Mammalian Reproductive Tract Research Articles

An alternative cell cycle coordinates multiciliated cell differentiation.

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one1. Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis2. Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium3,4. Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation5,6. Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which areregulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrantacquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation.

New insights into lumicrine secreted signalling that regulates the function of the mammalian male reproductive tract

New insights into lumicrine secreted signalling that regulates the function of the mammalian male reproductive tract

Spontaneous phase coordination and fluid pumping in model ciliary carpets

Ciliated tissues, such as in the mammalian lungs, brains, and reproductive tracts, are specialized to pump fluid. They generate flows by the collective activity of hundreds of thousands of individual cilia that beat in a striking metachronal wave pattern. Despite progress in analyzing cilia coordination, a general theory that links coordination and fluid pumping in the limit of large arrays of cilia remains lacking. Here, we conduct in silico experiments with thousands of hydrodynamically interacting cilia, and we develop a continuum theory in the limit of infinitely many independently beating cilia by combining tools from active matter and classical Stokes flow. We find, in both simulations and theory, that isotropic and synchronized ciliary states are unstable. Traveling waves emerge regardless of initial conditions, but the characteristics of the wave and net flows depend on cilia and tissue properties. That is, metachronal phase coordination is a stable global attractor in large ciliary carpets, even under finite perturbations to cilia and tissue properties. These results support the notion that functional specificity of ciliated tissues is interlaced with the tissue architecture and cilia beat kinematics and open up the prospect of establishing structure to function maps from cilium-level beat to tissue-level coordination and fluid pumping.

EFFECTS OF TRIBUTYLTIN EXPOSURE IN THE MALE MAMMAL’S REPRODUCTIVE SYSTEM: A SYSTEMATIC REVIEW

Objective: To conduct a systematic review of the literature describing male mammals' reproductive and sexual parameters exposed to the biocide TBT. Method: A formal computer-assisted search was performed independently by two authors using five online databases and keywords. A manual search of the reference list of the articles found for relevant original articles was also used. We initially identified potentially eligible publications related to the topic of interest through this procedure. The last systematic search resulted in adequate data on TBT toxicity in the mammalian male reproductive tract. Results: Decreases in testicular, epididymal, prostate and seminal vesicle weights were observed at higher TBT ranges. Decreases in serum testosterone levels were reported in some studies, with some histological changes in the surveyed tissues and decreased transcriptional expressions of steroidogenic enzymes. Notably, there were significant reductions in sperm count and motility and increased abnormalities. Conclusions: Further studies are needed to elucidate the precise manner of TBT's deleterious mechanisms of action on the spermatogenesis process. Therefore, a comprehensive survey of TBT levels in food and water sources should also be conducted better to protect susceptible populations from potentially deleterious reproduction effects.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

Extracellular vesicles in the male reproductive tract of the softshell turtle.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles that originate from the endosomal system or are shed from the plasma membrane respectively. As mediators of cell communication, EVs are present in biological fluids and are involved in many physiological and pathological processes. The role of EVs has been extensively investigated in the mammalian male reproductive tract, but the characteristics and identification of EVs in reptiles are still largely unknown. In this review we focus our attention on EVs and their distribution in the male reproductive tract of the Chinese softshell turtle Pelodiscus sinensis , mainly discussing the potential roles of EVs in intercellular communication during different phases of the reproductive process. In softshell turtles, Sertoli-germ cell communication via multivesicular bodies can serve as a source of EVs during spermatogenesis, and these EVs interact with epithelia of the ductuli efferentes and the principal cells of the epididymal epithelium. These EVs are involved in sperm maturation, transport and storage. EVs are also shed by telocytes, which contact and exchange information with other, as well as distant interstitial cells. Overall, EVs play an indispensable role in the normal reproductive function of P. sinensis and can be used as an excellent biomarker for understanding male fertility.

A Study of Histochemical Characteristics of Mucosubstances in Normal Endocervical Glands

Background: The apical epithelial surfaces of mammalian respiratory, gastrointestinal, and reproductive tracts are coated by mucus, a mixture of water, ions, glycoproteins, proteins, and lipids. Mucins are mainly of two types- Neutral and Acidic. Mucus plays an important role in reproductive function and defence of the female reproductive tract. Objectives: To know the mucin distribution in normal endocervical glands. Methodology: This is a retrospective, observational , analytical, case control study aimed to evaluate mucin histochemical pattern in normal endocervical glands. Ten histologically proven blocks of normal endocervical glands were taken. Tissue sections were stained by, PAS, PAS-diastase, Phenylhydrazine-PAS, Alcian blue pH 2.5, Alcian Blue pH 1, combined Alcian blue-PAS, Aldehyde fuschin and combined Aldehyde fuchsin Alcian blue techniques. Results: Results were tabulated according to colour intensity into different grades ranging from + to ++++. Regarding mucin histochemistry of normal endocervical glands; the epithelium and glands show mixture of both neutral and acidic mucins. Acidic mucins are more in amount than neutral. In acidic mucins, sialomucins are predominantly seen. Conclusion: Mucin histochemical patterns serve as valuable, cost-effective tool for diagnosis in histopathology and for the researchers in histology, where a slight change in the mucin pattern may help in the early diagnosis of the disease process.

Self-organization and multi-line transport of human spermatozoa in rectangular microchannels due to cell-cell interactions

The journey of sperm navigation towards ovum is one of the most important questions in mammalian fertilisation and reproduction. However, we know very little about spermatozoa propagation in a complex fluidic, chemical and topographic environment of a fertility tract. Using microfluidics techniques, we investigate the influence of cell-cell interactions on spermatozoa swimming behavior in constrained environment at different concentrations. Our study shows that at high enough cell concentration the interaction between boundary-following cells leads to formation of areas with preferential direction of cell swimming. In the microchannel of a rectangular cross-section, this leads to formation of a “four-lane” swimming pattern with the asymmetry of the cell distribution of up to 40%. We propose that this is caused by the combination of cell-cell collisions in the corners of the microchannel and the existence of morphologically different spermatozoa: slightly asymmetric cells with trajectories curved left and the symmetric ones, with trajectories curved right. Our findings suggest that cell-cell interactions in highly folded environment of mammalian reproductive tract are important for spermatozoa swimming behavior and play role in selection of highly motile cells.

Brain-derived neurotrophic factor (BDNF) expression and function in the mammalian reproductive Tract.

Neurotrophins of the nerve growth factor family are soluble polypeptides that are best known for their role in nerve growth, survival and differentiation in the central nervous system. A growing body of literature shows that neurotrophins and their receptors are also expressed throughout the reproductive tract. Neurotrophins are key regulatory proteins in reproductive physiology during development and throughout adult life. Of the neurotrophins, the literature describing the expression and function of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, neurotrophin receptor kinase-2 (NTRK2), has been expanding rapidly. We therefore conducted a systematic inductive qualitative review of the literature to better define the role of the BDNF in the reproductive tract. We postulate that BDNF and NTRK2 are central regulatory proteins throughout the reproductive system. An electronic search of Medline (PubMed) and Web of Science for articles relating to BDNF and the reproductive system was carried out between January 2018 and February 2019. In the ovary, BDNF expression and levels have been linked with follicle organisation during ovarian development, follicle recruitment and growth and oocyte maturation. In the endometrium, BDNF is involved in cell proliferation and neurogenesis. In contrast, literature describing the role of BDNF in other reproductive tissues is sparse and BDNF-NTRK2 signalling in the male reproductive tract has been largely overlooked. Whilst estradiol appears to be the primary regulator of BDNF expression, we also identified reports describing binding sites for glucocorticoid and myocyte enhancer factor-2, a calcium-response element through activation of an N-methyl-D-aspartate (NMDA) receptor, and aryl hydrocarbon receptor nuclear transporter protein-4 (ARNT) response elements in promoter regions of the BDNF gene. Expression is also regulated by multiple microRNAs and post-translational processing of precursor proteins and intracellular shuttling. BDNF-NTRK2 signalling is modulated through tissue specific receptor expression of either the full-length or truncated NTRK2 receptor; however, the functional importance remains to be elucidated. Dysregulation of BDNF expression and circulating concentrations have been implicated in several reproductive disorders including premature ovarian failure, endometriosis, pre-eclampsia, intra-uterine growth restriction (IUGR) and several reproductive cancers. We conclude that BDNF and its receptors are key regulatory proteins central to gonadal development, ovarian regulation and uterine physiology, as well as embryo and placenta development. Furthermore, dysregulation of BDNF-NTRK2 in reproductive diseases suggests their potential role as candidate clinical markers of disease and potential therapeutic targets.

Bone Marrow-Derived Cells Trafficking to the Oviduct: Effect of Ischemia-Reperfusion Injury.

The oviduct/fallopian tube is a crucial organ in the mammalian reproductive tract; it plays a critical role in gamete transportation and early embryo development. In women, torsion of the fallopian tubes can cause ischemia and reperfusion (IR) injury. In this study, we tested the effect of this injury on recruitment of bone marrow-derived cells (BMDCs) to the oviducts of reproductive age female mice. Bone marrow-derived cells were collected from ubiquitin-green fluorescent protein-positive male mice and transplanted into wild-type female mice. Ischemia and reperfusion injury was performed in half of the mice, while controls received equivalent surgery without oviduct injury. Two weeks following injury, recruitment of BMDCs to the oviducts was analyzed in both groups. Ischemia and reperfusion injury caused a greater than 2-fold increase in BMDC recruitment to the injured oviducts compared to those without injury. Specifically, the recruitment of BMDCs was localized to the stroma of the oviduct. We demonstrate that IR injury to oviduct recruits BMDCs to this tissue and suggest that BMDCs have function in the healing process.

LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization.

Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract.

Identification of CRISP2 from human sperm as PSP94-binding protein and generation of CRISP2-specific anti-peptide antibodies.

Cysteine-rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94-CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti-CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti-CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Effect of Recombinant Bovine Somatotropin on Libido, Pre and Post Freezing Semen Parameters in Cholistani Bulls

Growth hormone (GH) is secreted by the anterior pituitary gland and presence of growth hormone receptors has been reported in the mammalian reproductive tract. Growth hormone through these receptors has direct and indirect action on the mammalian reproductive system and affects the semen parameters. This study was aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on libido, pre and post freezing semen parameters of Cholistani bulls. For this experiment six Cholistani bulls were selected from Semen Production Unit, karaniwala Bahawalpur. The animals were randomly divided into two groups, a treatment (n = 3) group and b control (n = 3) group. The experimental group was given 500 mg s/c of rbST (Boostin) weekly from 1 nd week up to 13 th week. Libido of each bull was assessed in terms of reaction time. The semen samples were collected throughout the experiment twice a week and were evaluated for volume, sperm motility, viability, functional membrane integrity and concentration. The mean values of Cholistani bulls for libido (sec), semen volume (ml), mass activity (1-5) and sperm concentration (million/ml), were (50.1±10.8 vs 45.12 ± 9.5), (4.83±0.13 vs 3.83±0.1), (2.83 ± 0.03 vs 2.29 ± 0.02) and (1138.1±27.7 vs 920.8±21.2) in treated and control groups respectively. Pre-freezing % sperm motility, post-freezing % sperm motility, pre-freezing % live spermatozoa, post-freezing % live spermatozoa, pre-freezing functional membrane integrity and post-freezing functional membrane integrity were (70.97 ± 1.07 vs 67.15 ± 1.46), (49.82 ±1.08 vs 45.62±1.36) (77.35 ± 0.42 vs 72.07 ± 0.37), (62.94 ± 0.41 vs 54.06 ± 0.45) (76.06 ± 0.33 vs 69.85 ± 0.43) and (59.05 ± 0.48 vs 55.27 ± 0.44) in treated and control groups respectively. All semen parameters of Cholistani bulls affected by rbST treatment significantly.

Structural and functional analysis of BB0689 from Borrelia burgdorferi, a member of the bacterial CAP superfamily.

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Sperm selection in the female mammalian reproductive tract. Focus on the oviduct: Hypotheses, mechanisms, and new opportunities

Research over the past 3 decades has caused a major shift in the way that the oviduct, or fallopian tube, is perceived. Previously, it was regarded as little more than the anatomic site for fertilization, where spermatozoa and oocytes meet as they travel in opposite directions. However, this view has been radically altered by the realization that both spermatozoa and oocytes elicit changes in the biochemical composition of oviductal fluid through the induction of novel gene expression. Moreover, it has also been shown that only a privileged sperm population, selected on the basis of multiple criteria, is permitted to enter the oviduct, where they are subjected to even more selection processes that control their motility and capacitation status, thus either inhibiting or facilitating their progress toward the oocyte. Even more recently, it has become apparent that the oviduct has some ability to differentiate the genetic signatures of X- and Y-bearing spermatozoa. Although how exactly this is achieved is unknown, it prompts us to speculate that the oviduct may also be capable of distinguishing other genetically encoded properties of individual spermatozoa and that there must ultimately be a huge payoff in terms of selective animal breeding.

Effects of T-2 mycotoxin on in vitro development and chromatin status of mouse embryos in preimplantation stages.

T-2 toxin is a mycotoxin produced by phytopathogenic fungi of the Fusarium genus and has many well-studied deleterious effects on mammalian cells and reproductive tract. Despite the wide scale studies, the effects on preimplantation stage embryos are lacking. The aim of our study was to investigate the impact of T-2 on the cleavage stage of mouse embryos with regard to development to blastocysts and nuclear chromatin status.Six-weeks-old BDF1 female mice were superovulated and placed together overnight with mature males. Zygotes were flushed 20 h after human chorionic gonadotropin injection and divided randomly into treated (supplemented with 0.5, 0.75, and 1 ng/ml T-2) and nontreated (control) groups. Embryos were cultured in vitro for 96 h. Developmental stage was evaluated in the 72(nd)- and 96(th)-h for assessment of development dynamics. At the end of culture period, blastocysts from treated and control groups with normal morphology were selected for nuclear chromatin analysis. Blastocysts were categorized (grade A, B, and C) depending on the proportion of blasomeres with micronuclei and/or lobulated nuclei.Our data show significant decrease in the proportions of blastocysts in the 0.75 and 1 ng/ml toxin-supplemented groups compared with the control group. Blastocyst rate did not differ in embryos treated with 0.5 ng/ml T-2 but 24 h delay was found in blastocoel formation in all the treated groups. Only grade A (21.1%) and B (78.9%) blastocysts were found in low-toxin-contaminated group similar to the control ones (50-50%). Grade C embryos appeared in the 0.75 ng/ml (10%) treated group and the rate increased significantly (33.3%) in the highest contaminated group.T-2 mycotoxin has a harmful effect on early embryo development which results in decreased blastocyst proportion, delayed blastulation, and increased rate of chromatin damage.

Optimizing the temperature of embryo culture in IVF: a randomized controlled trial

Significant animal data exist to indicate that the temperature in the mammalian reproductive tract is more than one degree cooler than core. Traditional IVF systems culture embryos at 37 C to mirror standard cell culture practice. No study to date has evaluated the relationship between different culture temperatures, embryo development, aneuploidy, and ultimately implantation and delivery rates. This study seeks to compare these outcomes following in vitro culture at 36 and 37 C.

Serotonergic Signaling by Preimplantation Embryos Impacts Subsequent Trophoblast Differentiation and Pregnancy Failure.

Serotonin (5-hydroxytryptamine, 5HT) is a monoamine neurotransmitter that is also abundant outside the central nervous system where it circulates in blood platelets and mediates a broad variety of neuronal and non-neuronal biological functions. 5HT is produced in the mammalian reproductive tract, oocyte and preimplantation embryo where it may participate in the regulation of fertilization and early embryonic development. Preimplantation mouse embryos express several G protein coupled 5HT receptors, as well as the 5HT reuptake transporter, and are potentially capable of serotonergic signaling. 5HT is generated from tryptophan by tryptophan 5-hydroxylase (EC 1.14.16.4), an enzyme encoded in mice by two genes, Tph1 and Tph2, which are restricted in their expression to tissues either outside or within the central nervous system, respectively. Interestingly, Tph2, but not Tph1, is expressed in oocytes and preimplantation embryos. Transgenic mice homozygous for the deletion of Tph2 had a significant (14-fold) increase in pregnancy failure that was also evident in periimplantation development between embryonic day 4.5 (E4.5) and E7.5. However, morula-stage embryo (E2.5) production was not significantly affected by a lack of embryonic TPH2. TPH2 and 5HT were detected by immunofluorescence in wild type embryos from the 1-cell to morula stage, but decreased abruptly at the blastocyst stage. Developmental rates in vitro were similar for wild type and Tph2 null morulae to the hatched blastocyst stage, but trophoblast differentiation into migratory cells was delayed by about 24 hours (p<0.05) in knockout embryos cultured to form blastocyst outgrowths. Tph2 null mothers are able to produce 5HT in the reproductive tract using TPH1, but this was insufficient to support normal development of Tph2 null embryos. However, pregnancy failure rates were intermediate in Tph2 null mothers crossed with heterozygous males or in heterozygous mothers crossed with Tph2 null males, suggesting at least a partial rescue in vivo by 5HT-producing sibling embryos. We conclude that in the absence of endogenous 5HT synthesis, fertilization and preimplantation embryo production is normal, but implantation and pregnancy success rates are both reduced. These findings suggest that autocrine or local paracrine serotonergic signaling during preimplantation development, prior to blastocyst formation, ensures on time differentiation of the trophoblast for implantation. Activation of embryonic 5HT receptors could mediate interactions among blastomeres or induce gene expression that is required for trophoblast functions that take place during or after blastocyst implantation to maintain a viable fetus. The Tph2 null mouse is a unique model of implantation failure arising from trophoblast dysfunction that might also provide insight into placental insufficiency and associated perinatal disorders. Supported by the Intramural Research Program of the NICHD, NIH, and the Department of Veterans Affairs.

Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

BackgroundCervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue.ResultsWe tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining.ConclusionsWe provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

Mucin Histochemistry in Tumours of Colon, Ovaries and Lung

Introduction: Mucins implicated in cancers of various organs. The apical epithelial surfaces of mammalian respiratory, gastrointestinal, and reproductive tracts are coated by mucus, a mixture of water, ions, glycoproteins, proteins, and lipids. The purpose of this study was to confirm the presence of mucin production using Haematoxylin and Eosin (H&E) stain as the gold standard and to describe the types of mucins produced in tumors of lung, colon and ovaries using various types of histochemical techniques. Methods: The resection specimens and biopsies from tumours of colon (n=16), ovaries (n=13) and lung (n=5) were included and stained with H&E to determin the histological diagnosis for selecting tissues with mucin production. Slides were stained with PAS, Alcian blue, High iron diamine-Alcian blue, Meyer’s mucicarmine and Alcian blue-PAS to demonstrate the mucin production and to identify types of mucins. Results: In the present study we observed predominance of acid mucins over neutral mucins. In addition in these cases we observed sulphomucin predominating over sialomucin. Conclusion: Mucin histochemistry can effectively determine the types of mucins.