compared gene expression in the shell glands from juvenile and laying hens using a chicken cDNA microarray. Average expression profiles of juvenile and sexually mature shell glands were compared resulting in the identification of 266 differentially regulated genes. Reverse transcription quantitative polymerase chain reaction confirmed expression differences. The differentially expressed genes included several with known involvement in shell gland function, including ion transport and shell matrix proteins. There were also many unpredicted differentially expressed genes, and for some we propose hypotheses for their functions. These include those encoding (a) osteoprotegerin, a decoy death receptor for receptor activator of nuclear factor NFkB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), that in the shell gland, may prevent apoptosis and/or may have an endocrine effect by preventing RANKL’s action on bone osteoclasts that mobilize stored calcium; (b) prostatic acid phosphatase (ACPP) and prostate stem cell antigen (PSCA) that could play a role in sperm physiology within the shell gland; (c) urea transporter (SLC14A2) that could provide a novel anti-microbial defence; (d) bactericidal/permeability-increasing protein-like 2 (BPIL2), and other potential anti-microbials that have not previously been documented in the chicken. These new hypotheses, if borne out experimentally, will lead to a greater understanding of shell gland function including the processes involved in eggshell formation and anti-microbial activity.

REVIEW ARTICLE: Mechanism of Prolonged Sperm Storage and Sperm Survivability in Hen Oviduct: A Review

A unique property of the avian oviduct is to store sperm for a prolonged period. The sperm storage tubules (SST) are located in the utero‐vaginal junction of the oviduct, where sperm can be stored and survived for a few weeks after insemination or natural mating. The immune system in the oviduct is essential to prevent tissue infection by various microorganisms, and it may also affect the fate and survivability of sperm in the oviduct. Anti‐sperm immunoresponses including infiltration of leukocytes may be induced in the vagina of the oviduct. Sperm that will participate in fertilization may be selected by these immunoresponses. However, sperm stored in the SST may be protected from the immunoresponse by SST structures and transforming growth factor β, whose expression is increased during sperm storage in the SST. In this review, the mechanism of sperm survivability with reference to the regulation of anti‐sperm immunoresponses in hen oviduct is emphasized.

Effect of estrogen and its antagonist on the expression of arginine vasotocin (AVT) and its oxytocic-like receptor VT3 in the shell gland of Japanese quail, Coturnix coturnix japonica

Avian neurohypophysial hormone arginine vasotocin (AVT) is known to regulate shell gland contractility during oviposition. While studying the role of estrogen in the expression and regulation of AVT and its oxytocic-like receptor VT3, using in situ hybridization and immunohistochemistry, it was observed that the expression of AVT and its receptor was not detected in the shell gland of sexually immature Japanese quail. However, administration of estrogen to these birds not only stimulates the growth and activity (as assessed by increased mucosal fold length, total protein content and alkaline phosphatase level) of the shell gland but also upregulates the expression of AVT and VT3. Further, administration of estrogen antagonist tamoxifen to sexually mature bird shows opposite results. On the other hand, localization of ir-AVT, observed in the ovary of sexually mature bird, was not detected in the estrogen treated sexually immature quail. It is concluded that estrogen not only affects the growth and differentiation of avian oviduct, but also regulates the expression of shell gland AVT and its receptor VT3. Present findings suggest that the locally synthesized AVT acts in a paracrine way to upregulate VT3 receptor and thus facilitates the endocrine function of neurohypophysial AVT during oviposition.