Talpa occidentalis, and present the molecular basis of abnormal lens development. This is the first embryological developmental study of the eyes of any fossorial mammal at the molecular level.Fossorial mammals face natural selection pressures that differ from those acting on surface dwelling animals, and these may lead to reduced visual system development. We have studied eye development in a species of true mole, the Iberian mole PAX6 is not down-regulated in developing lens fibre nuclei, as it is in other species, and there is ectopic expression of FOXE3, a putative downstream effector of PAX6, in some, but not all lens fibres. FOXE3-positive lens fibres continue to proliferate within the posterior compartment of the embryonic lens, but unlike in the mouse, no proliferation was detected anywhere in the postnatal mole lens. The undifferentiated status of the anterior epithelial cells was compromised, and most of them undergo apoptosis. Furthermore, β-crystallin and PROX1 expression patterns are abnormal and our data suggest that genes encoding β-crystallins are not directly regulated by PAX6, c-MAF and PROX1 in the Iberian mole, as they are in other model vertebrates.Lens fibre differentiation is not completed in the Iberian mole. Although eye development starts normally (similar to other model species), defects are seen after closure of the lens vesicle. In other model vertebrates, genetic pathways controlling lens development robustly compartmentalise the lens into a simple, undifferentiated, proliferative anterior epithelium, and quiescent, anuclear, terminally differentiated posterior lens fibres. These pathways are not as robust in the mole, and lead to loss of the anterior epithelial phenotype and only partial differentiation of the lens fibres, which continue to express 'epithelial' genes. Paradigms of genetic regulatory networks developed in other vertebrates appear not to hold true for the Iberian mole. Astyanax mexicanus) have provided valuable insights about evolutionary eye development. This species has become a useful model for studying the molecular biology of eye organogenesis in vertebrates. The eyes of the blind cave-fish begin to develop relatively normal but there is a loss of pax6 expression in the lens secondary to increased midline expression of the morphogen sonic hedgehog (shh), leading to apoptosis and degeneration of other eye structures [Notoryctes spp.) represent the most extreme case described. These animals exhibit vestigial closed eyes without lenses [Eremitalpa granti) and blind mole rats  also show very small eyes completely covered by skin, although a rudimentary lens with disorganised fibre cells, that have not extruded their nuclei, is present [Heterocephalus glaber) have eyelids, but they are generally closed unless the animals are alarmed. The lens of these subterranean rodents seems to float freely inside the eyeball and exhibits various irregularities in shape [Talpa europaea) have less degenerated eyes in which the main eye structures are present; however, the lens shows disorganised nucleated lens fibres [Reduced visual systems are common among vertebrates adapted to live in subterranean habitats . Studiesructures ,3. In mat lenses . Grant's present ,6. Perha present . The nakin shape . The Eurs fibres . In spits fibres . Althougs fibres .Very few molecular studies have been performed in fossorial mammals -8,11. FoLens differentiation is a key event in eye development. It has an important role in the formation of the anterior segment of the eye and loss of the lens leads to failure of anterior eye structures ,13. NormDifferentiating lens fibre cells elongate, lose their nuclei and organelles  and synthesise specific soluble proteins involved in lens function: α-, β- and γ-crystallins ,19,20. FPAX6/Pax6, which is highly conserved across metazoans [PAX6 expression in lens development is dynamic. This gene is highly expressed in the lens placode [PAX6 expression is normally maintained in proliferating cells of the anterior lens epithelium, and it is down-regulated in posterior lens fibres to enable terminal differentiation [Many genes involved in lens development have been identified, including etazoans -24. PAX6 placode ,26. PAX6ntiation -28. PAX6ntiation ,30. On ontiation -35. On tntiation ,28,36.FOXE3/Foxe3 encodes a transcription factor essential for lens epithelial proliferation and closure of the lens vesicle, and is regulated by Pax6 in mice [PAX6 haplo-insufficiency are consequences of a reduced expression of FOXE3 [ in mice ,38. It hTalpa occidentalis, Cabrera 1907), in order to determine whether the eye defects of these subterranean insectivores are a consequence of a degenerated or a developmentally retarded visual system. Our results show that the development of the lens is not completed in the Iberian mole and that changes in the regulation of both PAX6 and FOXE3 expression underlie the eye dysmorphology of this species. Furthermore, our data suggest that expression of β-crystallin genes is not directly repressed by PAX6 in the mole, unlike in other model species.In this paper we have studied the whole process of lens differentiation in the Iberian mole  showed that the lens is surrounded by a lens capsule and there are normal 'ball and socket' connections between lens fibres, although they are scarce Figure . The lenHaematoxylin staining of tissue sections showed that the mole lens begins to develop properly. The lens vesicle detaches completely from the overlying surface ectoderm and become polarised Figure . HoweverThe analysis suggested that although the mole lens was transparent, there was some failure of normal developmental processes, with incomplete differentiation of both lens epithelium and lens fibres. A molecular analysis was therefore performed.PAX6 was expressed in the optic vesicle as well as in the adjacent surface ectoderm. The optic cup and lens vesicle were strongly positive for PAX6 at the next stage . From stage s4c (E11 in mice) on, the posterior cells begin to elongate. At later stages, primary lens fibre elongation continues until stage s8 , in which the lens cavity is almost closed. In mice, expression of Pax6 is maintained in the lens epithelium, but not in lens fibres: differentiation of lens fibre cells has been shown to require the down-regulation of Pax6 [By immunohistochemistry, PAX6 was detected in the cornea, iris, lens, retina and their precursors during the whole eye development in the Iberian mole Figure , as expe of Pax6 . In contPAX6 expression in lens fibre nuclei was causal to a failure of full differentiation, and tested this by examining the expression of another lens epithelial gene, FOXE3. In the mouse lenses, Foxe3 expression is similar to that of Pax6, with early expression throughout the developing lens pit becoming restricted to lens epithelial cells only during development [FOXE3 is weakly expressed in the invaginating lens placode at s4a stage , this transcription factor was detected mainly in the germinative zone, although there were also individual positive cells in all part of the lens. We confirmed that Foxe3 is expressed in all the epithelial cells but not in the lens fibres at equivalent stages in mouse samples. In the s9 stage of mole development, some  cells of the lens epithelium exhibited FOXE3-specific immunoreactivity. FOXE3 was not detected during the rest of the mole lens development. As not all the lens fibre cells that expressed ectopic PAX6 also expressed ectopic FOXE3, the data suggested that the dysgenic phenotype of the mole lens was not solely due to PAX6-induced misregulation of FOXE3. The close link between Pax6 and Foxe3 expression found in the mouse was lost in the mole, suggesting that there were further adaptations of the genetic pathways controlling eye development.We hypothesised that retention of elopment . In the e Figure . Its expPROX1 in the lens of the Iberian mole clearly differs from that observed in mouse. In the mole, PROX1 was detected in the cytoplasm of the cells forming the invaginating lens placode at the s3 stage , although the main signal was in the nuclei  in both moles and mice , whereas PH3 labelling was observed in all mouse lens epithelia at all stages. Hence patterns of proliferation are spatially deregulated in the mole lens, and proliferation is prematurely terminated with lens growth after s7 presumably relying solely on the increasing size of lens fibres.Cell proliferation was studied by analysing the presence of the phosphorylated form of the histone H3 (PH3). In wild-type mouse samples, this protein was observed in some cells throughout the lens vesicle at E12 Figure , but wasFOXE3 (N ≥ 5 lenses)  Figure . This suN ≥ 6 lenses). This situation is the opposite of that observed in E18 mouse embryos. The apparent apoptotic event is restricted to the immediate postnatal stages, because the TUNEL reaction was negative in all the mole adult lenses examined (N ≥ 3). The abnormal apoptosis profile observed in the lens epithelium of infant moles is very similar to that described in Foxe3-mutant mice [FOXE3 in many lens epithelial cells could be responsible for the apparent loss of the fully differentiated epithelial cell type phenotype in the mole.Immunohistochemical data, combined with the morphological analysis of lens abnormalities above, suggested that the robust genetic pathways, which distinguish the lens epithelial and lens fibre cells, are beginning to break down in the Iberian mole, with epithelial cells exhibiting some aspects of the fibre cell phenotype, and vice versa. This hypothesis was tested further. Apoptotic-like pathways have been described during lens fibre differentiation in mice, but no apoptosis is normally observed in the wild-type lens epithelium . This obant mice . TherefoPax6, but not in epithelial cells where Pax6 expression is maintained. Pax6 is reported to be a transcriptional repressor of genes encoding β-crystallins [Pax6 and β-crystallin is mutually exclusive – lens fibres were β-crystallin-positive and Pax6-negative, whereas all lens epithelial cells were Pax6-positive and β-crystallin-negative. During mole lens development, β-crystallins were first detected at the s5c stage , the expression of β-crystallins was normal, with expression in lens fibre cells but not the epithelium. Nevertheless, weak immunoreactivity against the anti-β-crystallin antibody was observed later, in epithelial cells at the last prenatal stage (s8) . These data suggest a breakdown of the inhibitory effect of PAX6 expression on lens fibre differentiation, consistent with the results above.The function of fibre cells in mole lenses was investigated using β-crystallin as a marker for terminal differentiation. In mice and chickens, β-crystallin genes are expressed only in lens fibre cells that have down-regulated stallins ,28,36. Ie Figure , but not) Figure , and sim) Figure  and 10B.Our results show that the eyes of the Iberian mole, although permanently closed, are morphologically relatively normal and we do not believe that this species is totally blind. The eyelids of the Iberian mole are thin and translucent , and their eyes have an apparently functional anterior segment with only minor overt morphological abnormalities of the cornea, such as the reduced thickness of the corneal epithelium, suggesting that the barrier function of this tissue may be redundant. The pupil diameter is probably fixed due to the absence of iris musculature, but the retina is well developed and our pilot data (not shown) indicate that the optic nerve successfully projects to the visual cortex. Molecular analyses of the developing retina and cornea will be presented elsewhere . However, it is the lens that shows the most overt abnormal phenotype, and, given the known role of the vertebrate lens as an organising centre for eye development, it is plausible that the other anterior segment defects are to some extent secondary to the failure of paracrine signalling from the lens to anterior eye tissues ,44.Iberian moles are closely related to the European mole, which has open eyes, with potentially better visual acuity ,46. CurrThe data presented here show that the abnormal lens in Iberian moles represents a series of primary developmental defects and is not solely an adult degenerative condition. Fibre cell differentiation and maturation in mouse and human lenses is associated with denucleation. In the mouse, the denucleation process is largely completed by postnatal day 1, so that there are no nucleated fibres in the centre of the lens at birth . Our resPAX6/Pax6 expression is restricted exclusively to the lens epithelium, and alterations in this profile lead to severe lens anomalies [Pax6 is ectopically expressed in lens fibres, exhibit abnormal fibre cell elongation and incomplete denucleation [Pax6 expression in fibre cells induces a reduction in the protein levels of c-Maf and β-crystallins, indicating that Pax6 inactivation is necessary for lens fibre differentiation and maturation [Pax6 in the mouse also causes cataracts, with partial failure of lens fibre differentiation and abnormalities in fibre shape as well as fibre cell/lens capsule and fibre cell/fibre cell interactions [PAX6 plays a crucial role in lens development in all vertebrate eyes ,49. In mnomalies ,26. Trancleation . Their lturation . Similarractions ,51.pax6 expression precedes lens apoptosis and subsequent eye degeneration in the blind cave-fish [Pax6 in fibre cells . However, no cataracts were observed in any of the mole lenses analysed, probably due to a higher β-crystallin levels in the lens fibres, in comparison to the overexpressing transgenic mice. In the 'PAX77' transgenic mouse line, which overexpresses human PAX6 from a YAC transgene, occasional pockets of PAX6-positive cells are seen within the lens fibre region, and some of these can be labelled with bromodeoxyuridine, indicating ectopic proliferation [PAX6 expression in this species. Nevertheless, the proliferation rate in the eye of the Iberian mole is very low in relation to the mouse, which may be the cause of its reduced size.Loss of ave-fish . In contferation ,52. Our Lens epithelial cells are flattened and lack apparent mitochondria in the adult mole, and TUNEL labelling showed that DNA double-breaks were present in most lens epithelial cells in infant moles, with no apoptotic cell detected in the posterior region of the lens. The data suggest that lens epithelial cells are lost during development in the mole and that the surviving lens anterior cells are fibre-like. TUNEL-positive cells were first detected, just when the vacuoles between the lens epithelium and the lens fibres are formed and correlated with loss of FOXE3, a winged helix-forkhead transcription factor. This abnormal apoptotic profile seems not to be conserved in other fossorial mammals with naturally reduced eyes, like the naked mole rat, in which TUNEL reaction is not detected in the lens epithelium at any postnatal stage .Foxe3 is thought to be activated by Pax6 at around 9 dpc in the mouse lens placode. This gene remains active in all the cells of the lens vesicle until 12.5 dpc, when it is down-regulated in the elongating primary lens fibres [Foxe3 continues to be expressed in the mouse lens epithelium until adulthood, being indispensable for a complete closure of the lens vesicle and for the maintenance, proliferation and survival of the lens epithelial cells [Foxe3 is very sensitive to Pax6 dosage, and it has been proposed that many of the ocular malformations associated with Pax6 haplo-insufficiency are consequences of a reduced expression of Foxe3 [Foxe3 are responsible for the dysgenic lens mouse mutant phenotype [Foxe3-null mice have small lenses as a result of a diminished proliferation in the lens epithelium. Fibre cells do not differentiate properly, showing irregular shapes and persisting nuclei. The lens eventually develops several vacuoles and cataracts [Foxe3 down-regulation in lens fibres is crucial for proper differentiation of these cells. Persistent expression of this gene in the posterior region of the lens leads to a partial epithelialisation of fibre cells, with severe consequences for lens function [Foxe3 is ectopically expressed in fibre cells exhibit lenses with numerous vacuoles and cavities, irregular shape of lens fibre and disorganised cytoskeleton [s fibres ,38. Foxeal cells . Foxe3 ihenotype ,38, whichenotype . Foxe3-nataracts . Foxe3 dfunction . Mice inskeleton .Foxe3 resemble in many respects the abnormal phenotype seen in the mole, and are in accordance with the loss of FOXE3 expression seen in many lens epithelial cells and the ectopic expression observed in lens fibres. Consistent with the known roles of Foxe3 in mice, the mole lens has a low proliferation rate and all proliferating lens fibres expressed FOXE3. Furthermore, no FOXE3 expression was detected in the mole lens at any postnatal stage, with vacuoles appearing just when this gene is down-regulated.These anomalies in mice mutant for FOXE3, the expression of PAX6 continues in all lens cells throughout the whole life of the mole, and hence, unlike in mice, patterns of FOXE3 expression do not parallel those of PAX6. Accordingly, we propose that PAX6 may not directly regulate FOXE3 in the Iberian mole.In contrast to Studies on crystallin regulation have been mainly carried out using mice and chickens as animal models. However, there are remarkable differences regarding the crystallin gene expression profile between both species. For example, the chicken lens does not express the γA-F crystallin cluster and contains two taxon-specific δ1 and δ2-crystallins. Moreover, different studies have shown that some regulatory regions of the mouse and chicken crystallins are not highly evolutionary conserved . NeverthPax6 in lens fibre cells, suggesting that the βB1-crystallin gene is also negatively regulated by Pax6 in mammals [PAX6. It would be worthwhile to perform a proteomic analysis of lens crystallins in the Iberian mole to determine which β-crystallins are present.PAX6 represses the chicken βB1-crystallin by displacing PROX1 and MAF transcription factors (which act as positive regulators) from the βB1-crystallin promoter . Cui et  mammals . NeverthProx1-/- mice [Similarly, the results described in this paper in relation to c-MAF and PROX1, which are reported to cooperate in the activation of β-crystallin gene expression ,36,59, a-/- mice . Its fun-/- mice . The PRO-/- mice . Once inc-MAF is only weakly expressed in epithelial cells in the Iberian mole. This fact could explain the absence of β-crystallins in the mole lens epithelium during almost the whole prenatal development, because c-Maf up-regulation in lens fibres has been described as essential for β-crystallin expression in mice [ in mice  and chic in mice . HoweverTaken together, our results clearly suggest that there is no evolutionarily conserved relationship between PAX6, PROX1 and c-MAF in relation to β-crystallin expression in the Iberian mole. A possible explanation for this could be the presence of specific mutations in promoters of mole crystallin genes, something that needs to be investigated in the near future. Another possibility is that all members of the complex network of transcription factors involved in the regulation of crystallin production are not yet completely elucidated, and that other unknown factor(s), which may be lacking or mutated in the Iberian mole, is (are) also necessary in this process.Talpa. To understand how the adaptation to dark habitats has modified the visual system of these fossorial mammals, it would be necessary to examine several taxa in a phylogenetic context. However, the significant difficulties associated with obtaining fresh embryonic material from such evasive wild animals are likely to make this fascinating challenge a more long-term project.Owing to the lack of molecular approaches on eye development in true moles, it is not possible for us to establish whether the anomalies observed in the Iberian mole are more severe than would be observed in mole species with open eyelids. Nevertheless, as stated above, we do not believe that Iberian moles are totally blind and regard it as unlikely that the European mole, which is evolutionarily very close to the Iberian mole but has open eyes, shows great visual acuity. The eyes of European moles are less sensitive to light, by several orders of magnitude, than those of humans , and oneFurther studies on other animal models of eye degeneration, similar to that reported in the present paper, may shed more light on the molecular pathways that control the mammalian lens development and function. Furthermore, research on these natural systems can provide useful information about the evolutionary mechanisms that lead to the atrophy of an organ as important as the eye, and how natural selection worked at the molecular level thus inducing morphogenetic and functional changes in evolving organisms.PAX6 and FOXE3 seems to be the cause of this abnormal phenotype. Furthermore, our data clearly show that the role of PAX6, c-MAF and PROX1 in relation to the control of genes encoding β-crystallins is not conserved in the Iberian mole. The presence of crystallins in the undeveloped mole lens may explain the fact that there are no acute cataracts, and suggests that the lens of the Iberian mole is probably partially functional. Studying different talpid species would clarify whether the abnormalities observed during lens development in the Iberian mole are specifically associated with the permanent closure of the eyes, or are conserved and shared in moles with open eyes. Furthermore, more research on these naturally modified visual systems would contribute current knowledge about human eye degeneration and disease.Fossorial animals represent natural models of dysgenic eyes in which molecular pathways involved in eye development have been modified by natural selection. This paper reports for the first time a molecular study of the entire process of lens development, from the establishment of the lens placode to adulthood, in a species of subterranean mammals, the Iberian mole. Our results show that there are primary developmental defects in the lens of this insectivore: lens fibre differentiation is not completed and there is loss of the lens epithelial phenotype, with most epithelial cells undergoing apoptosis. As a result, the adult lens is composed of a disrupted lens epithelium and a disorganised mass of immature and nucleated fibre cells. An impaired regulation of both Talpa occidentalis were analysed in this study. As moles do not breed in captivity, all samples were obtained from wild animals captured in poplar groves in the localities of Santa Fe and Chauchina , as described in Barrionuevo et al [A total of 64 embryos, foetuses, infant and adult moles of the species vo et al .Embryos and foetuses were obtained from pregnant females. Pregnancy was detected by abdominal palpation of females and the developmental stage of the embryos according to the size of the uterine swellings. Infants were collected directly from their nests by radio-tracking lactating females (recognisable by the size and colour of their teats). After dissection, precise developmental staging was carried out based on crown-rump length, body mass values and on the morphology of major external structures, according to Barrionuevo et al  and CarmCaptures were performed under annual permission granted by the Andalussian Environmental Council, and animal handling followed the guidelines and approval of the University of Granada's 'Ethical Committee for Animal Experimentation'.Four litters of CBA/Ca mouse embryos  and several adult mice, provided by the Medical Research Facility of the University of Aberdeen, were used as a positive control.Embryonic and s9 newborn infant heads and eyes of the rest of the stages were dissected out and fixed in paraformaldehyde solution (4%) overnight at 4°C. Samples were then washed with phosphate buffered saline (PBS), dehydrated, embedded in paraffin wax and sectioned in a microtome, following standard procedures.For gene expression analyses, sections were de-waxed using histoclear, rehydrated and washed with PBS. Slides were placed in 0.01 M citrate buffer pH6 and boiled for 20 minutes in a microwave for antigen retrieval. Samples were subsequently washed with Tris-buffered saline (TBS) , blocked with PBS, 0.3% bovine serum, 4% normal serum (according to the species in which the secondary antibody was developed) and incubated overnight in specific primary antibodies at 4°C.3,3'-Diaminobenzidine tetrahydrochloride (DAB) immunostaining was performed using secondary biotinylated-antibodies, previous incubation with 3% hydrogen peroxide in PBS for 20 minutes to block endogenous peroxidise activity. After washing with TBS, sections were exposed to ABC-streptavidin horseradish peroxidase complex  for 25 minutes at room temperature, washed again in TBS and then incubated in 0.83 mg/ml DAB, 20 mM Tris, pH 7.6, 1/6000 hydrogen peroxide in the dark for up to 45 minutes at room temperature. Subsequent haematoxylin staining was carried out in DAB-stained samples.Secondary fluorochrome-conjugated antibodies were used for immunofluorescence assays and photomicrographs taken using a red/green single bandpass fluorescence mirror unit. Table in situ TUNEL reaction was carried out using the 'In Situ Cell Death Detection Kit, Fluorescein' .To identify apoptotic cells, Samples for TEM were processed by the Histology and Electron Microscopy Facility of the Institute of Medical Sciences, University of Aberdeen. Electron micrographs were obtained using a Philips CM10 TEM microscope.DAB: 3,3'-diaminobenzidine tetrahydrochloride; dpc: days post-coitum; PBS: phosphate buffered saline; TBS: Tris-buffered saline; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling.FDC performed all experiments. JMC and FDC jointly planned the project and wrote the manuscript. RJ was responsible for the preparation of the samples as well as for reviewing the manuscript.


Monitoring and evaluation (M&E) of HIV care and treatment programs is impacted by losses to follow-up (LTFU) in the patient population. The severity of this effect is undeniable but its extent unknown. Tracing all lost patients addresses this but census methods are not feasible in programs involving rapid scale-up of HIV treatment in the developing world. Sampling-based approaches and statistical adjustment are the only scaleable methods permitting accurate estimation of M&E indices.In a large antiretroviral therapy (ART) program in western Kenya, we assessed the impact of LTFU on estimating patient mortality among 8,977 adult clients of whom, 3,624 were LTFU. Overall, dropouts were more likely male , and younger than non-dropouts , with lower median CD4 count at enrollment  and WHO stage 3–4 disease . Urban clinic clients were 75.0% of non-dropouts but 70.3% of dropouts (p<0.001). Of the 3,624 dropouts, 1,143 were sought and 621 had their vital status ascertained. Statistical techniques were used to adjust mortality estimates based on information obtained from located LTFU patients. Observed mortality estimates one year after enrollment were 1.7% (95% CI 1.3%–2.0%), revised to 2.8% (2.3%–3.1%) when deaths discovered through outreach were added and adjusted to 9.2% (7.8%–10.6%) and 9.9% (8.4%–11.5%) through statistical modeling depending on the method used. The estimates 12 months after ART initiation were 1.7% (1.3%–2.2%), 3.4% (2.9%–4.0%), 10.5% (8.7%–12.3%) and 10.7% (8.9%–12.6%) respectively.Assessment of the impact of LTFU is critical in program M&E as estimated mortality based on passive monitoring may underestimate true mortality by up to 80%. This bias can be ameliorated by tracing a sample of dropouts and statistically adjust the mortality estimates to properly evaluate and guide large HIV care and treatment programs. In resource-rich settings such as North America and Europe, use of antiretroviral therapy (ART) has vastly improved the prognosis of persons living with HIV/AIDS Essential to the identification of the most effective antiretroviral treatment (ART) delivery and cost-effective HIV management strategies for resource-constrained settings is appropriate and efficient monitoring and evaluation of ART care and treatment programs. However, accurate estimates of patient survival and other clinical outcomes have been difficult to obtain, as they are significantly impacted by patient loss to follow-up In the past, several approaches have been used to ascertain the vital status of patients who have not returned to clinic. The basic level of information gathering, and most common method utilized in resource-poor settings, is a passive surveillance system which relies on family and friends to report patient deaths to clinic personnel. To obtain more comprehensive information, some form of active patient surveillance has been used. This includes telephone contact with the patient or close relatives and acquaintances, home visits, reviews of obituaries, vital statistics registries (where these are available), or a combination of the above In two seminal reports, one from the Antiretroviral Treatment in Lower Income Countries (ART-LINC) CollaborationBy comparison, attempting to trace only a statistical (random or non-random) sample of the lost patients is feasible regardless of the size of the patient population and is thus a scalable alternative to census patient tracing approaches. Combined with statistical adjustment of the information gathered, this approach will generate much improved estimates compared to passive patient follow-up.The primary objective of this paper is to describe how the use of statistical sampling techniques, coupled with medical record infrastructure and a patient tracing program, can be used to a) enable more accurate estimation of mortality particularly in large HIV treatment programs, and b) identify subsets of patients who are at higher risk of being lost to follow-up (LTFU) and who may benefit more from active patient tracing in order to improve their clinical care.A partnership initially established between the Moi University School of Medicine in Eldoret, Kenya Indiana University School of Medicine and Brown Medical School in 2001 Included in this analysis are HIV-positive patients aged 18 years and over, who were enrolled between January 1, 2005 and January 31, 2007 at either of two clinics within the AMPATH system where an active program for tracing patients who had missed clinic visits was initiated. Patients were included regardless of whether they were ART-naïve or they had initiated combination ART (CART).Active outreach to patients who miss scheduled appointments started in January 2005 at two of the AMPATH Clinics. These were the clinics at Moi Teaching and Referral Hospital (MTRH), an urban referral hospital located in Eldoret, Kenya's fifth largest city, and Mosoriot, a rural health center which serves a catchment area of approximately 6,000 located about 30 Km from Eldoret. This program has now been extended to the entire AMPATH system.Outreach workers fill out a locator card for all patients enrolling in the clinical care program. The locator card includes contact information and a map to the patient's residence and is used to find the patient in the event of a missed appointment. The AMRS produces a daily list of patients scheduled for appointments and patients that miss their appointment are listed for outreach based on a three-tier triage algorithm. Adult patients on CART for less than three months are given priority. Outreach efforts for these patients are to commence within 24 hours of a missed appointment with a goal of locating the patient within seven days. For patients receiving CART for over three months, outreach is activated within seven days after a missed appointment. Individuals who do not receive CART are allowed a grace period of 28 days from the missed appointment prior to initiation of outreach activities. At the time of this study, the outreach program maintained a standalone MS Access database that contained data pertaining to every outreach encounter including vital status of located patients and date of death for patients found to be deceased. This database has since become part of the AMRS.In this study, a patient is considered to be a dropout if the patient has been declared as lost to follow-up by the program. This happens when a patient receiving CART has not kept a visit for more than three months or a patient that is not receiving CART has not come to clinic for more than six months. In addition, any patient for whom outreach was initiated, regardless of whether they had been declared as lost to follow up, is considered a dropout for the present analysis. This includes all patients that have missed appointments that AMPATH attempted to contact. We have not distinguished between patients declared as lost to follow-up and patients that missed appointments and were outreached. This is because the loss-to-follow-up status is not known among patients that have been outreached after a missed appointment but before they have been declared as lost to follow-up . For this reason, dropout rates reported here are much higher than the loss-to-follow-up rates previously reported for the AMPATH program All patients in the study provided locator information as described above. This is part of the standard of care at AMPATH. Use of these data, which were routinely collected as part of the AMPATH care protocol, was approved by both the Indiana University Institutional Review Board (IRB) and the Moi University Institutional Research and Ethics Committee (IREC).Descriptive statistics were produced. Patient subgroup sizes  were compared between dropouts and non-dropouts by chi-square tests. Continuous measures such as CD4 count were compared via the Kruskal-Wallis test. Estimates of time until an event  were produced by the method of Kaplan and Meier, with time zero (baseline) being defined as the date of enrollment in the AMPATH program. Comparisons of these times between dropouts and non-dropouts were performed by the log-rank test. A Cox proportional-hazards model was used to assess the impact on time to loss-to-follow-up of a number of measures obtained from the patients prior to dropping out. These are WHO stage, CD4 count at enrollment, gender, age and type of clinic attended . In addition, we have stratified the analysis to account for CART start status  because this factor summarizes a number of patient and disease-related issues that may not be captured by the other factors considered above. These analyses were implemented with SAS version 9.1 .Statistical estimation of mortality follows methods described by Frangakis & Rubin For patients that were deceased, we calculated survival time as the time from enrollment to the date of death. For deceased patients with missing death dates, we imputed a death date from other patients with a known death date and matching CD4 counts at enrollment. If no matches were found and the patient had a baseline WHO stage, then we searched among the deceased patients with available death dates and matching WHO stage. We then imputed a survival time from among this subset. If the above step found no matches, then we matched based on available death information from patients that started CART at a similar time from enrollment.For patients who dropped out, were double-sampled, were alive and returned to clinic, we defined “censoring time” to be the time from enrollment to the date of the most recent visit after outreach. For those found alive but did not return to clinic we imputed time from last visit (before dropout) to last visit after outreach from observed data, matching in a similar manner to that described for the death date above. We then added this imputed time to the observed time from enrollment to last visit to obtain an imputed date of last visit after outreach. Otherwise, “censoring time” was the time from enrollment to the end of the study (1/31/07), the usual administrative censoring time.A CD4 count and WHO stage measurement were considered to be a “baseline” measurement if the measurement were taken within 3 months after enrollment. If there were multiple measurements during this period, the one closest to enrollment was selected as “baseline”. Missing CD4 count and/or WHO stage were imputed according to the following:If CD4 count but not WHO stage were missing, CD4 count was imputed from among patients with similar WHO stage and matched CART start status .If WHO stage but not CD4 count were missing, WHO stage was imputed from among those with similar CD4 (stratified as <200 or ≥200 cells/ml) and matched CART start status.If both CD4 and WHO stage were missing, imputation of WHO stage and CD4 count was obtained from among patients with matching CART start status.We compared four methods for estimating mortality. The first method is the usual passive follow-up Kaplan-Meier estimate that is based on observed deaths only (Method 1). The mortality estimates produced by Methods 1 would typically be those reported from passive follow-up programs. In addition, a Kaplan-Meier estimate based on all deaths (both those recorded passively as well as those discovered through patient tracing) was produced (Method 2). In this method all vital status information is pooled without consideration whether it was obtained by passive or active follow-up efforts. These results would be routinely reported by active-follow-up programs with no statistical adjustment. We also produced a mortality estimate as the weighted average of the Kaplan-Meier survival estimates between dropout and non-dropout groups (Method 3). This method should be an improvement compared to Methods 1 and 2 because it assigns the proper weighting (invariable higher weight) to deaths discovered through patient outreach. This method however has been shown by Frangakis and Rubin to be biased There were 8,977 adult patients enrolled in the two participating sites between January 1, 2005 and January 31, 2007. Of these, 3,624 (40%) had missed clinic visits over this period, including 64% on CART and 36% not on CART. Outreach efforts were initiated for 1,143 (31.5%) of these and 621  were successfully located. Reasons why patients were not contacted through the Outreach Program include insufficient or inaccurate locator information or the patient returned to clinic prior to the initiation of outreach procedures. Unfortunately, the reason for not initiating outreach was not included in the outreach database at the time. It has been added to the newer version of the database.Patient characteristics at enrollment into AMPATH are shown in We generated a dropout risk score based on the Cox proportional hazards model presented in We present two analyses based on two definitions of “baseline” as the date of enrollment and the date of ART initiation. Results from the four methods of estimating patient mortality are shown in We present data from AMPATH, a large PEPFAR-funded HIV clinical care program in western Kenya. AMPATH has implemented an active follow-up program that attempts to locate as many patients as possible among those that have discontinued care. Analysis of these data allows adjustment of mortality estimates based on information obtained on patient dropouts that were located as part of the AMPATH outreach program.Comparison between mortality estimates reveals some interesting results. A significant number of patients miss at least one clinical visit . The majority of these will be lost to follow-up. Since risk factors for patient dropout (lower CD4 count or higher WHO stage at enrollment) are also risk factors for mortality, estimates that do not account for patients lost to follow-up are bound to severely underestimate the true extent of mortality and conversely overestimate the positive impact of a care and treatment program. This is not a novel observation. The significance of this finding is the size of the differences in mortality estimates produced by statistically adjusted methods. The mortality estimate produced by relying on passive follow-up (Method 1) was 1.7% one year after enrollment in the program compared to 2.8% when all known deaths are incorporated into the analysis (Method 2). This almost two-fold difference strongly supports inclusion of active follow-up for at least a sample of the patient dropouts. This has been consistently reported in similar settings A further example in which unadjusted data can skew program estimates of mortality concerns AMPATH's contribution to a recent ART-LINC collaboration Estimated mortality according to Methods 1 and 2 is significantly different from Methods 3 and 4. These differences do not abate with time but instead become increasingly pronounced as shown in The impression from mortality estimation adjustments one year after ART initiation is similar. The naïve estimate produced by Method 1 is 1.7%, revised to 3.4% when all deaths are pooled (Method 2), and revised further to 10.5% and 10.7% by using statistical adjustment (Methods 3 and 4 respectively). The mortality one year after ART is slightly higher than the estimate obtained one year after enrollment because a sizable proportion of the patient population did not require CART at entry because of less advanced disease and thus had lower risk of death during that period. By contrast, patients requiring ART had more advanced disease (a prerequisite for being eligible for treatment) and, at least in the short term, higher risk of death.To further understand the impact of dropout on estimates of mortality, we refer to ANRS protocols 059 and 1203, both initiated prior to the wide availability of CART Our results have both similarities with as well as important deviations from published reports in similar settings. The results from Method 1 are similar to the mortality estimate of 2.3% one year after CART initiation in a “passive follow-up cohort” reported by Braitstein, Brinkhof and colleagues from the ART-LINC collaboration There are several limitations in our study. The most important one is that the Frangakis & Rubin method we used to produce the adjustment of one-year mortality estimates assumes that a random sample was selected among the dropout patients and everyone from that sample was located. By contrast, AMPATH attempted to locate 1,143 patients out of the 3,624 dropouts (31.5%) of whom 621 were successfully located . We have made the tacit assumption that the group of patients that was outreached and the subgroup that was successfully located were both representative of the entire dropout population. The ANRS 059/1203 study results Our findings lead to the inescapable conclusion that both active follow-up and appropriate statistical modeling must be employed in combination to account for measurable and non-measurable factors affecting mortality. With rapid scale-up of HIV care and treatment programs, census approaches that canvass all lost patients (such as those reported by Anglaret and colleagues


It is only after their travel in the epididymis that sperm gain their motility and fertility. Epididymis is a crescent shaped organ adjacent to the testis that can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda). It contains a long and unique convoluted tubule connected to the testis via the efferent ducts and finished by joining the vas efferens, VE), nine distinct successive epididymal segments and the deferent duct  of four adult boars of known fertility were isolated and their mRNA extracted. The gene expression of each of these samples was analyzed using a pig generic 9 K nylon microarray  spotted with 8931 clones derived from normalized cDNA banks from different pig tissues including testis and epididymis. Differentially expressed transcripts were obtained with moderated t-tests and F-tests and two data clustering algorithms based either on partitioning around medoid (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue clustering defined seven transcriptomic units: testis, vas efferens and five epididymal transcriptomic units. Meanwhile transcripts formed only four clusters related to the tissues. We have then used a specific statistical method to sort out genes specifically over-expressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). The specific regional expression of some of these genes was further validated by PCR and Q-PCR. We also searched for specific pathways and functions using available gene ontology information.In this study, the testis, the efferent ducts (vas efferens and the vas deferens on the same species. It described new genes or genes not yet reported over-expressed in these boar tissues, as well as new control mechanisms. It emphasizes and fulfilled the gap between studies done in rodents and human, and provides tools that will be useful for further studies on the biochemical processes responsible for the formation and maintain of the epididymal regionalization and the development of a fertile spermatozoa.This study described for the first time the complete transcriptomes of the testis, the epididymis, the  Testicular spermatozoa are non functional, it is solely before reaching the end of the epididymis, (where they will be stored from days to weeks upon the species) that they will acquire their physiological functions: the possibility of motility, of undergoing the post-ejaculatory events , the ability to recognize and to bind to the oocyte investments and plasma membrane, and in short their natural fertilizing ability [Spermatozoa are produced in the testis as the result of a complex assembly line that makes a highly shaped cell, morphologically and biochemically specialized. These cells are released in the seminiferous tubules fluid and then begin a long journey by "floating" passively through the rete testis to leave the testis, entering the efferent ducts and thereafter moving to the  ability ,2.This post-testicular maturation results from changes in the sperm membrane composition and metabolism in response to the profound alterations of the surrounding fluid composition. The fluid components  are changing all along the different ducts through which the sperm transit -4. It isseptae of the supporting tissue [vas efferens and vas deferens transcriptomes and the human studies have been limited to the three gross morphological parts of the epididymis . Here we have used a microarray approach to analyze the transcriptome of nine defined regions of adult fertile boar epididymis, efferent and deferent ducts and testis. These epididymal regions were chosen according to previous studies done on this species [The epididymal secretome and proteome of several mammalian species including pigs as well some of the mechanisms involved in the sperm surface alteration have been recently described -18. The g tissue ,25. Howe species .Pigs provide good model for biomedical research, as pigs and humans show in many aspects physiological, biochemical, pathological and pharmacological similarities. Recently it has been demonstrated that the porcine and the human genome evolutionary distance is smaller than the distance between mouse and human . For exaThis study then fulfill the gap between the rodent and human by addressing a domestic species with an agronomical interest where the epididymal duct measure more than 50 meters long and the transit of the sperm is at least three weeks.We have used a microarray that was made from clones issued from a multi-tissue cDNA normalized library including the testis and the three main gross regions  of the epididymis . We havevas efferens, epididymis (zones 0 to 8/9) and vas deferens . Between the 12 tissue samples, 2115 transcripts showed a differential expression. At this step, we expected that the main housekeeping genes or the genes that were expressed at the same level in all the tissues tested have been removed from the analysis.We first analyzed how these 2115 differentially expressed transcripts could classify our biological samples. A clustering of the 12 tissue conditions was then performed with the combinative use of the PAM and the HAC. The PAM gives a graphic profile to help in the choice of the number of classes, which is a critical step in unsupervised methods Figure . In thisThe result obtained by the HAC analysis is shown in Figure versus the other tissues transcripts) and we were able with a threshold to separate 4 clusters identical to the PAM , ididymis ,35, and vas efferens in our analysis had consequence on numbers of transcripts retrieved as epididymal, we performed a second analysis on the 7676 qualified spots without these tissues. The Limma moderated F-test  gave 850 transcripts differentially expressed between the nine epididymal zones and the vas deferens. When we compared this new list with the list of differentially expressed transcripts between the 12 conditions, we observed that 838 transcripts were common and only 12 were new ones. The classification on the nine epididymal zones and the vas deferens was performed with 2 algorithms (PAM and the HAC) gave 5 tissue clusters corresponding to (i) the anterior caput of the epididymis (zones 0–1), (ii) the median caput (zone 2), (iii) the posterior caput (zones 3–4), (iv) the corpus (zones 4–7) and (v) the caudal region (zone 8/9) including the vas deferens. A clustering of co-expressed transcripts with the combinative use of the PAM and the HCL on the 850 differentially expressed transcripts found only 3 clusters: (i) the head of the epididymis, (ii) the corpus, (iii) the caudal region.Because including the testis and the vas deferens).A contingency analysis showed that 721 transcripts among the 850 are clustered in the same way by the 2 statistical approaches used. Among them, 220 have a preferential expression in the head of the epididymis, 343 in the corpus and 158 in the caudal region , for the median head (zone E2) 8 potential markers, for the posterior head (zones E2-3-4) 14, for the corpus (zones E4-5-6-7) 90 and for the cauda (zone E8/9 and deferent duct) 43 transcripts . As control we also amplified two genes (RERG and APLP2) that were not found in the common list of the two statistical approaches but had a statistically significant differential expression on microarray. Again the results of microarray and RT-PCR were in agreements.Almost all transcripts were found effectively expressed within the transcriptomic unit assigned by the statistical approach Figure . Only foB2M) gene that was not found differentially expressed. The primers for this gene were designed in such way that they were on two successive exons. The RT-PCR showed only the waited amplicon of 189 bp (instead of 757 bp when the intron is included) indicating also the absence of genomic DNA contamination. As predicted this gene showed no differential expression by PCR whatever the number of PCR cycles.We also analyzed the beta 2-microglubulin , SPP1, TSPAN1, CPE, AWN). The expression of these genes was measured by the intensity of the amplicon on gel after RT-PCR on the twelve different tissues . Meanwhile when the level of PCR expressions is mathematically pooled to reproduce the seven tissue clusters, this gene expression was clearly higher in the vas efferens compared to the proximal caput.Among the transcripts, we have also chosen for each one of our transcriptomic units, one gene found as a marker by the statistical analysis  and one uncharacterized lipocalin (UNQ2541) were spotted on the microarrays and showed a differential expression. We designed primers . LCN2 expression was detected in boar epididymis but did not show a differential expression  a low sDEFB109, DEFB111, DEFB113, DEFB121, DEFB129, SPAG11B_C, SPAG11B_E). The identity of each of these pig defensins was ascertained by sequence comparisons and cysteine positions and spacing  and two were miss-annotated . We also identified two new boar defensins homologous to DEFB109 and DEFB113.Specific expression of a large number of defensins has been shown by previous epididymal transcriptomic studies ,21,38. Tg Figure . Five ofDEFB109 is mainly restricted to the anterior caput  is an "epididymal specific" gene expressed in the caput and corpus region in mouse [Cst11Δ2) has been suggested from "in silico" genomic sequence analysis but not shown in vivo [CST11 and one of these clones had a sequence deletion  was described in mouse epididymis as a specifically caput/corpus over-expressed gene [sed gene . DNA seqAmong our over-expressed genes we choose two genes that were not previously observed in epididymis and four not yet described in boar.SPINK5L2, a Kazal type serine protease inhibitor, is a marker of the corpus with a maximum intensity in zones 6 and 7  gene not yet described in epididymis, is highly expressed in the anterior caput in boar , is a new member of the galactosidase beta family. We found a high expression of this enzyme in the boar testis but also a differential expression in the epididymis with a maximum in corpus  and generated a list of over-represented GO terms using either the five transcriptomic clusters or only the three epididymal clusters. From this analysis, several over-represented categories were identified Figure . As expeDifferent epididymal transcriptomic studies have been done in mouse, rat and human. Comparison of the data between rat and mouse have been made and showed the difficulties to compare the different zones of these two mammalians epididymis, even using similar microarray technology ,45. In hmus musculus homologous UniGene IDs. We searched transcripts that have their higher level of expression for the 4 species studied, in the same epididymal unit  or in segments corresponding to these units. For mouse, the 3 units were defined based on anatomical and clustering information given by Johnston et al., [However, we tried to compare our data with those of these three studies. We have first obtained the list of expressed genes in the different studies. One obstacle to this comparison was the low level of gene annotation in boar or homogeneity of genes annotation between species. To encompass this problem, we have transformed all the genes in their  et al., . The cap et al., . The cap et al.,  study. TWe then found that 768 mouse UniGene IDs were common between the 4 studies. Among these 768 genes IDs, 236 unigene IDs were found over-expressed both in one region of boar and one of the regions of the three other different species . Meanwhile when we looked for the common over-expressed genes in the same unit in the four different species we ended with only 8 IDs in the caput, 4 in corpus and 35 in cauda  and the vas deferens. The pig epididymis is a large organ of more than 20 cm long containing about 50 meters of a convoluted tubule and its internal organization is much complex than in rodent. It is then more difficult to individualize part of the boar epididymis between connective tissue septae as it has been done in mouse and rat [vas efferens that is more difficult in rodents. Meanwhile anatomical differences between two individual boars may exist, and although we have drawn a special attention to be very reproducible when collecting the samples from different animals, we observed some differences in the gene expressions from some epididymal zones between animals resulting in zones shuffling during membrane clustering (see further). Because acquisition of motility and fertility by the spermatozoa is progressive along the epididymis and secretion of proteins is regionalized, we postulated that genes expressed in a tight regionalized manner might play a major role in this process. For this reason we have focused our attention on differentially expressed genes. In doing this we expected to gain information on how this regionalization is maintained (common transcription factors controlling the genes expressed in one transcriptomic unit), get clues on specific mechanisms  and deduced factors that may be important in the sperm fertility acquisition and epididymal regionalization. Then we have not analyzed major known epididymal genes such as clusterin or PTGDS since we already known that these genes are widely expressed or present at several levels of the genital tract. These genes that are over-expressed in more than one epididymal transcriptomic unit may have important roles for the cellular functions but may be not directly related to the sperm maturation. Meanwhile the microarray approach we used offer the possibility to analyze also these genes, since they could be retrieved in the list of expressed genes that is deposed on the GEO database (GSE15614).Here we used a 9 K Nylon microarray made during the French AGENAE program which is spotted with 8931 PCR amplified clones. Although this represents only a subset of all genes expressed from the complete genome, one of its advantages is that cDNA clones libraries from the testis and the epididymis were included at the initial step of normalization. Arrays were hybridized to compare gene expression between the testis,  and rat . Doing t and rat  and dataBecause our type of array is not widely used and the biological question more complicated than a simple comparison between two conditions on the same tissue, few statistical tools were available. We had then adapted classical microarray statistical approaches in order to compare the different conditions with a low number of replicates. We have also decided to cross the results from different statistical analysis to ascertain and strengthen our results, especially to define epididymal markers. Our analysis found 2115 transcripts with a differential expression between the 12 conditions; this represents about 24% of the transcripts spotted and 27% of our 7676 qualified spots. In rat about 13% (4000 genes) from the 30000 transcripts of the microarray were found highly regulated along the epididymis and comparable results were obtained in mouse and human.vas deferens). The vas efferens formed one independent cluster.Tissues clustering indicated a good biological reproducibility since arrays from the same tissues from different animals clustered together with few exceptions where one zone could shift from one cluster to the previous or the following one. We did not observe an effect of the breed induced by the 3/4LW-1/4Landrace boars. The clustering procedure builds 7 informative tissue array clusters from our data. The testis was always well separated from the other tissues and the epididymal zones were clustered in anterior-, median-, posterior-caput, corpus and cauda  could be explained by the number of objects included in each analyze: for classifying the 48 microarrays the procedure used 2115 transcripts, while transcripts were classified using only 3 or 4 values for each of the 12 different tissues which reduced the statistical power of the analysis.In contrast, transcripts clustering defined only 4 different co-expression clusters: testis was separated from the rest of the tissues then vas efferens/cauda/vas deferens may have been clustered together because they have some common genes involved in common functions (see GO terms) such as ions binding and transport, or response to the same regulatory factors (for ex: retinoic acid receptor RARRES2 or steroid hormone receptor) or more strikingly gene such as AWN that codes for a specific boar sperm associated protein described mainly in the seminal vesicle [vas efferens showed also a unique and different set of genes expression in agreement with its mixed embryonic origin (embryonic glomerulus and upper Wolffian duct) compared to the epididymis (upper Wolffian duct) and the vas deferens (middle Wolfian Duct) [In that analysis  vesicle , althoug vesicle . Meanwhian Duct) .vas efferens were included. This approach also explains why some of the gene known to be highly expressed by the boar epididymis were not found in the list of markers; these genes were either expressed in two different clusters (ie testis and epididymis) or their high expression spanned over different adjacent clusters and then they were not retained as differential between the epididymal transcriptomic units. This was the case for example for RNase 10 (formerly described as train A), previously found in the pig anterior caput [This discrepancy in the number of clusters between tissues and transcripts has conducted us to analyze the data with particular statistical approaches to assign specific differential transcripts for each of the 7 groups of microarrays. The use of two different approaches has also allowed us to strengthen our results and choose the transcripts with more confidence. Moreover the search for differential genes in each epididymal transcriptomic unit brought us to re-do the analysis excluding testis and VE units. In doing that, we increased the number of transcripts in some of the epididymal clusters but this was not general since some clusters have also lost some genes that became non statistically significant over the epididymis as they were when the testis or the or caput , that waWe have ended with lists of significant over-expressed transcripts and markers for each of the tissue clusters that were further used to validate our microarrays data. For almost all transcripts chosen, we find a very good agreement between the level of expression obtained either by microarrays, RT-PCR and real time PCR. Among these genes used for this validation we have selected a certain number because information on expression in male reproductive tract were available and we have also analyzed new genes and genes not yet reported in epididymis.CLU). The mRNA is present both in the testis and the epididymis as well as different isoforms of the protein. This protein as many different functions  but its exact role in these organs is not yet known. This dual expression was retrieved in boar by microarray and PCR, and CLU was classified in our analysis in the testis due to its higher level of expression in this tissue. This gene is expressed over more than one transcriptomic unit of the epididymis  where the protein secretion and presence in the fluid has already been described [INSL3 and INHA were genes already known to be testicular and were found over-expressed in the boar testis. INSL3, expressed mainly in Leydig cells, mediates the first phase of testicular descent and mutation of this gene leads to cryptorchidism [INHA encodes for the inhibin alpha subunit, mRNA and protein chain have been localized to Sertoli cells, which is the major body source of this protein.The first one was clusterin  was analyzed. This gene has been found in the upper part epididymis of dog and mouse epididymis (named ME9) but not in human epididymis [For the anterior caput, the boar homologue to ididymis ,50. It hididymis . This geGPX5 gene has also been extensively study in the epididymis where it codes for an androgen-dependent glutathione peroxidase specific in the distal caput epididymis of mouse and rat [GPX5 over-expressed in zones 0 to 3. Secreted Phosphoprotein One  is widely distributed and was found over-expressed in vas efferens and caput and at a lower level in cauda epididymis. This protein is involved in different cellular processes such as calcium transport, bone resorption and extracellular matrix (ECM) remodeling. In rat, it has been expressed and localized in testis, vas deferens, and caput epididymis where it was found associated with cellular vesicles and may be involved in fluid calcium depletion [OPN gene region have been significantly associated with male fertility traits, mainly motility and acrosome reaction [ and rat ,52. A ro and rat ,53. In aepletion . In bullepletion . In boarreaction .MAN2B2 is a lysosomal type D-mannosidase secreted in porcine epididymal fluid  [MAN2B2 was mainly epididymal in boar with a caput-corpus specific over-expression. Recently we have described the CES7 carboxylesterase  in the epididymal fluid of various mammals, and its mRNA was found in ram corpus epididymis. Here we showed that like in ram, CES7 expression is restricted to the corpus epididymis where the protein was immunolocalized in the boar fluid [CPE) is a pro-hormone processing enzyme  in neuro/endocrine cells and a peripheral membrane protein of secretory granules where it plays also a receptor role that directs the pro-hormone to the regulated secretory pathway [CPE mRNA in both neural tissues and some non-neural tissues has been shown during embryonic development [CPE gene is over-expressed in the corpus epididymis where the main enriched GO terms are related to reticulum endoplasmic functions. Whether this region secreted specific factors or hormones that need to be processed by carboxypeptidase E remains to be elucidated.osidase) ,58 and wosidase) . Recentlosidase) . Our datar fluid . Carboxy pathway ,62. The elopment  but not We have also focused our attention on several gene families that have been previously described in different epididymal studies.LCN5 ), LCN6, LCN8, LCN9, LCN10 or the PTGDS (prostaglandin D2 synthase) and numerous studies have shown their regional intensities of expression both at mRNA and proteins levels [LCN5, LCN6, LCN8, PTDGS) and shown their regional expressions in boar and we have described new lipocalin members that were not yet observed in mammalian epididymis (UCAL-P19 and APOD (apolipoprotein D)) and a new lipocalin member matching with the human UNQ2541 sequence. UCAL-19 is a 19 kDa lipocalin that has been described in the horse endometrium before embryo attachment where it is secreted abundantly suggesting a role between maternal and foetal tissues dialogue [LCN2, neutrophil gelatinase-associated lipocalin NGAL) which is a 25-kDa lipocalin originally purified from human neutrophils and highly expressed in rodent caput epididymis [UCAL-P19 showed a differential expression by microarray that could not be clearly confirmed by RT-PCR, certainly due to its low level of expression. PCR indicated also that, contrary to rodent, LCN2 had a low and not differential expression in boar. Apolipoprotein D (APOD), a 29-kDa glycoprotein associated with high-density lipoproteins, is predicted to be a member of the lipocalin family based on its primary structure [vas efferens and vas deferens than other epididymal regions. Human UNQ2541 sequence, for which no role or localization information is available, was located on chromosome 9 close to a lipocalins cluster containing LCN6, LCN8 and LCN10. Its expression was very similar to LCN6 and LCN8 expression in boar caput epididymis suggesting they could be controlled by the same factors [Lipocalins are found in vertebrates, insects and plants. They are involved in the transport or storage of hydrophobic and/or chemically sensitive organic compounds, especially vitamins, lipids, steroids and other secondary metabolites . Severals levels . Their edialogue . This geididymis ,66. In btructure . The pro factors .SPAG11) are able to use different exons to produce multiple isoforms of beta-defensin-like sequences [pBD4, pBD108 were found mainly in proximal epididymis while pBD3 was mainly in cauda. pEP2C was found in testis and epididymis while pBD104 and pBD2 only in testis. Other defensins were also expressed , pBD125) in testis and/or epididymis but without differential expression in this last tissue. We have ascertained the identities by sequencing and cysteine motifs alignments of the seven beta-defensin clones found spotted on our microarray. Five of them showed an anterior caput over-expression while the SPAG11B_E isoform had a posterior caput expression. Only DEFB129 was found in corpus. The discrepancy with the previous study could be explained by the fact that these authors used a 45 cycles RT-PCR that is certainly not as discriminative as our microarray analysis. Moreover for this defensin and the two other (DEFB109 and DEFB113), that were also analyzed by RT-PCR, we found a very good agreement with the microarray data. Interestingly the expression profiles of the defensin genes in boar were closely similar to those observed in mouse and rat, even for the SPAG11B isoforms.Defensins, a family of secreted antimicrobial cationic peptides with molecular weights of 4–5 kDa, have a distinctive sequence pattern of six to eight cysteine residues. They are categorized into alpha-, beta-, and theta-defensins according to the arrangement of their three-four disulfide bonds ,69. Defeequences . Specifiequences ,73. In tequences  analyzedequences  in a blaWFDC2  is also over-expressed in different normal and malignant tissues [WFDC2 was mainly found in caput epididymis in mouse and rat and it is a distal cauda epididymal gene in human. In boar, it is present all along the epididymis with an over-expression in cauda. WFDC3 is a testicular over-expressed gene in boar while it is found in testis (round spermatids) and highly expressed in caput/corpus in rat. SLPI is expressed in wide range of tissue and it has been immunolocalized in human epididymis and found in seminal plasma. It has been shown highly expressed all along the rat epididymis and slightly in mouse [SLPI appears as a specific vas efferens gene. We analyzed also an uncharacterized WAP domain-containing protein with an unknown function mainly expressed in the boar cauda epididymis and that was named WFDC10A-like from the closest human sequence found (WFDC10A). The exact role of all these wap domains proteins as inhibitors in epididymis remained to be elucidated as well as their potential role as bacterial defenses. Interestingly, WFDC2, WFDC3, WFDC10A and SLPI are found on the same gene cluster in human chromosome 20. The mouse WFDC10 homologuous gene has also been found highly expressed in mouse cauda epididymis [Proteases and proteases inhibitors have an important role in proteins processing both in tissues, fluids and at the sperm plasma membrane levels during the maturation process. We have focused on several members of different type of protease inhibitors. WAP protease inhibitors contain the WAP four-disulfide core domain structure, a motif first described in whey acidic protein present in the milk of rats and mice. WAP motifs consist of about 50 amino acids with eight highly conserved cysteine residues forming four disulphide bridges. Then WAP protease inhibitors possess like defensins specific cystein motifs and exhibit antimicrobial and antifungal activities (may be due to this wap domain) indicating also a role in the innate immunity ,76. Seven 2, HE4 ,79) and 1, EPPIN ) and mayin mouse ,21. In bididymis .SPAG11), we analyzed two genes for which spliced mRNA forms were found in our data.Because a large number of gene are translated in several spliced mRNAs coding for different proteins and this different spliced mRNAs may be differentially expressed along the epididymis (i.e Cst8 (CRES) in proximal caput where it is secreted in the fluid [CST11) known as CRES2 in mouse is a member of the CST type 2 family. Its mRNA was identified in Macaca epididymis where it is expressed almost exclusively in the epididymis. The protein is most abundant in the initial segment, but is detected throughout the epididymis and on ejaculated sperm [Cst11Δ2) and both recombinant proteins were produced. Interestingly both form of this protein exhibited antibacterial activity. We found the full length and the deleted mRNAs in our spotted clones, and observed by PCR that both mRNA exist and were expressed in the epididymis. Both had a very similar pattern of expression with two maximum  although the deleted transcript showed lower expression intensity. This caput over-expression of CST11 is also reported in mouse and in rat.Cystatins are reversible inhibitors of cysteine proteases. They are found in all living organisms and are involved in various biological processes and also take part in disease pathology ,81. Sevehe fluid . Cystatied sperm . These aEG627821 mRNA (previously EST AV381130) that has been found in mouse epididymis [We also found in our microarray two clones that were similar to the mouse ididymis . This geididymis .SPINK5L2, which is a putative Kazal type serine protease inhibitor 5-like 2. SPINKs are a family of inhibitors with a characteristic signature consisting of six consensus cysteines forming three specific disulfide bonds [SPINK5L2 [SPINKL5 homologue is highly expressed in the corpus epididymis. We found also a gene that is similar to MON1B, a SAND family protein. These proteins have recently been shown to function at the tethering/docking stage of the vacuole fusion as a critical component of the SNARE complex [FXYD2, also known as the gamma subunit of the Na, K-ATPase, regulates the properties of that enzyme. In mouse epididymal transcriptome this gene was not reported and in rat it was found mainly over-expressed in the cauda. In boar it is mainly in the VE where it may play a role in fluid/ion balance.At least we have also analyzed newly discovered genes and genes not yet reported in boar. The first one was de bonds . SeveralSPINK5L2 . None of complex . The MONGP2, Glycoprotein 2 (Pancreatic zymogen granule membrane protein GP-2) gene encodes for a GPI-linked membrane protein that plays an important role in assembly of vesicular compartments targeted to apical plasma membranes via regulated exocytosis. This gene was not found differentially expressed in mouse, rat and human epididymal transcriptome. MUC15 is a novel mucin-like protein that showed structural characteristic of membrane-bound mucins including an extracellular region with several potential glycosylation sites, a putative transmembrane domain, and a short cytoplasmic C-terminal. mRNA presence has been reported in human testis [GLB1L3 mRNA was not previously reported in epididymis but it is mainly found expressed in testis and prostate using the ESTProfile viewer at NCBI. The protein has recently been reported in mice acrosome by proteomic study [n testis  and trann testis , but it ic study . In boaric study .vas efferens or only with clustered transcripts in the three epididymal units. In testis our GO analysis revealed function related to gametogenesis, this is not surprising but gave a good indication on the validity of our data and analysis. As already described some function were common between the vas efferens and vas deferens (fluid and ion transports), but the cauda and vas deferens had functions specific and related to the extracellular matrix remodeling. This enrichment in these GO terms has also been observed in human, mouse and rat, suggesting that an important plasticity of this region exists, may be related to the storage of sperm in that part of the epididymis. In the corpus the main terms were associated with genes involved in the endoplasmic reticulum function and membrane/vesicles trafficking, suggesting protein synthesis and secretory/reabsorbtion pathways. In boar, immunological and histological studies have described the different pathways of secretion and reabsortion of vesicles at the cellular level [The use of microarrays allows also analyzing differential functions through the use of gene ontology terminology. In boar it was difficult to use all the information due to the few genes with a valid GO annotation. We had to transform genes in their mouse, human, rat, dog or bull homologous genes and retrieved when possible the GO term associated. This results in the loss of a large number of transcripts. However we were able to found several functions over-represented in the different transcriptional units when we compared them together. We analyzed the functions either with four clusters including testis and ar level . Althougar level ,15.vas efferens was clustered with cauda and vas deferens, the GO enriched term "coated pit" was associated with the VE. This term was related with four different transcripts over-expressed in this tissue with homology to DAB2_HUMAN, LRP2_HUMAN, LRP1_HUMAN and CUBN_MOUSE. LRP1 and LPR2 are members of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes, and cellular entry of bacterial toxins and viruses. The presence of low-density lipoprotein receptor-related protein-2 (LPR2) has been shown in mouse where LPR2 plays a role in endocytosis of testicular clusterin by the vas efferens [In the caput the main enriched term was receptor activity. Receptors for different modulators such as activin, progesterone and FGF were present and among the 9 receptors forming this category at least four were retinoic acid responders. This is in agreement with previous studies in rodent that have shown the important role of acid retinoic in the functions of the caput epididymis . Althougefferens . Other tDAB2 (disable 2 protein), LRP2  and CUBN (cubilin)) are involved in vitamin D uptake, which is considered as a steroidal hormone. The vitamin D metabolite (25(OH)D3) is synthesized in the liver and circulate in complex with its binding protein (DBP). The messenger for this protein is present in boar testis and zone 2 of the epididymis . The 25(OH)D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin (LPR2), cubilin (CUBN) and the adaptor protein disabled-2 (DAB2), the expression of these proteins is in turn modulated by retinoids [CYP27B1 is expressed all along the boar epididymis and VDR is expressed in the boar testis, vas efferens and epididymis . VDR has also been shown in mouse, rat and rooster epididymis [vas efferens  and then regulates genomic expression of a large number of genes. Vitamin D plays a pivotal role in maintaining serum calcium homeostasis by modulating calcium/phosphate absorption via its action on calcium binding proteins, such as SPP1 (osteopontin) that is over-expressed in boar VE and caput epididymis. Vitamin D is also a regulator of the aromatase in female and male gonad but also in the epididymis [vas efferens region, and control of these mechanisms by estrogen has been demonstrated since ERalphaKO mice are infertile primarily due to a defect in efferent ducts development and function related to fluid reabsorbtion [We noticed that three of the four genes , in the corpus three genes with cell morphology, assembly and development while in the cauda, two main groups of genes formed first a network in cell to cell signaling, morphology and connective tissue development (24 genes) and a second including 11 genes involved in cell death and endocrine system. This demonstrated, if needed, that the epididymis is highly regionalized and this regionalization, regulated by the endocrine system, shows also an important plasticity.CYP17A1, ferritin light and heavy chain (FTL and FTHI), CYB5, DHRS4).We did not attempt to do the same analysis with the testicular transcripts with different species. Meanwhile, although both approaches were quite different, we compared the list of the most intense transcripts from our study with the one recently published in a differential boar testis transcriptomic study between two breeds . Among tvas efferens and the caput might have a certain number of genes regulated by retinoid receptors. Moreover our analysis suggests also that secosteroids (such as vitamin D) certainly carried by the testicular and epididymal fluid may have a role in the efferent ducts and epididymal function. It is interesting to note that in rat, epididymis presents the second body level of this compound just behind the kidney [In conclusion, the present study and transcriptomic studies in other species clearly demonstrated that the high degree regionalization in epididymis is linked to the regionalization of gene expression. This is also well in agreement with the previous secretomic and fluid proteins analysis. This regionalization appears as the results of a specific and subtle regulatory mechanisms from the endocrine system involving androgens and also estrogens. Different studies suggested also a numbers of other regulatory components such as retinoid derived from vitamin A. Our study indicated that the e kidney . Then thAll the transcriptomic studies have found that family of genes such as defensins or different type of protease inhibitors are over-expressed in different places of the epididymis. These proteins have role in the innate immunity response against infection from bacteria and fungus. The presence of such large amount of these compounds may be a necessity to reduce or prevent the presence in the fluid or within the spermatozoa from cells of the immune system that could after developed autoantibodies deleterious for reproduction. It is likely that different defensins are much more than antimicrobial, and further functional work will uncover different roles for what is increasingly regarded as a multifunctional gene family. Anti-proteases may also be involved in the regulation of the sperm and fluid proteins processing that occurs during maturation and are part of this maturation process.At least all these transcriptomic studies allow us to see through the high complexity and specialization of epididymis and differences between species. Numerous genes are under the same common regulators and then could be targets for future studies to understand the mechanisms involved in epididymal regionalization and those implicated in sperm maturation.Four adult boars ) aged from 18 to 36 months were used in this study (average weight was 301 ± 17 Kg). These animals were provided by insemination centers where they were used for genetic improvement. For each animal, different criteria of the semen quality  were followed during their reproductive life and considered as good or very good. Their fertility calculated on at least 150 artificial inseminations ranged from 88 to 94% .vas efferens (VE), nine define epididymal zones (zero to 8/9) and vas deferens (VD) were snap frozen and used further for mRNA extraction . The animals were sacrificed at our local slaughterhouse and the testis and epididymis were removed after bleeding. The testicle and epididymal weights were 626 ± 53 g and 135 ± 7 g respectively. The tissues were carefully dissected in less than 30–40 min after the death of the animal in order to minimize the mRNA degradation. The maximum of interstitial tissue and blood vessels were removed from the tubule. Samples from the testis, Frozen tissue (150 to 200 mg) was homogenized in 2 ml of Tri-Reagent  using an Ultra-Turrax. The RNA extraction was performed according to the manufacturer's instructions with small modifications. A second extraction was performed on the aqueous phase to optimize the RNA quality. For all samples, RNA quantity and quality were measured by absorbance ratio at 260/280 nm  and the integrity of the mRNAs was determined by both standard agarose gels and mRNA-chip gel system using an Agilent Bioanalyzer . Only samples passing these tests were kept and for sample that failed, extraction was redone on a different piece of sample in order to meet these criteria of quality. under the reference number GPL3729.The set up of the AGENAE cDNA library and of the 9 K cDNA nylon microarray used in this study has been published in detail previously . The mic33P-labelled 19-mer oligonucleotide (CACTATAGGGAATTTGGCC), which matches a sequence present at the extremity of each PCR product to estimate the amount of cDNA dropped on each spot. Only membranes with a good pattern of spotting were retained and were attributed at random for the hybridization with the 33P-cDNA from the tissues. After stripping , hybridizations were carried out as previously described [33P-dCTP, 2.4 μM dCTP, 0.4 mM each dATP, dTTP, dGTP, 40 units RNasin® Ribonuclease Inhibitor  and 200 units MMLV SuperScript RNase H-reverse transcriptase  in 30 μL final volume. RNAs were degraded by treatment at 68°C for 20 min with 1 μL 10% SDS, 1 μL 0.5 M EDTA and 3 μL of 3 M NaOH, and then equilibrated at room temperature for 10 min. Neutralization was done by adding 10 μL 1 M Tris-HCl, 3 μL 2 N HCl, 2 μL of an 80 polyA oligomers (1 μg. μl-1) and 50 μL of hybridization solution then by incubating during 5 min at 99°C and 1 hour at 65°C.Forty-eight RNA samples originating from the four animals were used for microarray hybridization according to the following procedure. A first hybridization was performed using a escribed , with miArrays were then incubated with the corresponding denatured labeled cDNA for 18 h at 65°C in hybridization solution. After 3 washes , they were exposed to phosphor-imaging plates (24 or 48 hours) before scanning using a FUJI BAS 5000 . Each array image was inspected using AGScan software ,105 to e2-transformed, normalized by the median of intensities by block then by array. Finally, signal processing was performed using vector oligonucleotide data to correct the relative amount of cDNA present in each spot. The microarray data from this research has been deposited in the NCBI Gene Expression Omnibus data repository under accession number GSE15614.Spots for which the vector oligonucleotide data had intensities lower than a cut-off value established as a mean level intensity of water control spots plus 3 times the standard error deviation were excluded from analysis. This cut-off value was calculated for each microarray independently. Then data corresponding of all hybridizations (vector and labeled cDNA) were log.Statistical analyzes were performed using the R software version 2.4.1  and packages that can be downloaded either from the CRAN or the BioConductor site The search for differentially expressed transcripts had two purposes: first, find transcripts that clustered our data and second, find transcripts that were preferentially over-expressed in a particular condition  or between 10 samples , the moderated F-test available in the Limma R package was used [To determine differentially expressed transcripts between 12 samples  hierarchical clustering algorithms are the most commonly used. Hierarchical methods are especially useful, because they enable to simultaneously examine groupings of the data into many small clusters (e.g. repeat samples from the same tissue) and a few large clusters (e.g. different treatment groups). The use of a single hierarchical model ensures that groups at different levels are nested, facilitating interpretation. Bottom-up clustering, which successively joins objects, is good at identifying small clusters, but can provide sub-optimal performance for identifying some large clusters. Conversely, top-down methods are good at identifying a few large clusters, but weaker when there are many small clusters. Because we had no a priori on the number of classes expected, but a major weakness of the hierarchical algorithm is that an improper fusion of data at an early stage cannot be corrected later. In order to correct this weakness, at least partially, we decided to add a second clustering step with the partitioning method. We choose the PAM algorithm because, compared to the k-means approach, it: (a) accepts a dissimilarity matrix , (b) is more robust as it minimizes a sum of dissimilarities instead of a sum of squared Euclidean distances, (c) provides a graphical display, the silhouette plot, which allows the user to select the optimal number of clusters [We then defined an original combination of statistical methods using: (1) a hierarchical method  and (2) a partitioning method (Partitioning around medoid: PAM). The hierarchical method is an unsupervised method: the analysis of the data is done without any clusters . The PAMTo identify some transcriptomic units defined by the agglomeration of membrane arrays inside of which the majority of the studied transcripts have similar expression profiles, we used the list of differentially expressed transcripts obtained from the Limma analysis. To determine the number of classes for which our data are the best classified, we used the PAM from the Cluster R package . The PAMThe aim of this analysis was to identify some characteristic evolution of gene regulation between the different tissues studied. This classification was performed similarly as the zone clustering, using the list of differentially expressed transcripts obtained with the Limma package.Sus scrofa (Build #33), Homo sapiens (Build #213), Bos Taurus (Build #92), Mus musculus (#173), Rattus norvegicus (Build #175) and Canis familiaris (Build #22) using BLASTN (v2.2.18) [Sus scrofa database, we kept only sequences (subjects) with a global High-scoring Segment Pair (HSP) length of at least 200 nucleotides and 80% of identities with the cDNA sequence (query). For other databases, only sequences with a global HSP length of at least 200 nucleotides and 70% identities with the cDNA sequence were kept. For EST sequences with a length around 200 nucleotides that were still not annotated, we used the following criteria to filter their blast results: for Sus scrofa database, we kept only sequences with a minimum global coverage of 50% of the cDNA sequence with 80% of identities. For other databases, we kept only sequences with a minimum global coverage of 50% of the cDNA sequence with 70% of identities. Then for cDNA sequences that pass these filters, we kept the UniGene ACC corresponding to the sequence with the best score for each species to annotate the corresponding clone.Because pig genome is not yet entirely sequenced and assembled, in order to obtain the name of the gene coding for the cDNA clones spotted on the microarray and hence the maximum of physiological information, we have compared the complete (when available) or partial sequence (EST) of clones with the NCBI UniGene "unique" database  of different mammalian species. cDNA sequences were compared to UniGene v2.2.18) . Two cri. When a UniGene ACC mapped to several UniProt ACC, we kept the one corresponding to the SwissProt databank. These UniProt ACC were used to annotate the corresponding clone.The UniGene ACC were further used to obtain the corresponding UniProt ACC using the ID Mapping tool on the UniProt web site .Gene ontology annotations associated to the UniProt ACC were obtained on the Gene Ontology Annotation (GOA) Database To have the maximum information, we worked with a multi-species approach. For each clone, we have kept the Gene Ontology information available for the UniProt ACC corresponding to the Homo UniGene ACC hit, otherwise we have kept the Gene Ontology information available for the UniProt ACC corresponding to the mouse UniGene ACC, then to the rat, then to the bovine, to the dog and at least the pig. The Gene Ontology term enrichment analysis was done with the data corresponding to transcripts clustered by classification analyses with 12 and 10 tissues for the 3 main GO terms independently . For each cluster, we have retained a unique UniProt ACC (to eliminate multiple spotted genes). We have searched for Gene Ontology terms that were enriched in one cluster compare to all the clusters obtained by the classification analyses with 12 and 10 zones using EASE program . A Benja15 primers. PCR was performed at the temperature specified for the primer sets  and oligo(dT)RPL19.For each set of primers . Column 4 gives the transcript cluster in which it was enriched. The last 5 columns show the index on the array, UniGene ACC, UniProtKB ACC, UniprotKB ID and UniprotKB definition of transcripts corresponding to GO terms enriched, respectively.Click here for fileTranscripts corresponding to analysis of Gene Ontology term enrichment for clustering with 10 tissue samples. For each GO term defined as enriched, the first column gives its definition, columns 2 and 3 show its GO ID and the class it belongs to . Column 4 gives the transcript cluster in which it was enriched. The last 5 columns show the index on the array, UniGene ACC, UniProtKB ACC, UniprotKB ID and UniprotKB definitions of transcripts corresponding to GO terms enriched, respectively.Click here for fileCommon over-expressed genes in the same epididymal unit between mammal species . For each gene found over-expressed in the same epididymal unit between the 4 species, column 1 gives the unit for which it was found to be over-expressed. Column 2 shows its index on the pig array and the last 2 columns give its UniGene ACC and UniGene definition in mouse species.Click here for fileList of boar potential markers for which a knockout has a reproductive effect on mouse (MGI database). For each potential marker for which a knockout has a reproductive effect on the mouse, column 1 gives the transcriptional unit for which it is a potential marker. Column 2 shows the UniGene ACC corresponding to this marker used to obtain the Mouse.Ensembl.Gene.ID (column 3). Column 4 gives the MGI ACC associated with the marker and columns 5 and 6 show the symbol and the name of this marker, respectively. The last column gives a short description of the reproductive system phenotype.Click here for fileStatistical methods. This flowchart summarizes our statistical methods in 3 steps. First (in blue), we search for differentially expressed transcripts between our 12 or 10 tissue samples in order to discover the classes of co-expressed transcripts and transcriptional units. Results of sample classification were used to search for over-expressed transcripts in a single transcriptional unit (orange). Finally, potential markers were transcripts which were common for one unit in cluster and over-expressed lists (purple).Click here for fileGene-specific primers used for realtime PCR study. For each primer pair is given the Gene Symbol, the Gene name, sequences of forward and reverse primers, the length of amplified fragment and the GenBank Accession of the sequence used to design primers.Click here for file